Bottom‐Up Synthesized Glucan Materials: Opportunities from Applied Biocatalysis

Linear d‐glucans are natural polysaccharides of simple chemical structure. They are comprised of d‐glucosyl units linked by a single type of glycosidic bond. Noncovalent interactions within, and between, the d‐glucan chains give rise to a broad variety of macromolecular nanostructures that can assemble into crystalline‐organized materials of tunable morphology. Structure design and functionalization of d‐glucans for diverse material applications largely relies on top‐down processing and chemical derivatization of naturally derived starting materials. The top‐down approach encounters critical limitations in efficiency, selectivity, and flexibility. Bottom‐up approaches of d‐glucan synthesis offer different, and often more precise, ways of polymer structure control and provide means of functional diversification widely inaccessible to top‐down routes of polysaccharide material processing. Here the natural and engineered enzymes (glycosyltransferases, glycoside hydrolases and phosphorylases, glycosynthases) for d‐glucan polymerization are described and the use of applied biocatalysis for the bottom‐up assembly of specific d‐glucan structures is shown. Advanced material applications of the resulting polymeric products are further shown and their important role in the development of sustainable macromolecular materials in a bio‐based circular economy is discussed.


Introduction
The "defossilization" of the material sector of industrial chemistry is a cornerstone of all current roadmaps to a bio-based circular economy. [1]Polymers derived from renewable feedstocks enable the carbon-neutral production of materials with reduced environmental footprint. [2]They represent the sustainable macromolecular materials of the future.Carbohydrate-based DOI: 10.1002/adma.202400436polymers (polysaccharides) and derivatives thereof constitute a large class of bio-based materials.They are versatile in chemical and supramolecular structure and offer a diverse range of functions important for industrial applications. [3]The wide spectrum of uses of different nanocellulose materials can serve here as a prominent example. [4]Besides applications unique to them, polysaccharide materials also have significant potential to replace a notable fraction of the synthetic polymers currently in use. [5]As an example, compostable bio-based plastics made from starch blends with polyesters are emerging materials for flexible packaging applications. [6]atural polysaccharide materials are derived from homo-or heteroglycan polymers and can be composites of several polymers, including other glycans.Even in homoglycans that are built from just a single type of monosaccharide, there exists broad variation in the chemical structure due to differences in glycosidic linkage used. [7]he linkages of the glycan chain can be uniform as in cellulose (a -1,4-d-glucose polymer) [8] or they alternate along the chain in a repetitive manner, as for example in pullulan, which is comprised of -1,4-dglucose trisaccharide units linked by -1,6-glycosidic bonds. [9]lycan structural diversity is further increased by branching or by side-chain substitution of the polysaccharide backbone. [7]4a,10] Here we are primarily interested in linear homoglycans built from d-glucose, so-called d-glucans.The enormous structural diversity of natural and synthetic glycans necessitates a suitable restriction of the scope of this article.The focus on "simple" d-glucans arises here from considerations of both relevance and application potential in the modern material sciences.10c,d,11d,12] Despite notable efforts, such relationships are exceedingly complex to unravel for heteroglycans (e.g., glycosaminoglycans, [13] hemicelluloses, [14] and others).Lastly, approaches of bottom-up synthesis of glycan represent a center   b) The linkage of branched glucan is shown as -b-, with the main chain underlined; c) Pullulan is produced aerobically from starch by A. pullulans; d) Pullulan is composed of consecutive maltotriose (-1,4 linkage) units that are connected to each other by an -1,6-glycosidic linkage.
point of this study and these have been developed primarily for d-glucose polysaccharides. [15]able 1 summarizes the natural d-glucose disaccharides and the different polysaccharides derived from them.Applications of these polysaccharides span the full variety of industrial materials [3a,4a] and further include nonmaterial uses in the food sector (e.g., nondigestible fibers [16] ) as well as in medicine (e.g., ingredients with immunomodulating bioactivity [17] ).
4a,19] The properties of the isolated glucan vary strongly with the processing conditions used during isolation.Processing usually requires multiple steps and the unit operations used are often energy-intensive (e.g., milling) and can generate large amounts of waste (e.g., when hydrolysis with strong acids is used [19a] ).11b,19b,20] Various glucans from plants and microorganisms have been considered as ingredients for high-fiber food products but their extraction from the natural source is complex and costly. [21]20b,21,22] Plant-derived nanocelluloses typically contain hemicellulose (∼5%) and lignin (∼1%). [22]Fungal glucans (e.g., -1,3-d-glucan) are often isolated as a mixture with proteins and other structurally diverse (poly)saccharides. [23]Additionally, the top-down processing can involve a severe disruption of the original d-glucan structure via depolymerization of the polysaccharide chain, chemical modification of monosaccharide units, or both. [24]All these features can make it difficult to control the quality of the final d-glucan product obtained by the top-down approach (Figure 1).

Bottom-Up Synthesis of Glycans
15a,b,e,25] The d-glucan materials are generally obtained in two steps.First, the oligo-/polysaccharide chain is synthesized through iterative polymerization from suitable precursor monomers or oligomers.12a,15a,b,d,e,26] The bottom-up approach usually offers a higher synthetic precision of polymer structure than achievable through top-down processing.15b,e,27] The morphology of the resulting material can often be fine-tuned by the conditions of bottom-up synthesis.15d,27,28] The top-down approach necessitates chemical modification of the glycan chains within the structurally organized material, which is usually more difficult to attain. [21,22]15a,29] Overall, therefore, the bottom-up approach of polysaccharide synthesis is of great interest in the field of functional glycan materials.Synthetic precision is key for the studies of structurefunction relationships and ensures product quality.10c,12a,30] Material production may become more efficient due to better control, fewer steps used, and reduced waste generated.
31b,32] Glycosyl donor reactivity versus selectivity is a long-standing issue of chemical glyco-sylation.31b,33] The well-established procedures of chemical glycosylation preclude an aqueous solvent. [34]As the synthesized glycan grows in length, however, its solubility in the most widely used organic solvents (e.g., dichloromethane, methanol) decreases dramatically. [35]This can limit the size and concentration of the glycan attainable in the synthetic reaction.
31b] The protocols minimize the number of process steps by aligning the reactivity of the glycosyl acceptor group to that of the glycosyl donor leaving group.Additionally, they eliminate largely the need for intermediate purification.Programmable one-pot approaches integrate modern machine learning (e.g., software Auto-CHO) to guide the design of building blocks for an efficient synthesis. [36]15d,37] It is relatively flexible concerning the structure of the target product and can be transformed into an automated process.The synthetic scale is however limited due to the excess amount of building blocks required in each glycosylation step.Application of automated glycosylation strategies was demonstrated in the preparation of polysaccharides (DP ≥ 100) [38] and various oligosaccharides of complex molecular structure (e.g., heparin [39] ).In summary, despite the important advances made in chemical glycosylation for diversity-oriented glycan synthesis, the transferability of the method(s) for development of a production process at larger scale is not clear.In this review article, we show that biocatalysis with enzymes offers promising solutions to the challenges of bottom-up synthesis of homoglycan materials.
Enzymes, mostly polysaccharide hydrolases, are well-known for use in top-down processing of natural glycan materials. [40]15b,c,26] The excellent stereo-and regiocontrol afforded by many carbohydrate-active enzymes would greatly benefit the glycan synthesis, by enabling one to work in water without the requirement for protecting groups.15a,41] The overall conversion is also stepeconomic in that it does not require the isolation of intermediates.Solvent consumption is low and reagents used are nontoxic.The reactions are well scalable in principle and can be optimized for production.With technologies available to also reduce the enzyme costs of synthesis (e.g., protein engineering for enhanced activity and stability; [42] immobilization for enzyme catalyst recycling [43] ), the bottom-up production of glycan materials is becoming an increasingly feasible option.In the extent that the polymerization-driven self-assembly of the glucan chains happens under kinetic control, the parameters of the enzymatic synthesis can affect the nano-and mesoscale structural features of the glycan material. [44]This presents interesting opportunities to fine-tune the material structure and function by the conditions of the biocatalytic synthesis.
Despite authoritative reviews on enzymes for glycan synthesis, [15b,f,28b,45] the important interconnection between enzymatic polymerization of the polymer chain and the property of polysaccharide material has not been worked out in detail.The present study focuses on d-glucan materials synthesized enzymatically.However, the here discussed strategies of bottomup assembly of simple polysaccharide chains are applicable in principle to other homopolysaccharides and structurally more complex heteropolysaccharides.Current limitation however is that the glycosyl donors for the synthesis of "nongluco" glycans are not readily available.

