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Keywords:

  • chitooligosaccharides;
  • N-acetylhexosaminidase;
  • Stenotrophomonas maltophilia

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The Stenotrophomonas maltophilia k279a (Stm) Hex gene encodes a polypeptide of 785 amino acid residues, with an N-terminal signal peptide. StmHex was cloned without signal peptide and expressed as an 83.6 kDa soluble protein in Escherichia coli BL21 (DE3). Purified StmHex was optimally active at pH 5.0 and 40 °C. The Vmax, Km and kcat/Km for StmHex towards chitin hexamer were 10.55 nkat (mg protein)−1, 271 μM and 0.246 s−1 mM−1, while the kinetic values with chitobiose were 30.65 nkat (mg protein)−1, 2365 μM and 0.082 s−1 mM−1, respectively. Hydrolytic activity on chitooligosaccharides indicated that StmHex was an exo-acting enzyme and yielded N-acetyl-d-glucosamine (GlcNAc) as the final product. StmHex hydrolysed chitooligosaccharides (up to hexamer) into GlcNAc within 60 min, suggesting that this enzyme has potential for use in large-scale production of GlcNAc from chitooligosaccharides.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Chitin, a linear polysaccharide composed of β-1, 4 linkages of N-acetyl-d-glucosamine (GlcNAc) that can be obtained mainly from crustacean shells. To use chitin as a carbon source, microorganisms require at least three types of enzymes, namely endo-chitinase, exo-chitinase and N-acetyl-β-d-glucosaminidase (NAGase). Among these enzymes, NAGase hydrolyses the terminal, non-reducing GlcNAc residues in N-acetylchitobiose and N-acetylchitooligosaccharides with a high degree of polymerization (DP).

According to the CAZy database, NAGase belongs to glycosyl hydrolase family 3, 20 or 84. These three families are functionally related but differ significantly in their amino acid sequences. Family 20 NAGases use a classical substrate-assisted mechanism in which the carbonyl oxygen of the 2-acetamido group of the substrate acts as a catalytic nucleophile that attacks the anomeric C1 to form an oxazoline intermediate (Slámová et al., 2010). In bacteria and fungi, NAGase is required for nutritional purposes because chitin can be utilized as a source of nitrogen and carbon. As chitin is absent in plants, bacteria and vertebrates, NAGase has been used for developing species-specific pesticides (Yang et al., 2008; Liu et al., 2010; Neeraja et al., 2010). Bacterial N-acetyl-β-hexosaminidases (Hex) or N-acetyl-β-glucosaminidases have been intensively studied due to their important physiological role in cell-wall recycling. In marine chitinolytic bacteria such as Vibrio furnissii or Alteromonas species, N-acetyl-β-hexosaminidases participate in chitin degradation and chitinase inducer formation (Tsujibo et al., 1995; Keyhani & Roseman, 1996). In humans, deficiency of this enzyme causes severe disorders such as Tay–Sachs and Sandhoff disease (Gutternigg et al., 2009).

GlcNAc, the end product produced by the cooperative action of chitinases and N-acetyl-β-d-glucosaminidase, has pharmacological applications. GlcNAc possesses anti-inflammatory activity and has been used to treat ulcerative colitis and gastrointestinal inflammations and also as a nutritional substrate for paediatric chronic inflammatory bowel disease (Salvatore et al., 2000). The deacetylated derivative of GlcNAc helps regeneration of joint cartilage and in the treatment of osteoarthritis (Huskisson, 2008). Exploring the utilization of chitin has been important for industrial applications such as functional foods, nutraceuticals, cosmeceuticals and pharmaceuticals (Lee, 2009). Chemical or enzymatic degradation of chitin is necessary to generate GlcNAc from resilient polymer chitin. Production of GlcNAc by chemical methods is hampered by its poor repeatability and the toxic nature of oligosaccharides, necessitating enzyme hydrolysis.

Stenotrophomonas maltophilia (Stm) is an aerobic Gram-negative bacterium with strong chitinolytic ability. The CAZy database indicates that several genes in the genome sequence of S. maltophilia could be potentially involved in chitin turnover. The present study describes cloning, expression and characterization of a recombinant StmHex from S. maltophilia k279a, which has the ability to release GlcNAc from chitooligosaccharides.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Bacterial strains, plasmids, media and biochemicals

Stenotrophomonas maltophilia k279a (Microbial Type Culture Collection, IMTECH, Chandigarh, India) grown in Luria–Bertani (LB) medium at 37 °C for the extraction of genomic DNA (QIAgen, Duesseldorf, Germany). The plasmid pET 22b (+) (Novagen, Darmstadt, Germany) and Escherichia coli BL21 (DE3) were used for heterologous expression of StmHex. E. coli BL21 was grown in LB broth with ampicillin (100 μg mL−1). Restriction enzymes, T4 DNA ligase and Pfu DNA polymerase were from MBI Fermentas. Oligonucleotide primers were from Eurofins (Bangalore, India). Isopropyl-β-d-thiogalactoside (IPTG), ampicillin and all other chemicals used were purchased either from Sigma-Aldrich or Merck. The polymeric substrates α- and β-chitin were provided by Mahtani Chitosan (Veraval, India). Chitooligosaccharides with different DP were purchased from Seikagaku Corp (Tokyo, Japan), through Cape cod, USA.

Cloning of N-acetyl-β-d-hexosaminidase from S. maltophilia k279a

The 2.26 kb StmHex (GenBank accession no. CAQ47422) gene was PCR-amplified using gene-specific forward (5′-CATAACCATGGTGGCCGACCCCACGCCCGCCAC-3′) and reverse (5′-CCCAAGCTTTTTCCCCAGGGTCACCTCATCC-3′) primers with Pfu DNA polymerase. Expression vector pET 22b(+), and the amplicon were separately digested with NcoI and HindIII (restriction sites underlined in the primers), gel purified and ligated using T4 DNA ligase at 16 °C for 16 h. Transformants were selected on LB plates containing ampicillin.