Enzymes for d-Glucan Synthesis
Four types of enzymes are known for d-glucan synthesis: glycosyltransferases, [46] phosphorylases, [47] transglycosidases, [45c,d] and glycosynthases [48] (Figure 3).Glycosyltransferases (EC 2.4) are the natural enzymes of biological synthesis.46c] The primer is generally not a free d-glucose but a short oligosaccharides.The oligosaccharide primer is often anchored on another molecule such as lipid (e.g., sitosterol-cellodextrins [49] ) and protein (e.g., glycogenin [50] ).The mature polysaccharide is later cleaved off. [49,51]Based on the similarity in amino acid sequence, glycosyltransferases are classified into families in the CAZy (carbohydrate-active enzymes) database. [52]The glycosyltransferases of d-glucan synthesis are found in glycosyltransferase families of GT2, GT5, GT48, and GT84.Enzymes are specific for glycosyl transfer with inversion or retention of the -anomeric configuration of d-glucose nucleotide in the product, to give a or -d-glucan polymer, respectively. [53]Invertive or retentive reaction is a conserved property of the enzymes within a particular GT family. [54]hemically, the glycosyl transfer is a nucleophilic substitution at the anomeric carbon of the glucosyl residue that proceeds in an axial-to-equatorial or axial-to-axial reaction. [54]ccording to EC categorization (EC 2.4), the phosphorylases are also transferases of a glucosyl residue. [55]45e,56] In the reverse direction of reaction, sometimes referred to as synthesis, phosphorylases use dglucose 1-phosphate for d-glucan polymerization. [57]Phosphorylases operate with inversion or retention and there exist enzymes specific for the or -configured form of d-glucose 1-phosphate.The stereochemical course of the phosphorylase reaction from d-glucose 1-phosphate can be  →  (e.g., glycogen/starch phosphorylase), [58]  →  (e.g., cellodextrin phosphorylase), [15a] and  →  (e.g., maltose phosphorylase). [59]Within the CAZy database, phosphorylases are classified in glycosyltransferase families (GT4, GT35) and glycoside hydrolase families (GH13, GH94, GH149, and GH161).
45d] These are glycoside hydrolases (EC 3.2.1)that transfer a glucosyl residue from substrate not just to water as the EC class implies, but also to a glucose or gluco-oligosaccharide acceptor.Transglucosi-dases are distributed across the CAZy glycoside hydrolase families.15b,28a,45b,60] Each type of enzyme involves distinct opportunities but also notable issues in material synthesis, as discussed later.
Thermodynamic versus kinetic control of synthesis.46a,b] A glucose nucleotide is typically higher in energy than glucose 1-phosphate.46b] The d-glucan product can be hydrolyzed as it accumulates.Control of reaction time is critical in the optimization of synthesis with transglucosidases.The product yield is usually lower than it is in the reactions of glycosyltransferases and phosphorylases.Reactions of glycosynthases are thermodynamically controlled in principle.46a,62] Chemoselectivity and regiocontrol.Glycosyltransferases and phosphorylases are highly chemoselective for glucosyl transfer from the sugar nucleotide or d-glucose 1-phosphate donor to the acceptor. [53,56]Hydrolysis of the donor is absent or represents just a scant side reaction.Transglycosidases hydrolyze the donor substrate in an extent that depends on the enzyme and the conditions used. [63]Hydrolysis competes with transfer.63b,64] Unreacted acceptor must be removed during product isolation. [65]46a,66] For native and engineered transglycosidases, product yields of greater than 50% are typically considered to be sizeable by most authors. [67]The transfer/hydrolysis ratio is an important parameter in transglycosidase discovery and engineering (Figure 3). [64,68]62a] A suitable transfer/hydrolysis ratio is critical for efficiency in synthesis.
Regiocontrol of the glucosylation is usually an enzyme-specific property.A suitable enzyme catalyst must be identified for the particular synthetic task.However, glycoside hydrolases in general exhibit a more relaxed regiospecificity, i.e., are less precise, than glycosyltransferases and phosphorylases.
Control of product DP and dispersity.The bottom-up synthesis of glycans by enzymes is generally believed to happen in the liquid phase when the nascent polymer chains are still somewhat soluble and have not yet self-assembled into solid material.The fully formed material may no longer be substrate for the enzymes to further polymerize the polymer chains. [69]Conditions of in vitro synthesis differ substantially from those of biosynthesis in the cell.As an example, the cellulose chain DP in biocatalytic synthesis ranges from ∼9 to 200 depending of the enzyme used (e.g., cellodextrin phosphorylase, cellulase, cellulose synthase) [69,70] whereas the natural cellulose chains produced by cellulose synthase in the plant cell wall are several thousands of dglucose units long.The example highlights the fact that polysaccharide materials obtained from bottom-up synthesis and topdown processing of natural feedstocks can be very different, and potentially be highly complementary, in the properties of structure and function offered.Generally, the maximum length of chain and the average DP in glycan synthesis depend on, and so can be tuned by, the reaction conditions used.61a,72] Processive polymerization means that a single glycan chain is elongated at the enzyme active site by adding multiple monomer units without intermediate release of the growing chain.Nonprocessive polymerization, by contrast, involves the intermediary release of the nascent glycan after each addition of monomer.The known enzymes of d-glucan synthesis act processively (e.g., cellulose synthase, [72a] cellulase, [72b] starch synthase [72c] ) or nonprocessively (e.g., cellodextrin phosphorylase [61a,72d] ).Glycogen phosphorylase may exhibit a mixed processive/nonprocessive behavior of activity due to the presence of a "glycogen storage" site in the enzyme (Figure S1, Supporting Information). [73]Dispersity of the resulting d-glucan oligomer/polymer is expected to be larger when the polymerization mode is nonprocessive compared to when it is processive.Assessment of the polymerization kinetics is thus an important element of reaction characterization and optimization in biocatalytic bottom-up synthesis of dglucans.Kinetically controlled reactions intrinsically involve the requirement of control of the reaction time or progress of conversion.
Availability and suitability of donor substrate.15b,26,28a,60] The donor substrate should be favorable from at least three points of view: availability/costs, kinetics/reactivity, and thermodynamics/internal energy.Activated glycosynthase donors such as d-glucose 1fluoride must be synthesized chemically and are prone to an uncatalyzed decomposition during the reaction (e.g., hydrolysis of -d-glucose 1-fluoride). [74]The released fluoride ion should not remain in the product.48b,75] Donor substrates of transglucosidases can be disaccharides (e.g., sucrose as mentioned [45c,76] ) or oligosaccharides. [77]Still, synthetic glucosides are used often.46a] The d-glucose 1-phosphate appears to be more expedient by comparison, but it would also be better provided from a different substrate source than be supplied directly. [66]Glycosyltransferase and phosphorylase reactions are therefore performed as cascade transformations that involve the in situ formation of the actual glycosylation donor from a simple "glucosyl sugar" substrate (Figure 3).Sucrose is a wellstudied substrate to achieve release of glucose nucleotide or -dglucose 1-phosphate. [57,78]The reactions are shown in Figure 3. Starch is an additional source of -d-glucose 1-phosphate. [79]The atom economy of glucosyl transfer from sucrose is only 50%.d-Fructose is released as a coproduct. [57,80]The atom economy in respect to supply of -d-glucose 1-phosphate can be improved when starch is used.However, depolymerization of starch by phosphorylase stops at the level of trisaccharide due to enzyme specificity. [81]Moreover, sucrose is higher in energy than maltose (the repeating unit of starch), [82] making sucrose usually the more efficient glucosyl donor for the synthesis of d-glucan via intermediary -d-glucose 1-phosphate.Nonetheless, starch is a valid alternative from the overall perspective of d-glucan production process. [79,83]omplexity and scalability of reaction.Enzyme application in the synthesis of d-glucan materials requires a robust biocatalytic process technology.Enzymes must be made available in suitable amounts, ideally by recombinant overproduction in microbial hosts.They must be stable under conditions of use (e.g., solvent) and should exhibit high specific activity/protein mass (≥10 U mg −1 ). [84]Synthetic reactions should avoid the requirement for extensive optimization and minimize the effort in product isolation.The four types of enzymes discussed differ in these characteristics of process technology.45c,76,85] Glycosynthases exhibit comparable process-related properties as transglucosidases with the exception that their specific activity can be lower considerably.15b,28a,60,86] In a very general assessment overall, [46a,87] glycosyltransferases are less robust in all relevant parameters than the other enzymes.Enzymes used to convert sucrose for supply of -d-glucose 1-phosphate (sucrose phosphorylase) and UDP-glucose (sucrose synthase) reflect the difference in robustness characteristically (Table S1, Supporting Information).Robustness is critical in process scale up.
The reactions catalyzed by transglucosidase and glycosynthase require only a single enzyme to proceed.46b,88] Glycosyltransferase and phosphorylase reactions are often designed as two-enzyme cascade transformations in which the glucosyl donor is an intermediate (Figure 3).61b,78,89] Application of these reactions is discussed for the different d-glucan materials.
Product recovery from the biocatalytic synthesis is critically important for process development.Relatively few studies examine it in detail with emphasis placed on the process technology.Inactivation (e.g., by heat treatment) and removal of the enzymes (e.g., by ultrafiltration) are often among the first steps of downstream processing that are carried out. [90]91b,92] The ultrafiltration can be performed in dead-end or tangential flow mode. [93]Standard methods of precipitation involve the use of alcohol cosolvent (e.g., ethanol, isopropanol) in concentrations typically in the range of 40-80 vol%. [41,94]The precipitated products are recovered by gravity separation, centrifugation or microfiltration.91b,95] Solvent removal from product involves freeze drying or spray drying. [96] Enzymatically Synthesized -Glucan Materials

Structure and Properties
-1,3-Glucans are water-insoluble polysaccharides.They are prominently found in the cell walls of filamentous fungi (e.g., Penicillium sp., Aspergillus sp.).They play an essential role for maintaining the cell integrity. [97]In Aspergillus species, the -1,3-glucans represent up to 32% of the total cell wall carbohydrates. [98]The glucans are soluble in alkaline aqueous so-lutions.This enables their isolation via alkaline extraction and neutralization-induced precipitation.The -1,3-glucan from A. wentii is comprised of -1,3-linkages exclusively and it has a molecular mass of ∼850 kDa (DP ∼5000). [98]The -1,3-glucan is a crystalline material that exists in several polymorphic forms, often referred to as I-IV. [99]The native form of the -1,3-glucan in the fungal cell wall is designated as polymorph I, based on X-ray diffraction analysis of tissue (trama) from fruiting bodies. [100]The alkali-extracted -1,3-glucan is referred to as polymorph II.It exhibits a two-chain orthorhombic structure. [100]The polymorph III structure is observed in anhydrous -1,3-glucan derived by dehydration of polymorph II material.It also involves a two-chain orthorhombic crystal structure.Polymorph II and III -1,3-glucan structures are reversibly interconverted into one another. [101]The polymorph IV structure is observed in an anhydrous -1,3-glucan regenerated from material previously acetylated, stretched, and deacetylated. [102]nly little work has been done to explore the biological activity of isolated -1,3-glucans.23b] The sulfated, thus watersoluble -1,3-glucan from Streptococcus mutans MTCC 497 was shown to exhibit fibrinolytic, anti-inflammatory, and antimicrobial properties. [103]The sulfated glucan had a molecular mass of 1.2-9.0kDa and a degree of substitution DS (the number of sulfate groups per glucose residue) of 1.2.The rather low molecular mass was attributed to the degradation of native glucans during sulfation at a temperature of 80 °C. [103]The -1,3-glucans from Boletus edulis (850 kDa, -1,3-linkage ratio 67%) and Lentinus edodes (-1,3-linkage ratio 86%) were considered for use as biosorbents, to achieve removal of heavy metals from wastewater (e.g., Pb 2+ , 50-90% efficiency). [104]ifferences in the biological raw material (e.g., fruiting body or mycelium of the fungus) and the procedure used for -1,3glucan isolation cause variation in the structure (e.g., presence of -1,4-linked glucose residues located in the center or at the reducing end of the chains, accounting for ∼9% linkage ratio, in the -1,3-glucan from Aspergillus sp.) [23b,97a] and purity (e.g., contamination with other sugars such as mannose and xylose) of the final preparation.This limits the efficiency of the top-down approach for -1,3-glucan production.

Bottom-Up Enzymatic Synthesis
To prepare pure -1,3-glucans, bottom-up synthesis is a promising approach.Different types of enzymes might be usable for the task.A membrane-bound glycosyltransferase referred to as -1,3-glucan synthase (AGS1; from Schizosaccharomyces pombe) uses UDP-glucose to elongate -1,3-glucans as the cell surface polysaccharides. [105]The Aspergillus species also contain such an enzyme. [106]Based on sequence similarity, the -1,3-glucan synthases have been classified into glycosyltransferase family GT5.The proposed function of the enzyme in -1,3-glucan biosynthesis has not yet been confirmed in vitro at the level of the isolated protein.The proposed glycosyl transfer from UDP-glucose proceeds with retention of anomeric configuration ( → ).The -1,3-glucoside phosphorylase from Clostridium phytofermentans (a member of glycoside hydrolase family GH65) catalyzes the synthesis of -1,3-oligomers (DP 3-4) using nigerose (10 mmol L −1 ) and -d-glucose 1-phosphate (10 mmol L −1 ) as the substrates. [107]he phosphorylase reaction involves inversion of configuration ( → ).The strategy of phosphorylase reaction coupling (see Figure 3) is promising here to provide the costly donor substrate in situ.The -d-glucose 1-phosphate can be released from ,trehalose or maltose using the relevant disaccharide phosphorylase and an approach of one-pot cascade reaction is suggested for the synthesis. [108]In a different approach, the strain Leuconostoc pseudomesenteroides G29 grown on sucrose (80 g L −1 ) was found to release an extracellular glucansucrase (∼1.5 U mL −1 ) into the fermentation broth.This enzyme is the functional equivalent of the glucosyltransferase GtfJ referred to below.A crude enzyme preparation was used for the synthesis of an insoluble -glucan product (3300 Da, -1,3-linkage ratio 93%) from 100 g L −1 sucrose (pH 5.5; at 30 °C in an overnight reaction). [109]n vitro -1,3-glucan production was shown by GH70 transglucosidases from Streptococcus sp.(referred to as -1,3glucosyltransferase GtfJ), as shown in Figure 4a. [110]Mechanistically, the enzyme uses a two-step double displacement-like reaction that proceeds through a covalent, configurationally inverted -glucosyl enzyme-intermediate.Two catalytic steps with inversion give retention of the -configuration of the sucrose donor substrate. [111]The enzymatic transglucosylation is largely regioselective at the O-3 of the terminal -glucosyl residue of the nascent -1,3-glucan chain.111b,c] Glucose is preferred when available in sufficient amounts. [112]During polymerization, nigerose and nigerotriose (DP 2-3) are initially generated.110b,113] The as-synthesized -1,3-glucan is lacking branches entirely.Its weight-average molecular weight (M w ) is 700 000 with a PDI of 1.9, received from reaction with 1 mol L −1 sucrose at pH 5.5 and 15 °C for 14 d. [114]The -1,3-glucans, manufactured through the GtfJ reaction, are now produced at an industrial scale by International Flavors & Fragrances Inc.The resulting insoluble -1,3-glucans are separated through centrifugation or filtration, and the dry materials are acquired through a vacuum freeze-drying process. [115]The commercial product Nuvolve is reported to have average DP 800 and PDI 1.7-2.0. [116]Two forms of -1,3-glucan, a dry powder (88 wt% solids) and a wetcake preparation (40 wt% solids), are commercially available.It exhibits a bulk density of 1.5 g cm −3 in its dried state and is identified as a biodegradable material with degradation properties similar to cellulose (ASTM D5511 and ISO 17556 assessment).

Material Development
The large-scale availability of the -1,3-glucan has promoted the deeper characterization of its polymer properties in relation to other industrial polysaccharides (e.g., cellulose, starch).The processing-and formulation-dependent material characteristics of -1,3-glucans are crucial to consider these polysaccharides for industrial production.
Morphology.The -1,3-glucan from the GtfJ reaction (1 mol L −1 sucrose, pH 6.8; at 37 °C for 6 d) is a fibrillar material of a mainly wavy-fibril morphology (Figure 4b-i; width, 13 nm; thickness, 20 nm; length, up to 1 μm).Thin lamellae are also observed (Figure 4b-ii; thickness, 5 nm; width, 30-40 nm; length 0.1-0.5 μm), but to a smaller extent. [99]The two different materials showed the same hydrated crystalline form (polymorph II), which was changed upon dehydration (polymorph III).Peculiar feature of the fibrils is that the -1,3-glucan chains (M w 5400; DP ∼33) are not extended parallel to the fiber axis as with the other fibrillar polysaccharides (e.g., cellulose), but are folded perpendicularly to the long c-axis of the fibrils, giving a width of 13 nm (equivalent to DP 31; Figure 4b).In -1,3-glucan fibrils, the c-axis is the direction of crystal growth, where the glucose rings are hydrophobically stacked in the same manner as in other polysaccharide crystals.The minor lamellar morphology is assumed to feature chains assembled perpendicularly to the base plane of the lamellae.
The commercial -1,3-glucan material (average DP 800 and PDI 1.7-2.0)was found to exhibit variable morphologies, namely spherical aggregates, fibrids, and platelet. [117]The spherical aggregates (as in wet-cake material) are composed of randomly oriented -1,3-glucan chains that are precipitated into particles.The primary particles, in size of 10-30 nm, are highly aggregated with an agglomerated particle size (diameter) of 5-10 μm (Figure 4b-iii).The fibrids are composed of -1,3-glucan chains oriented in elongated fibril-like structures.The platelet-like particles are composed of a microcrystalline glucan with a crystallinity of up to 76%. [117]) Filler material.The commercial -1,3-glucan materials (particle size of 5-10 μm; zeta potential in the near-neutral range) are useful to form stable colloidal dispersions in aqueous systems.This can be exploited, for example, in order to stabilize Pickering emulsions. [117]The -1,3-glucans can be formulated into solventbased resin systems typically used in the coatings industry (e.g., acrylic latex, alkyd resins, and epoxy resins).The addition of -1,3-glucans (at 0.5-2.0wt%) improves the viscosity (by 30%) and imparts a thixotropic rheology to the paint system under a high shear rate. [118]he ability to form stable colloids, together with the features of low backbone density (∼1.5 g cm −3 ) and high surface area (several hundred m 2 g −1 ), makes the -1,3-glucan particles also promising filler materials.The -1,3-glucan material (particle size of 5-10 μm) was incorporated into ethylene vinyl acetate (DuPont Elvax 40 W; with vinyl acetate content of 40 wt%) via a melt fabrication process.The tensile properties (e.g., Young's modulus by 115%; flexural modulus by 15%) and the impact strength (by 71%) were improved in the composite with 30 wt% of -1,3glucan. [116]A similar effect of material strengthening was seen when -1,3-glucans were used for natural rubber film reinforcement.The -1,3-glucan particles (size of 0.2-1 μm, prepared by applying high shear to form a colloidal dispersion from the wetcake material with a size of 5-10 μm) were admixed as fillers into natural rubber, and the resulting mixture was used for casting on glass mold [119] and on paper (20 μm thickness). [120]A progressive improvement in the wet tensile strength (8-fold) and the elastic modulus (4.5-fold) as well as a 6-fold reduction in the water absorption were noted in the paper coated with natural rubber/-1,3-glucan (50 wt% of glucan).The coating on paper also showed interesting barrier properties (∼50% reduction of oxygen permeability). [120]More recently, epoxidized natural In the GtfJ reaction, the glucose released by hydrolysis serves as an acceptor (primer) for polymerization.The sucrose substrate is an acceptor as well.The degree of polymerization (or n) in the product is dependent on the enzyme and the condition used.b) Assembly of the synthesized -1,3-glucan chains into organized (crystalline) materials of varied morphologies, as shown in the transmission electron images depicted in (i)-(iii).Reproduced with permission. [99,117]Copyright 2017, Elsevier; Copyright 2021, Elsevier.

Table 2. Esterified synthetic 𝛼-1,3-glucans and their chemical and material properties. Material a)
DP n (PDI) Degree of substitution Refs.  a) The glucan esters prepared in homogeneous reaction are marked with asterisks.
2) Aerogels.More recently, -1,3-glucan-based aerogels were reported.Dissolution (-1,3-glucan at 5.6 wt%) in 8 mol L −1 NaOH followed by neutralization was used to induce gelation.The inclusion of inorganics (e.g., TiO 2 ) during the neutralization step enhanced the mechanical strength of the hybrid hydrogels, which were then converted into aerogels through lyophilization.The aerogels displayed a high surface area (82 m 2 g −1 ).They also showed high yield stress (3.0 Pa), which was ∼2-fold increased as compared to the neat glucan system.Moreover, an almost doubled water absorption capacity was found in TiO 2 -aerogels.These aerogel properties suggest potential applications as absorbent materials for hygiene products. [122]) Polysaccharide-based films and fibers.The synthetic -1,3-glucans are considered for development of polysaccharide films and fibers.The materials are esterified so that they can be dissolved in organic solvents and become thermally processable.The thermoplasticity, the mechanical properties as well as the moldability of material are expected to be improved as result of the derivatization. [123]Table 2 shows esterified -1,3-glucans along with their chemical and material properties.The T d.5% , represents the thermal decomposition behavior.It is higher (>50 °C) in all the ester derivatives than in neat -1,3-glucan.123a] The -1,3-glucan acetate (DS 3.0) shows a T m of 339 °C, which is higher than those of the commercial polymers (e.g., polyethylene terephthalate PET, 265 °C; and cellulose acetate, 293 °C).
For industrial processing, materials are preferred that exhibit low T m (∼200 °C and higher) and high T g (glass transition temperature; ∼150 °C or higher) due to broad operational window and favorable dimensional stability offered.123a] Besides thermoplasticity, postfunc-tionalization (e.g., alkylation to alter wettability) of esterified glucan films is attainable. [124]sterification of the -1,3-glucan was performed in heterogeneous reaction that involved the glucan as a solid. [123,124,127]lternatively, the -1,3-glucan was dissolved in solvents such as N,N-dimethyl acetamide (DMAc)/LiCl.The DS achieved varied with the ester group and the conditions used: for example, benzoate ester (DS 1.7-2.0),and fatty acid esters (DS 1.0-2.2). [125] The corresponding melt products were shaped into films and could thus serve as the basis for hot-melt adhesives. [126]Table 2 suggests that the DS obtained in homogeneous esterification (DS 1.0-2.2) was relatively low as compared to the heterogeneous esterification (DS ∼3.0).35b] -1,3-Glucan esters are promising for fiber development, especially in melt-spinning processes (Figure 5).123b] However, concerns arise regarding the biodegradability of polysaccharide ester derivatives, necessitating exploration of wet-spinning approaches using unmodified -1,3-glucans (Figure 5).The as-produced fibers had tensile strength of 138 MPa, Young's modulus of 3.5 GPa, and elongation at break of 12%, which could be enhanced further through subsequent stretching and heating treatments. [128,129]) C-6 modified -1,3-glucan.C-6 site-specific modification of -1,3-glucan was conducted to develop materials.For instance, the C-6-amino-glucan (DS 0.97; M w 3.7 × 10 4 ; PDI 4.6) was chemically crosslinked to form hydrogels.The resulting hydrogels, highly swellable in water, show promise for biomedical applications (Figure S2, Supporting Information). [130]dditionally, the C-6 azido-glucan (DS 0.94; M w 1.4 × 10 5 ; PDI 5.2) was used for gelation via an azide-alkyne cycloaddition reaction, forming a cationic gel suitable for protein immobilization over a wide pH range (4.5-8.0), with a point of zero charge at 8.8. [131]igure 5. Spin fibers produced from plain -1,3-glucans or esters thereof using wet-spinning and melt-spinning approach.Photograph (upper) and polarized optical microscope image (bottom) of the fiber are reproduced with permission.123b,128] Copyright 2021, American Chemical Society; Copyright 2021, Elsevier.

Structure and Properties
Starch is one of the most abundant polysaccharides in nature.It exists in two structural forms, the linear amylose and the branched amylopectin.Most starches are composed of 20-30% amylose and 70-80% amylopectin.The linear chain is an -1,4-glucan polymer.The branching points involve -1,6-linkage. [134]Unless noted, linear -1,4-glucan is referred to here.
The weight-average M w of amylose varies considerably with the source.It ranges from around 10 5 to 10 6 g mol −1 , corresponding to a DP of 500 to 6000.Amylose chains can be disordered or adopt two types of helical conformation in the crystal structure. [134,135]One type, referred to as allomorph A and B, involves the interaction of two polysaccharide chains to form a parallel-stranded typically sixfold left-handed double helix.Organization into solid crystalline material is under discussion, but it likely involves parallel packing of the double helices and includes water molecules in substantial number (e.g., 36 water molecules are reported to be in the unit cell of B-amylose). [136]The second type of helical structure is referred to as V-amylose.As its formation is connected to host-guest interaction of the amylose with another molecule (e.g., iodine, hydrophobic organic compounds), the V-amylose involves considerable variation in crystal structure. [137]The number of glucose units per turn can vary between 6 and 8, as indicated in the name of the amylose as V 6 -, V 7 -, and V 8 -amylose.Multiple helices of V-amylose form a central channel where the guest molecules are accommodated.Varied arrangements of chains are observed in the unit cell of V-amylose crystals, depending on the crystallization conditions used. [137,138]136b,139] Amylose has been widely studied for its interesting solution properties related to possible applications (e.g., emulsion stabilizer, [140] and gelling agent [141] ).Of particular interest is its ability to entrap molecules in the V-helix form, [137] where the amylose acts as a one-dimensional supramolecular host molecule.Amylose-lipid complexes are suitable carriers for a controlled release of lipids. [142]The interest in amylose hybrid materials is rapidly growing considering their numerous potential uses in food, biomedicine, and material industry applications. [137,142]

Bottom-Up Enzymatic Synthesis
Top-down approaches of amylose production generally involve leaching from natural sources of raw starch. [143]To achieve it, aqueous suspensions of starch are heated to above the gelatinization temperature.Outcome of the leaching in terms of amylose DP, yield, and purity depends on several parameters, including the heating rate, the shear forces applied, the final leaching temperature, and the botanical origin of the starch. [143]At the commonly used temperature of 80-85 °C, pure amylose in DP ∼10 3 is expected from considerations of solubility. [144]Using a higher temperature (∼95 °C), the DP gets higher (∼10 4 ) but the purity is lowered due to amylopectin being also leached out. [145]In addition, fractionation of raw starch into amylose and amylopectin can be achieved by selective complex formation with suitable agents (e.g., n-butanol and isopentanol), after dispersion in water followed by precipitation. [146]Prior to the complex formation, effective dispersion of amylose is critical to obtain Figure 6.Enzymatic synthesis of -1,6-glucan-graft--1,3-glucan from dextran and sucrose by using GtfJ and application of the grafted glucans for hydrogel and film development.The image of hydrogels and films are reproduced with permission.Reproduced with permission. [132,133]Copyright 2021, American Chemical Society; Copyright 2022, Elsevier.
amylose of high purity. [143]The properties of the as-isolated amylose is technically difficult to control via the top-down procedure.
Bottom-up synthesis of amylose was considered via different chemical and enzymatic routes.Chemical synthesis is challenging due to the requirement to form 1,2-cis glycoside linkages.Synthesis of an amylose-like polysaccharide (DP 25-30) was reported from a protected 1-thio--maltooctaoside derivative, offering a single position (O-4) at the nonreducing end for polycondensation. [147]Automated glycan assembly was previously used to prepare amylose of DP 16. [148] However, despite the progress in chemical methods, enzyme-catalyzed assembly of glycans remains the preferred choice for synthesis.Enzyme systems relevant for amylose (bio)synthesis are the following.
1) In plants, starch synthase (EC 2.4.1.21;glycosyltransferase family GT5) catalyzes polymerization of an -1,4-linked glucan precursor.The enzyme uses ADP-glucose as the donor substrate.The catalytic reaction proceeds with retention of anomeric configuration.Among the starch synthases in plants, six enzyme isoforms (soluble form I-V; granule-bound form) are known. [149]149a] Starch branching enzymes introduce -1,6 linkages by simultaneous cleavage of some short -1,4-glucan chains and connecting them to other chains, pro-viding amylopectin molecules as well as increasing the number of nonreducing ends for further elongation by the starch synthases.The structure and size of amylopectin clusters are mainly controlled by three soluble starch synthases I-III.The short -1,4-glucan chains of DP 6-7 are presumably the substrates for enzyme isoform I.They get extended to a length of 8-12 glucose residues. [150]These glucan chains (DP 8-12) are subsequently used by starch synthases II and III to preferentially synthesize longer -1,4-glucan chains in DP 12-28 and DP 25-36, respectively. [151]The granule-bound starch synthase (EC 2.4.1.242)72c,152b] The oligosaccharide substrate of the enzyme is generated in the plastid by the actions of branching enzyme, disproportionating enzyme or starch (-1,4-glucan) phosphorylase.Due to its small size, the oligosaccharide may diffuse into the starch granule where they are elongated by the granule-bound starch synthase.72c,152b,154] The studies, however, addressed the starch biosynthesis mechanism and were not designed for production.
An enzyme functionally related to starch synthase is glycogen synthase.The glycogen synthases are classified into glycosyltransferase family GT3 (enzymes from prokaryotes and nonplant eukaryotic sources) and GT5 (enzymes from bacteria).Besides the differences in sequence that give rise to assignment to different GT families, the enzymes also differ in specificity for the sugar donor substrate and in the regulatory mechanisms. [155]he family GT3 glycogen synthases use UDP-glucose and the biosynthesis of glycogen happens through the combined actions of glycogenin and glycogen branching enzyme. [156]The initial glycogen primer (DP 8-12) for glycogen synthase is provided through the auto-glycosylation reaction of glycogenin. [157]Glycogenin and glycogen synthase interacts physically for glycogen biosynthesis. [156]Recent studies also found that glycogen can be synthesized in the absence of glycogenin, both in human and mice.Glycogenin inactivation resulted in an increased amount of glycogen. [158]The direct connection to glycogen synthase mechanism is however unknown.
2) Amylosucrase (EC 2.4.1.4)is a catalytically versatile family GH13 transglycosidase.The enzyme uses the canonical two-step catalytic reaction of retentive glycoside hydrolases via a configurationally inverted -glucosyl enzyme intermediate.Besides hydrolysis of sucrose, the enzyme catalyzes glucosyl transfer from sucrose to different acceptors, including glucose and sucrose itself. [159]The glucosyl transfer is nonprocessive.Due to the specificity of amylosucrase, a linear -1,4-glucan chain is released (Figure 7a). [159]In the course of polymerization, the initial phase of the reaction primarily yields maltose and maltotriose (DP 2-3).Following this, iterative growth takes place, resulting in the generation of soluble products with a DP ≤ 25.161a] The -1,4-glucan product involves considerable distribution in DP (e.g., 3-35, [160] 3-60, [161c] and 4-90 (PDI 3.0) [139a] by the amylosucrase from Neisseria polysaccharea (NpAS); and 3-46 [161b] by the amylosucrase from Deinococcus geothermalis (DgAS), all using 100 mmol L −1 sucrose).The synthetic product is obtained as partly insoluble material since amylose of DP greater than 25 shows low solubility in water. [160]he solid materials can be separated using either centrifugation or filtration.Concurrently, the soluble fraction can be precipitated using ethanol, usually at a concentration of 50-60 vol%, [94a,162] and then subjected to centrifugation or filtration.The dry materials can be acquired through a vacuum freeze-drying process.
Among the amylosucrases reported, the DgAS and NpAS are well characterized in regard to their crystal structure, catalytic mechanism, and synthesis applications (e.g., glycosylation of bioactive compounds, modification of oligosaccharides, and sucrose isomerization), as summarized in a recent review. [76]Here, emphasis is placed on the amylosucrase-catalyzed synthesis of -1,4-glucan materials.
In the NpAS-mediated reaction (0.6 mol L −1 sucrose, 30 °C), synthetic amylose chains in average DP 35 (PDI, 2.3) selfassembled spontaneously in a process referred to as retrogradation to form ovoidal particles with a size of ∼5 μm.139a] The DgAS was also used for the production of amylose (0.5 mol L −1 sucrose, 30 °C).The product in average DP 47 also formed as solid particles in size of 2.8 μm. [163]he as-synthesized discrete amylose microparticles are promising biomaterials for drug delivery.For example, in the DgAS-mediated reaction (0.5 mol L −1 sucrose, 30 °C), -carotene (0.45 mg mL −1 ) was added and encapsulated into the amylose particles with a yield of 65%.The -carotene embedded amylose particles, with an average DP of ∼40, exhibited a size of ∼8 μm.The particle size was about twice that of blank amylose microparticles, and it was due to a toroidal morphology pattern formed during assembly. [164]The encapsulated -carotene is more resistant to stressors such as photodegradation and chemical oxidation, and can be slowly released into the intestine (∼25% after 1 h) due to the degradation of amylose microparticles. [164]urther, superparamagnetic amylose microparticles were prepared by introducing iron oxide nanoparticles during the DgASmediated reaction (0.5 mol L −1 sucrose, 30 °C).The iron oxide nanoparticles (0.1-0.3 wt%) were effectively encapsulated into the amylose particles, giving a mean size of composite ∼2.5 μm and a magnetization value of 4.5 emu g −1 .The composite microparticles are amenable to magnetic separation and could be employed for purification of protein tagged with the maltose binding protein.The captured target protein can be eluted from the particles by free maltose that competes for the binding site on the surface of microparticles.The composites maintained their purification capacity (88%) after three rounds of recycling. [165]) -Glucan phosphorylase (GP; EC 2.4.1.1)belongs to glycosyltransferase family GT35.47a] The GP catalyzes the polymerization of linear -1,4glucan chains using -d-glucose 1-phosphate (Glc1P) as the donor substrate (Figure 7a).The acceptor substrate is an -1,4oligosaccharide of minimum DP 3. [44c] The reaction is reversible and proceeds with retention of configuration ( → ).In the process of polymerization, iterative growth occurs from a low DP, typically ranging from 3 to 7.This ongoing growth yields a product with a DP of up to 60, marking ∼70% completion of the overall conversion.Subsequent to this stage, elongation continues, albeit at a slower rate, until amylose chains assemble caus-ing precipitation. [166]The average DP is dependent on reaction conditions used, typically falling within the range 100-300 (see later).GP is peculiar among enzymes catalyzing glycosyl transfer in its use of vitamin B6 (pyridoxal 5′-phosphate) as cofactor in catalysis. [167]Here the 5′-phosphate group is crucial for the activity and mechanistic analogies between GP and retentive sugar nucleotide-dependent glycosyltransferases have been pointed out. [54,55]The role of GP in vivo is degradation of storage -1,4-glucan in the presence of phosphate.For synthesis in vitro, the phosphate released from Glc1P can be precipitated to shift the equilibrium to the amylose product. [168]94a,169] Synthetic amylose-based material engineering has received considerable interest.Structural derivatives of amylose (e.g., hetero-oligo/polysaccharides of -1,4-glycan) and amylose complex/hybrid materials are important directions pursued in this field.Diversification of amylose materials is achieved in the form of block and graft copolymers.15g] Two strategies of GP-catalyzed polymerization are used for synthesis.First, the donor or the acceptor substrate is varied to obtain an -1,4-glycan chain that features substitution of d-glucose in the growing chain or at/near the reducing end.Change in the polymer structure is expected to result in altered material properties.For example, -1,4-linked glucosamine chains (average DP 23) do not form double-helix assembly [168b] as the native amylose chains do.In addition, partially 2-deoxygenated amylose (average DP ∼103; 2-deoxy-Glc:Glc ratio 2.6:7.4)exhibited an enhanced hydrophobicity, and the film prepared from it showed a water contact angle of 96.2°as compared to 38.5°for native amylose starch. [170]15g,29a,c,139d] Here, important properties of the synthetic amylose materials are highlighted along with possible applications promoted by them.

Amylose Material Engineering
1) Amylose-grafted materials.The overall idea is to have the acceptor substrate (the "primer") of the GP-catalyzed polymerization attached to another polymer.Subsequent chain elongation of the -1,4-glucan results in amylose-grafted hybrid materials. [171]139d] Maltoheptaose was attached with reductive amination to chitosan and C-6 aminated cellulose (Figure S3a, Supporting Information), and the amylose chains with average DP of 310 and 115 on chitosan and aminated cellulose, respectively, was synthesized by using potato GP. [172]Another approach involved coupling a maltoheptaose derivative with a reducing-end amino group to anionic polysaccharides (e.g., alginate, xanthan), and the amylose side chains in DP of ∼100 was obtained using the GP from Aquifex aeolicus VF5. [173]The amylose DP was controlled by adjusting the molar ratio of Glc1P donor to maltoheptaose acceptor, typically in range of 50:1 to 300:1.Similarly, the GPcatalyzed chain elongation of glycogen was achieved, resulting in hydrogelation. [174]n addition to enzymatic reaction in solution, amylose chain polymerization can also be performed on solid surfaces.Maltoheptaose was linked to silicon wafer and gold with surface derivatized by amino-silane and l-cysteamine, respectively.Reaction with potato GP yielded amylose-grafted surfaces (Figure S3b, Supporting Information) with layer thickness of ∼20 nm and amylose DP of ∼150.Due to its hydrophilic nature, the amylose layer has promising antifouling properties. [175]) Block copolymer.Another GP-based application is the synthesis of block copolymers.The maltoheptaose or maltopentose, each having the relevant polymers attached to the reducing end, are used for GP mediated polymerization.
The hetero--1,4-oligosaccharides and glycans have been considered for material development.The A. aeolicus VF5 GP was for synthesis due to its broad substrate scope and robustness. [168,193,194]168a] Interchain crosslinking via reductive amination led to hierarchically controlled chain assemblies of these glycans (Figure 9), where the morphology of the resulting material depended on the feed ratio of reductant (e.g., NaBH 3 CN) to the reducing end of -1,4-linked polymer chains. [196]Furthermore, maltooligosaccharides with carboxylate groups on both ends were synthesized and crosslinked with water-soluble chitin to form cryogels. [197] Additionally, partially 2-deoxygenated amylose with an average DP of ∼103 was synthesized, where the ratios of 2-deoxygenated glucose units was dependent on the molar ratio of donor d-glucal to Glc1P. [198]

Structure and Properties
Dextran is a bacterial exopolysaccharide.It is an -1,6-linked linear glucan, with branches of -1,3 linkages.Occasionally, the branches can involve -1,4 or -1,2 linkages.The degree of branching varies broadly from 3% to 50%. [199]Over 80% of the branched chains in dextran contain less than two glucose units. [200]Dextran is produced by certain lactic acid bacteria, including the genus Leuconostoc and Streptococcus in particular. [201]199a] There are some commercial dextran available: dextran 40, dextran 60, and dextran 70, whereby the number indicates the average molecular mass in kDa. [202]The dextran is generally considered to be well soluble in water (>30 g L −1 ).The dextran produced by Leuconostoc mesenteroides NRRL B-1149 was insoluble in water. [203]Possible explanation is that the product contained 40% -1,3 branching linkages.
Dextran has been widely used for biomedical applications.Dissolved in saline, dextran exhibits similar colloid osmotic pressure and viscosity as human blood.The dextran 40, available as 10% solution, and dextrans 60 and 70, available as 6% solution, have been used as plasma volume expander for several decades. [204]extran 40 and 70 are also prescribed for the treatment of shock or impending shock due to hemorrhage, burns, or trauma. [205]esides these applications, the dextran-based drug delivery systems have been developed.The esterified dextran form micelles in aqueous medium.These micelles are useful for hydrophobic drug encapsulation and controlled release. [206]In addition, dextran-based microgels have recently been proposed. [207]207b] The fabrication strategies and medical applications of dextran-based drug carriers have been reviewed. [208]

Bottom-Up Enzymatic Synthesis
The dextran molecular weight and its degree of branching are challenging to control in the microbial production.199a] Commercial dextran of certain average molecular masses (40, 60 or 70 kDa) are widely used in medicinal applications. [204,205]Rather than produce them by chemical depolymerization of the native dextran of ∼10 3 kDa mass, bottomup synthesis is a promising alternative.Degree of branching control, or elimination of branching entirely, is facilitated by polymer synthesis.Enzymatic and chemical routes of synthesis have been proposed. [148,210]extransucrase (EC 2.4.1.5)is a family GH70 transglycosidase reported for the in vitro synthesis of dextran from sucrose.The enzyme operates by covalent catalysis (-glucosyl enzyme intermediate) and its reaction involves retention of configuration. [112]everal dextransucrases have been characterized from lactic acid bacteria. [210,211]Interestingly, the degree of branching of the synthetic dextran product varies strongly with the enzyme used.Branching can be effectively zero but also reach unusually large values of up to 25%. [212]The type of linkage in branches is also variable. [212,213]The branching of dextran does not occur from the action of a separate dextran-branching enzyme, instead it is done by the dextransucrase itself via a so-called "acceptor reaction."This reaction involves the transfer of d-glucosyl moiety of sucrose to a dextran chain to give d-glucosyl branch linkages, and the transfer of dextranyl chain to another dextran chain to give dextranyl branched dextran chains. [214]The extent to which these branching reactions occur is dependent on reaction conditions.For example, in the reaction of dextransucrase from L. mesenteroides B-512FMCM, the degree of -1,3 branching increased from 5% to 16.6% with change in sucrose concentration from 0.1 to 4.0 mol L −1 ; and from 4.8% to 14.7% with increase in tempera-ture from 4 to 45 °C. [215]Besides, the dextransucrases from Streptococcus sp. and Leuconostoc sp.45c,199a] Recently, dextransucrase DSR-M, discovered in L. citreum NRRL B-1299, has gained interest for selectively producing low-molecular-weight dextran (∼23 kDa). [216]The polymerization kinetics of DSR-M exhibits a distinctive profile.The initial reaction phase involves a rather low conversion rate and oligosaccharides of DP not exceeding 20 are released.Subsequently, the reaction accelerates about fourfold and remains constant until sucrose conversion approaches ∼70%.The product DP reaches ∼150.Following this main phase, a deceleration in elongation kinetics is observed, concluding upon sucrose depletion and resulting in a modest increment in the final DP. [93a,216] In a recent study, enzymatic synthesis of dextran using DSR-M was examined in a lipidic mesophase prepared from 70 wt% monolinolein and 30 wt% aqueous buffers.The mesophase was applied with the idea of generation of a soft nanoconfinement for the enzyme reaction.Applying the compartmentalized system, reaction at 50 g L −1 sucrose (30 °C) gave a sixfold increase in polymer molecular mass (49 kDa; PDI 1.7) and showed twofold faster conversion compared to reaction in buffer.The mesophase conditions (91 wt% phytantriol and 9 wt% aqueous phase) enabled the enzymatic reaction to be carried out in cryogenic condition (−20 °C), generating dextran in a molecular mass of 6 kDa. [217]93a] Important parameters of the ultrafiltration are molar mass cut-off of the membrane, temperature (typically in the range of 25-40 °C), and water washing. [219]94b,218,220] Recovering precipitated products involves gravity separation or centrifugation.93a,218] The final step in the process is solvent removal from the soluble or precipitated products, accomplished by freeze-drying.

Material Development
The microbially produced dextran and top-down processed derivatives thereof are well studied materials.Their properties have been reviewed. [208,209]Here we therefore focus on the synthetically obtained and effectively branchless -1,6-glucan and its material properties.

Cyclic 𝛽-1,2-Glucan
-1,2-Glucans are natural glycans from bacteria that exist predominantly in a cyclic form. [226]Their DP is typically in the range of 17-28, but higher DP of up to 40 has also been reported. [227]he cyclic -1,2-glucan is very well soluble in water (232 g L −1 ). [228]Physiologically, the cyclic -1,2-glucans are critical for bacteria to establish host-microbe interactions, as in symbiosis or pathogenesis. [229]They are found mainly in the periplasmic space of the microbial cell and have been reported from various species of Gram-negative bacteria, including Agrobacterium sp., Brucella sp., Rhizobium sp., and Sinorhizobium sp. [229,230]yclic -1,2-glucans are naturally synthesized by cyclic -1,2glucan synthase (CGS). [226]The CGS from B. abortus is the most studied enzyme from this class. [231]231c] The N-terminal domain belongs to glycosyltransferase family GT84.231a] The glycosyltransferase reaction proceeds with inversion of anomeric configuration ( → ).The C-terminal domain is a -1,2-oligosaccharide phosphorylase that belongs to glycoside hydrolase family GH94.231b] The phosphorylase reaction is reversible.Trimming (depolymerization of the chain) requires phosphate to present in excess.The product released by chain cleavage from the nonreducing end is -d-glucose 1-phosphate.The reaction proceeds with inversion of configuration, characteristic of enzyme of glycoside hydrolase family GH94.The cyclization which is performed by a separate domain puts an end to the catalytic  [221] Copyright 2020, American Chemical Society.
interplay between enzymatic elongation and trimming of the -1,2-glucan chain.Cyclization involves an intrachain transglycosylation that proceeds with retention of configuration ( → ).231c] The production of cyclic -1,2-glucans is currently done mainly by microbial fermentation.The cyclic -1,2-glucans released from the cells can be isolated from the culture supernatant via ethanol precipitation and size exclusion chromatography. [232]The yield of cyclic -1,2-glucan, in range of 0.1-10.9g L −1 , is dependent on the strain used and is also affected by the culture medium and the fermentation time. [233]233b] Physiologically, the cyclic -1,2-glucan is noncytotoxic and nonimmunogenic.It can however activate mammalian dendritic cells, thereby triggering antigen-specific CD8 + T cell responses in vivo. [234]The glucan may therefore act as an adjuvant to a) The N-terminal domain belongs to glycosyltransferase family GT84.
The exploitation of material applications of linear -1,2glucans is currently limited by the product amount obtainable from the reported syntheses.Scale up and intensification of the enzymatic production will be necessary.Phosphorylase cascade reaction for -1,2-glucan production from sucrose and glucose seems promising.The characteristic of being highly water-soluble makes -1,2-glucans feasible for specific applications.For example, the -1,2-glucans can be interesting for homogenous hydrogel preparation without requirement of prior solubilization of glycan used.With waterinsoluble materials, the hydrogel preparation usually necessitates solubilization in 2-4% (w/v) NaOH solution. [221,223]The soluble -glucans can be applied to carbohydrate microarrays for enzyme screening and to study of carbohydrate-recognition events. [243]

Bottom-Up Enzymatic Synthesis
94c,267] However, several points need to be considered.267b] Moreover, there are issues of long extraction time, high process costs, and impurities of product. [251,268]ottom-up synthesis of -1,3-glucan was achieved by chemical means to a DP of 16. [269] However, the -1,3-glycosylation is made difficult by steric hindrance from the protecting group (e.g., benzylidene group) at the C-4 position.Also, the chemical polymerization to a higher DP is challenging.Applied biocatalysis (Table 4) can present a useful alternative.
1) Glycosyltransferase. -1,3-Glucan synthase belongs to glycosyltransferase family GT48.The enzyme uses UDP-glucose for glucan chain polymerization with inversion of configuration.The synthase is associated with plasma membrane through its multi-transmembrane domains.The -1,3-glucan as-synthesized is extruded into the cell wall space through a channel formed by the transmembrane domains during synthesis. [270]. cerevisiae Fks1 gene was first identified to encode the putative catalytic subunits of -1,3-glucan synthase. [271]The Fks gene family was found also in other fungal species such as Neurospora crassa [272] and Paracoccidiodes brasiliensis. [273]S. cerevisiae Fks1 shares ∼88% identity to Fks2 and 56-86% identity to the Fks family in major pathogenic fungi species.Recently, the cryo-electron microscopy structure of S. cerevisiae Fks1 was solved at 2.47 Å resolution, which is the first structure of a GT48 family member.The core domains of Fks1 and cellulose synthases share a conserved fold containing a cytosolic glycosyltransferase domain and a transmembrane glucan-transporting domain.This suggests that -1,3-glucan synthases may use a similar mechanism as cellulose synthases. [274]-1,3-Glucans have been synthesized by the cell-free -1,3-glucan synthase preparations.During polymerization, iterative chain elongation proceeds rapidly initially.Slowing down of the reaction in the late phase of conversion may be caused by inhibition from the accumulated UDP (∼2 mmol L −1 ). [275]The -1,3-glucan synthases are large in a molecular mass from 180 to 280 kDa. [198]Their extraction-purification from the cellular membranes for biocatalytic applications is however challenging.
Considering the benefit of a hydrolytically stable product, the glycosynthase approach was pursued.For example, the E231G variant of the -1,3-d-glucanase from Hordeum vulgare catalyzed -1,3-glucan polymerization using -laminaribiosyl fluoride as the substrate (37 °C, 12 h).The polymeric product of DP 30-34 was obtained from 58 mmol L −1 substrate (75% yield).The as-prepared -1,3-glucan adopts a parallel, triple helical conformation, and the triple helices are orientated perpendicularly to the plane of lamella. [284]In another study, the glycosynthase E134A from Bacillus -1,3-1,4-glucanase was used to produce mixed-linkage -1,3/1,4-glucans from the polymerization (selfcondensation) reaction using 150 mmol L −1 glucooligosaccharide fluoride substrate (e.g., (Glc 1,4 ) 2 Glc 1,3 Glc-F) at 35 °C for 12 h.The reaction with 84% yield generated a product with molecular mass of ∼30 kDa (number of condensation, 47) and PDI of 1.8. [286]Stability of the glycosyl fluoride donor substrate under the conditions used (>40 °C) was a concern.Alternative mode of donor substrate supply in the glycosynthase reaction was considered. [287]) Glycoside phosphorylase.-1,3-d-Glucan phosphorylases (EC 2.4.1.97)catalyze the elongation of -1,3-glucans using Glc1P as the donor. [279]They are classified into glycoside hydrolase family GH149 or GH161.The sequence-based classification reflects enzyme specificity in terms of acceptor substrate used.The GH149 enzymes use glucose, [288] while GH161 enzymes require laminarin acceptor with DP ≥ 2. [279] In the course of polymerization, iterative elongation from laminaribiose proceeds rapidly until the reaction attains equilibrium, reaching 70-80% donor conversion.The product has a DP of up to 35.The released product is soluble when the DP is below 25, while precipitation occurs as a result of the assembly of the longer chains. [277,278]To isolate the product, the solid material can be separated through centrifugation or filtration.Furthermore, the soluble fraction can be precipitated with ethanol or isopropanol, typically at a concentration of 75-80 vol%.94c,267] The use of phosphorylases for the branchless -1,3-glucan synthesis and relevant material development is summarized below.

Material Development
1) By using the phosphorylase from E. gracilis, -1,3-glucan with an average DP of 30 (PDI 1.2) were prepared from 200 mmol L −1 sucrose and 0.1 mmol L −1 glucose in reaction at 37 °C for 4 d.Sucrose phosphorylase was used to release the Glc1P donor for glucan polymerization.The synthetic glucan chains self-assembled into a triple-helical conformation and formed as hexagonal lamellar crystals. [276]In another study, -1,3-glucan was prepared from 200 mmol L −1 Glc1P and 50 mmol L −1 glucose using the phosphorylase from Thermosipho africanus.The product synthesized at 50 °C showed an average DP of 30 (PDI 1.0) and formed as single crystals with macroscopic appearance of hexagonally shaped lamellar structure and hierarchical chirality (Figure 11b).With a large available surface area (∼300 μm 2 ), well-defined structure (hexagonal particles), and spacing between layers (∼15 nm), the synthetic material was used as the carrier for the drug substance acetaminophen.In the form of compressed tablet (80 mg -1,3-glucan and 5 mg drug), a maximum drug release of ∼75% was obtained in phosphate buffer (pH 7.0) after 50 min. [277]he triple-helical conformation of the synthetic -1,3-glucan potentially supports different types of host-guest application.The native -1,3-glucan schizophyllan can form a triple-stranded helical structure comprised of two polysaccharide strands and one polynucleotide strand after being dissociated (as single strand) and subsequently reassembled in the presence of polynucleotides. [289]Following this concept, it is reasonably possible that a similar structure/complex can be generated through in situ synthesis of -1,3-glucan in the presence of polynucleotides (e.g., DNA).Besides, the triple-helical -1,3-glucan (e.g., schizophyllan) can include hydrophobic compounds into intrastrand hydrophobic cavity and solubilize the poorly watersoluble compounds. [290]With that, one can consider to entrap such molecules into the -1,3-glucan triple-helix during synthesis, potentially functioning as a drug carrier or solubilityincreasing strategy.
extracted from raw materials via successive chemical and mechanical treatments. [295]The fibers as-prepared exhibit a diameter generally of several micrometers.4a,b,10c,296]

Bottom-Up Enzymatic Synthesis
To the extent that top-down processing alters cellulose structure and functionality, [4a,22,293] the bottom-up approach comes in as a promising additional option.15a,b,f,31c,298] Here, we focus on enzymatically synthesized cellulose and derivatives or composites thereof.Table 5 shows a comparison of material properties of cellulose obtained by approaches of top-down processing and bottom-up enzymatic synthesis.The resulting celluloses differ largely in molecular size, morphology, and strength.The top-down and bottom-up strategies are revealed as being complementary in scope of material application.In the following section, we discuss the role of synthetic cellulose for material development.
1) Enzymes for cellulose synthesis.Cellulose synthase (EC 2.4.1.12)belongs to glucosyltransferase family GT2.The enzyme uses UDP-glucose to polymerize the -1,4-glucan chains. [316]he enzymatic reaction is configurationally inverting.Cellulose synthase involves cyclic-di-guanosine monophosphate as an important activator. [316,317]Cellulose synthase is a membranebound multicomponent enzyme complex that adopts a rosettelike structure in the plasma membrane. [318]70a,321] The synthetic cellulose chains showed DP in range of 60-200.Recently, the glycosyltransferase LgtB (N.meningitidis) was found to catalyze -1,4-oligoglucose polymerization of glucose-terminated glycosides, using UDP-glucose as the donor substrate.Cello-oligomers in much smaller size (DP 7-9) were generated in the LgtB reaction, [322] as compared to the cellulose synthase reaction.Using LgtB, authors showed the synthesis of cellulose chains modified at the formal reducing end with azido and p-nitrophenol group, as well as the imidazoliumbased probes (e.g., 4-(1-methyl-3-methylene imidazolium) benzyl group). [322]ellulase-catalyzed transglycosylation was employed for cellulose synthesis.-d-Cellobiosyl fluoride was used as substrate and reactions were performed in the presence of organic solvent (e.g., acetonitrile/buffer mixture, 5:1).The enzyme(s) (note: cellulase is a mixture of different endo-and exo chain-cleaving activities) form a covalent -cellobiosyl enzyme intermediate that can react with the substrate to allow for an overall polycondensation reaction that yields polymerized cellulose chains.70b] Using optimized acetonitrile/buffer ratio (2:1), material of cellulose I crystal structure was received from the enzymatic synthesis. [323]A "surfactant-enveloped" cellulase prepared with the nonionic surfactant dioleyl-N-d-glucona-l-glutamate was used for cellulose synthesis from 146 mmol L −1 cellobiose in a nonaqueous solvent of LiCl and N,N-dimethylacetamide (37 °C, 24 h).The insoluble cellulose (yield ∼4.5%) was in DP close to 100. [324]Glycosynthase E197A was developed from the endo-cellulase CelB of Humicola insolens. [325]Cellulose was synthesized in a high yield (92%) from -cellobiosyl fluoride (174 mmol L −1 ) at 40 °C, 24 h.325a] Cellodextrin phosphorylase (CdP; EC 2.4.1.49)belongs to glycoside hydrolase family GH94.15a] It is soluble for DP 6 or smaller.Products of higher DP precipitate due to spontaneous chain self-assembly. [69]The polymerization kinetics is influenced by the type and the concentration of the acceptor used.The use of poor acceptors (e.g., glucose) generally results in the formation of long(er) cellulose chains.The "priming rate" of the acceptor is lower than the subsequent elongation rate.Using a good acceptor (e.g., cellobiose), the priming and elongation rates are similar and so shorter cellulooligosaccharides are formed.61a,301] The donor Glc1P can be provided from starch or sucrose using a coupled phosphorylase reaction. [15a,47b,56] Here, cellulose and hybrid materials prepared from the CdP reaction are pointed out.
To isolate soluble cello-oligomers, chromatography (e.g., size exclusion, ion exchange) and precipitation are used. [95,298]Chromatography is predominantly employed at the research scale.94d,328] Specifically, methanol was employed for successive solubilization to fractionate oligomers based on their molecular size. [329]ecovery of precipitated products involves either centrifugation or filtration.The resulting dried materials are obtained through freeze-drying.
2) Nanoarchitecture of synthetic cellulose.Cellulose was produced by Clostridium thermocellum CdP using cellobiose (2.5 mmol L −1 ) and Glc1P (100 mmol L −1 ). [330]The cellulose chains (average DP 8) self-assembled into cellulose II crystalline material and formed elongated platelet crystals with a lateral size of 0.1 × 1 μm and a thickness of ∼10 nm.The molecular axis of the cello-oligosaccharides was perpendicular to the plane of the platelet. [330]12a,15a,71b,301] Alternative crystalline forms of synthetic cellulose have been reported.Cellulose I  (DP 7) was received upon polymerization of hexyl or octyl -d-glucoside (50 mmol L −1 ) by C. thermocel-lum CdP using Glc1P at 200 mmol L −1 , [331] with a nanomorphology of bilayer helical nanorod (left-handed, periodical pitch of 290 nm) and distorted nanosheet, respectively. [331]In another study, 6-fluoro-Glc1P (200 mmol L −1 ) was used as donor of C. thermocellum CdP to polymerize cellulose from cellobiose (30 mmol L −1 ).The resulting C-6 fluorinated derivative of cellulose (average DP 10) was crystalline material but its structure differed from the known cellulose polymorphs. [332]Recently, a crystal structure of mixed cellulose II (main) and cellulose IV (minor) was observed in synthetic celluloses (average DP ∼10) assembled in the presence of guanidinium chloride (3-6 mol L −1 ). [333]he nanomorphology of synthetic cellulose materials varies strongly in dependence of the conditions used for synthesis.In water, the oligomerization-induced self-assembly of cellooligosaccharides normally results in the formation of plateletshaped crystals, often referred to in the literature as cellulose nanosheets. [326,334]44a] Cellulose chain polymerization and selfassembly/aggregation are mutually interlinked processes in the CdP-catalyzed synthesis of cellulose materials.Factors of specific relevance for the polymerization (e.g., donor/acceptor molar ratio [69,71b,335] ) thus also affect the self-assembly.Bulk factors such as temperature [44a] or solvent conditions [336] influence both processes.12a,15a,297]

Synthetic Cellulose-Based Hydrogels
Cellulose-based hydrogels have attracted interest for bio-based soft material applications in chemosensing, medical therapy (e.g., drug carrier and delivery systems), and tissue engineering. [337]While numerous reports on hydrogels prepared from top-down processed cellulose material exist, [337] the nexus between cellulose synthesis and hydrogel formation is less well explored.Hydrogel preparation under conditions of bottom-up synthesis coupled to gelation in situ offers unique opportunities to tune the composition and the properties of the resulting materials.Considering that cellulose chain assemblies are typically stable and robust to changes in bulk conditions (e.g., temperature, pH, ionic strength), [338] structurally durable biomaterials of versatile function might be obtained.12a,15a,297] Several studies have explored the kinetic requirements for gelation of the synthetic cellulose.Soluble precursors need to be formed at sufficient rate [44b] and in sufficient concentration [69] to support the gel formation.44a] Water-soluble macromolecular additives (e.g., dextran, PEG, poly(N-vinylpyrrolidone), Ficoll, gelatin) supplied in high concentration (≥100 g L −1 ) modulate the cellulose chain assembly and the associated gelation. [339]he gel formation is slowed down in the presence of the "macromolecular crowding" agents but cellulose precipitation is reduced/prevented. [339]44a,307,335,340] Cellulose composite hydrogel prepared with cellulose nanocrystals (0.1-0.5% w/v) as reinforcing filler material exhibited enhanced stability (Young's modulus of up to 21 kPa). [307]Enzymatic synthesis of cellulose oligomers in dispersions of graphene oxides (1 mg mL −1 ) facilitated the assembly of aggregation-prone graphene oxides (lateral size of 1-2 μm and thickness of 1 nm) into a stable and highly porous nanoribbon structure.340b] Small molecule additives have also been used to tune the gel formation by synthetic cellulose.336b] The ionogel thus obtained could be interesting for electrochemical applications.It is worth emphasizing that the CdP reaction was broadly applicable under conditions of small molecule addition.
Cellulose synthesis coupled to mineralization processes happening within, or directly on, the biomolecular assembly of cellulose chains was suggested for the sustainable fabrication of organic-inorganic hybrid materials, as hydrogels with desired mechanical properties.Due to the phosphate ions released from Glc1P during enzymatic polymerization, the presence of Mg 2+ cations in the reaction promotes a concomitant mineralization and gel formation. [69]The controls show the absence of gelation in the absence of Mg 2+ -induced mineralization, suggesting causal connection between the two processes. [69]More recently, Serizawa and co-workers [341] demonstrated a mechanically enforced hybrid hydrogel composed of nanoribbon assemblies of reducing-end carboxylated cellulose chains (average DP 8-9) and hydroxyapatite.The hydroxyapatite was formed through the mineralization of Ca 2+ cations with phosphate ions released in the reaction.It appeared as the hydroxyapatite granules attached to the cellulose nanoribbon network grown from the synthetic cellulose.The Young's modulus of the hydrogel was notably improved (57 kPa, ∼8-fold), suggesting a beneficial effect of material hybridization on the mechanical properties. [341]astly, chemically induced gelation of cellulose can be facilitated through structure design of the synthetic cellulose chains that are amenable to chemical crosslinking.334c] The chains were shown to self-assemble into crystalline nanosheets of cellulose II polymorph structure, featuring polymerization-active methacryloxyethyl groups exposed on the surface.Through thiol-ene Michael addition reaction, the synthetic cellulose (0.5-2.0 wt%) was incorporated into PEG matrix, forming a hybrid hydrogel that is swellable (swelling ratio, 2.8-8.6),334c] Reducing-end functionalization with a reactive moiety in the way shown can open up versatile routes toward property-tunable cellulose hydrogels via chemical design.

Reducing-End Modified Synthetic Cellulose
The relaxed specificity of several CdPs for the structure of the acceptor substrate for iterative glycosylation enables the synthesis of cellulose chains with variable chemical modification at the reducing end.The groups, introduced through -glycosidic linkage with glucose residue, include alkyl alcohols, oligo-ethylene glycol, vinyl alcohol, azido, thiol, and fluorine (Table 6).Unless mentioned later, the cellulose chains resulting from iterative -1,4glycosylation of these -glucosides self-assemble into crystalline material of cellulose II structure.The preferred cellulose polymorph implies antiparallel alignment of cellulose chains. [8]12a,71b,301,334a] The reducing ends bearing the functional groups are thus displayed both sides of the platelet surface.This generates surfacespecific chemical character and reactivity in the cellulose assemblies as-synthesized.
Synthetic cellulose with -linked alkyl groups at the reducing end assembles into variable nanomorphologies dependent on the alkyl group length (Table 6, Figure 12). [331]Interestingly, the cellulose chains (DP 5-9) prepared from octyl-glucoside assumed a bilayer structure and the cellulose was of polymorph I crystallinity.The bilayered material was explored for absorption of hydrophobic molecules, such as the fluorescent dye Nile red. [345]Controlled release of the loaded dye was shown in response to cellulose degradation by cellulases, suggesting a possible application in detection assays for enzyme activity. [345]The "n-octyl-cellulose" just described was also considered as a molecular surfactant which, through its ability to adsorb to aqueous-organic interfaces, can be applicable to the structuring of liquids in two-phase mixtures. [349]aterial prepared from the "n-hexyl-cellulose" (average DP 7) showed a lamellar assembly of cellulose II crystallinity [350] and promoted hydrophobic adsorption of protein. [351]Celluloses with a phenolic group (e.g., arbutin) [348] were synthesized and explored for polymer coating applications.The surface of polyvinylidene fluoride membrane was made more hydrophilic by coating with the "phenol-cellulose."The membrane exhibited improved characteristics regarding of antifouling performance, antibacterial activity, and antiadhesion to Staphylococcus aureus. [348]elluloses with oligo(ethylene glycol) groups at the reducing end were shown to form hydrogels comprised of cellulose II nanoribbon networks (Figure 12). [352]Recently, bifunctional oligo(ethylene glycol)-based primers were shown to enable enzymatic synthesis of di-or triblock co-oligomers of cellulose (Figure 13). [353]Oligo(ethylene glycol) featuring a reactive triazole group on each end was derivatized at the triazole with glucosyl or -cellobiosyl, as shown in Figure 13.Thus, a bifunctional acceptor substrate is generated that can be elongated at both ends by CdP reaction.Studies with C. thermocellum CdP show that the -glucosyl primer was polymerized only at one end, giving a diblock co-oligomer of average DP ∼9.Interestingly, the -cellobiosyl primer was elongated at both ends, giving a triblock co-oligomer of average DP ∼10.The co-oligomers self-assemble into nanoribbon networks of cellulose II crystallinity. [353]Syn-thetic block copolymers in the way shown can provide new entries into the preparation of nano-to macroscale materials featuring stable architecture and interaction of the polymer phases, as shown recently with organic oligo(ethylene oxide) biblock copolymers. [354]gure 12.CdP-catalyzed polymerization of reducing end-functionalized acceptor substrates and self-assembly of the resulting oligomer chains into crystalline nanostructures of cellulose.The illustrations of cellulose nanostructure are reproduced with permission. [331]Copyright 2016, American Chemical Society.Cellulose featuring -N 3 at the reducing end has attracted interest due to the convenient handle that the azido groups provide for further site-selective derivatizations of the material.The "azido-cellulose" (average DP 9) obtained from reaction with 1-azido--d-glucose assumed a 2D nanosheet (Figure 12) of cellulose II crystallinity.The material was functionalized with 1-ethynyl pyrene via Huisgen cycloaddition reaction. [342]hus, spectroscopic reporter group can be incorporated into the cellulose material.Recently, azido-cellulose (average DP 7-8) was used for surface functionalization of cellulose paper.An approach of neutralization-driven self-assembly of the azidecontaining cellulose chains (see Section 4.3.5)was used to coat the surface of the paper with an azido-cellulose of cellulose II crystallinity.The paper surface was immobilized with biotin through cycloaddition reaction and could thus be used for immunoglobulin G detection. [347]Methods based on synthetic azido-cellulose offer replication potential for versatile material applications.
Like azide, the thiol group is suitable for site-selective chemical modification (e.g., by thiol-ene "click" reaction). [355]Moreover, it provides a site for pH-dependent ionization, [356] for coordination of metal ions, [357] and for biological recognition. [358]In fact, thiolated celluloses have been reported for their interaction with biological tissues different from plain cellulose. [359]334b] The thiol surface groups enabled the templated assembly of silver nanoparticles (2.2 g silver/ g cellulose material) in a high yield of ≥95%.359b,360] Point recognized while studying the synthesis of thiol-cellulose [346] was that the CdP enzymes from different sources differ intrinsically (i.e., at the level of highly purified enzyme) in the degree that they can hydrolyze the Glc1P donor substrate.The glucose released by the hydrolysis can compete with the -glucoside acceptor for cellulose chain polymerization.Depending on the extent of hydrolysis and competition so generated, the thiol-cellulose was contaminated with plain (underivatized) cellulose.When working with slow acceptor substrates of CdP, therefore, the choice of enzyme for synthesis becomes important. [346]3.5.Neutralization-Induced Self-Assembly of Synthetic Cellulose Serizawa and co-workers developed an approach referred to as "neutralization-induced self-assembly" in order to expand the possible material applications of synthetic cellulose.The approach involves an alkaline dissolution of the cellulose material (pH ≥ 13) that is followed by careful neutralization to trigger chain self-assembly.[361] The setting and the conditions used for the self-assembly can differ largely from the ones used during enzymatic synthesis.This can facilitate the formation of new cellulose nanostructures and nanomorphologies.[350,362] Moreover, it provides opportunities to create hybrid materials that could not have been formed in conjunction with the cellulose synthesis.[361] Surface coating applications (e.g., azido-cellulose on paper, [347] phenol-cellulose on polyvinylidene fluoride membrane [348] ) represent the situations of material fabrication that benefit from neutralization-induced self-assembly.Alkyl-celluloses can be rearranged from the cellulose I crystallinity in the as-synthesized materials to the more common cellulose II crystallinity after neutralization-induced self-assembly under certain conditions of temperature and salt.[350] The nanomorphology of the alkylcellulose material can also be fine-tuned in that way.[351] The neutralization-induced self-assembly has been explored in combination with gelatin as macromolecular crowding agent, resulting in a double-network hydrogel that showed fourfold enhanced stiffness (force-stroke; 32 mN mm −1 ) supposedly because the cellulose chains become entangled with the gelatin.[362] The approach was applied furthermore to 3D cell encapsulation that showed promising results with mammalian cells, to improve the cell viability and processability (e.g., filtration).[361]

Conclusions and Perspective
Biocatalysis by natural and engineered enzymes provides a powerful toolbox of synthesis for the bottom-up assembly of dglucans as materials.The types of enzymes available for d-glucan synthesis are reviewed and the different requirements for their efficient and controllable use are discussed.Sucrose is the preferred starting material (donor substrate) for polymerization reactions by transglycosidases.Enzyme cascade reactions by phosphorylases or glycosyltransferases enable the use of sucrose via formation of the actual polymerization donor substrates in situ.Starch can also be used with phosphorylases.As exemplified by the -1,3-glucan products currently moving to commercial application (e.g., Nuvolve), sustainable production of d-glucans by enzymatic synthesis is showcased and opportunities for diverse applications (e.g., coatings, fibers and fillers, composites, packaging) are opened up.The synthetic d-glucans are here presented in particular for the versatile (soft) material applications considered for development.End-group functionalized d-glucans, block-copolymers of d-glucans and supramolecular hybrid dglucan materials are important directions of future development in synthetic d-glucan polymers.Controlling and engineering the nanostructural assembly of d-glucan chains into organized materials of tunable morphology is an important, yet still very challenging topic.Unraveling the interplay of d-glucan chain synthesis in solution and association of chains to form solid materials is another topic of considerable interest whose study is greatly facilitated by enzymatic bottom-up synthesis.The possibility of structural and functional diversification of the d-glucan chain in ways inaccessible to well-developed top-down routes of polysaccharide material processing is inspirational for new material design.
linear glucan (oligo-/polysaccharides) are shaded; and the branched glucan are marked with asterisks;

Figure 1 .
Figure 1.Comparison of different approaches for the preparation of glycans.The index for each category is present with the number of filled circles.

Figure 2 .
Figure 2. Orthogonal protecting group strategy for multistep iterative glycosylation.The glycosyl acceptor is protected for site-selective glycosylation.The  or  configuration in the glycoside formed is directed by the protecting group R/R′ at the O-2 position (neighboring group).

Figure 3 .
Figure 3. Glucan synthesis by different types of enzymes.The nascent oligosaccharide is framed and iterative cycles of its glycosylation lead to chain polymerization.Cascade reactions to provide glucose 1-phosphate or nucleotide-activated glucose in situ are shown.Hydrolysis of donor substrates by transglycosidases and glycosynthases are marked with asterisks.The R group on the sugar acceptor can be variable.

Figure 7 .
Figure 7. a) Enzymatic syntheses of amylose and b) preparation of amylose-based hybrid materials due to complex formation with guest polymers.

Figure 8 .
Figure 8. Block copolymer application of bottom-up amylose synthesis.a) Reducing end-modified amylose (or maltodextrin) primers for the synthesis of block copolymers.b) Self-assembly of amylose-block copolymers and the corresponding nanostructures formed.The microscopy image of amyloseb-polystyrene micellar aggregates is reproduced with permission.Reproduced with permission. [177b] Copyright 2005, American Chemical Society.

Figure 11 .
Figure 11.Structure and morphology of -1,3-glucan materials.a) Triple helical structure of curdlan.Each chain is represented in a different color.The triangular arrangement of interstrand H-bonds is presented.b) Structure of -1,3-glucan lamella and microparticle.Upper panel: hexagonally shaped lamellar structure of -1,3-glucan assembly.Lower panel: transmitted light and SEM images of -1,3-glucan microparticles synthesized at 50 °C.The structure of curdlan and images of microparticles are reproduced with permission.Reproduced with permission. [245b,277] Copyright 2022, MDPI; Copyright 2022, Royal Society of Chemistry.

Figure 14 .
Figure 14.Enzymatically synthesized reducing end thiol-modified nanocellulose and its applications as functional (composite) materials.

Table 5 .
Property comparison of cellulose prepared from top-down and bottom-up approach.

Table 6 .
Reducing-end modified cello-oligomers, and the crystallinity and morphology of their assembled materials.