Protein expression and purification

The cloned StmHex gene was expressed by taking a single colony of E. coli BL21(DE3), harbouring the respective recombinant plasmid. Culture was grown at 37 °C in LB medium containing ampicillin (100 μg mL−1) and chloramphenicol (25 μg mL−1). At an OD600 nm of 0.6 IPTG was added to a final concentration of 0.5 mM and incubated for 24 h at 18 °C and 200 r.p.m., followed by centrifugation at 9000 g for 10 min at 4 °C for harvesting the cells. The cell pellet expressing StmHex was suspended in Ni-NTA equilibration buffer (50 mM NaH2PO4, 100 mM NaCl and 10 mM imidazole, pH 8.0). Cells were lysed by sonication at 20% amplitude with thirty 15-s pulses on ice, with a Vibra cell Ultrasonic Processor (Sonics, Newtown, CT). The sonicated cell lysate was centrifuged at 15 200 g for 10 min at 4 °C to pellet the insoluble cell debris. Ni-NTA affinity chromatography was used to purify the C-terminal His-tag carrying StmHex as described by Suma & Podile (2013). After purification, the StmHex was buffer exchanged with 50 mM sodium acetate, pH 5.0, using Macrosep Centrifugal Devices (Pall Corp., Port Washington, NY), and stored at 4°C until use.

Protein measurement

Purified StmHex was quantified using a bicinchonic acid protein estimation kit (Novagen) using a standard calibration curve constructed from bovine serum albumin. The molecular weight of StmHex was estimated using the ExPASy compute pi/mw software tool. The amino acid sequence of StmHex was analysed for sequence homologies and a phylogenetic tree was constructed using mega 5 software.

Reducing end assay

A reducing end assay was performed in triplicate for chitinase activity. The reaction mixture contained 5 μg StmHex and 300 μM chitohexaose or chitobiose, and 50 mM sodium acetate buffer at pH 5.0 was incubated at 40 °C for 1 h; the reaction was stopped using colour reagent containing 0.5 M sodium carbonate and 0.05% potassium ferricyanide. The reaction mixture was boiled for 15 min and cooled to 30 °C. The reducing ends generated in the reaction were estimated at 420 nm, against a GlcNAc standard. Specific activity in nkat (mg protein)−1 was measured using graphpad Prism version 6.0 (GraphPad Software Inc., San Diego, CA). One katal is the amount of enzyme that converts 1 mole of substrate per second.

Kinetic analysis, temperature and pH optima

Kinetic parameters of StmHex were determined using 200–1800 μM chitohexaose and chitobiose as substrates and 0.5 μg StmHex in 50 mM sodium acetate buffer, pH 5.0. After 1 h of incubation at 40 °C, the generated reducing ends were measured by reducing end assay. Kinetic parameters were obtained by analysing average values of the triplicate data sets and fitted to the Michaelis–Menten equation to determine Vmax, Km and kcat using the nonlinear regression function available in graphpad Prism.

For optimum temperature and pH, the reaction mixture (40 μL) containing 300 μM substrate and 5 μg StmHex in 50 mM sodium acetate buffer, pH 5.0, was incubated at 40 °C for 1 h and subjected to reducing end assay as described by Suma & Podile (2013). The StmHex activity was assessed at pH 2.0–12.0 and 20–100 °C under standard assay conditions. Thermal stability of the enzyme was tested by preincubating the enzyme at 30–100 °C for 1 h followed by reducing end assay as described above. The data were subjected to statistical analysis by Student's t-test using sigmaplot Version 9.0.

Time-course analysis of chitooligosaccharide hydrolysis by HPLC

Hydrolysis of chitooligosaccharides (DP2–DP6) by StmHex was analysed by incubating 5 μg StmHex with 2.5 mM DP2–DP6 in 50 mM sodium acetate, pH 5.0, as described by Purushotham et al. (2012). Reaction mixtures were incubated at 40 °C for 0, 1, 2, 3, 5, 15, 30, 45, 60, 90, 120, 180 and 720 min. The reaction mixture (50 μL) was transferred to an Eppendorf tube containing an equal volume of 70% acetonitrile to stop the reaction and stored at 4 °C. Twenty microlitres of the reaction solution was injected into the HPLC machine equipped with a Shodex Asahipack NH2P-50 4E column (4.6 ID × 250 mm, Showa Denko K.K) using a Hamilton syringe. Reaction mixtures were analysed at 25 °C. The mobile phase consisted of 70% acetonitrile and 30% MilliQ H2O and the flow rate was set to 0.7 ml min−1. The eluted chitooligosaccharides were monitored by recording absorbance at 210 nm. The chitooligosaccharide mixture containing equal weights of oligomers ranging from DP1 to DP6 was used to plot a standard graph. The data points yielded a linear curve for each standard amino sugar with R2 values of 0.997–1.0, allowing molar concentrations of chitooligosaccharides to be determined with confidence.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Cloning and expression of StmHex

StmHex was predicted to contain an N-terminal leader peptide directing sec-dependent secretion. The signal peptide was predicted using the signalp server (http://www.cbs.dtu.dk/services/SignalP/). The Hex gene was cloned without the signal peptide-encoding portion (87 bp). The 2.26-kb amplicon of StmHex was cloned in the NcoI and HindIII sites of pET 22b(+) (Fig. 1a,b). The clone was checked by double digestion and confirmed by automated DNA sequencing (Eurofins). StmHex was expressed with a C-terminal His-tag in E. coli BL21(DE3) and obtained in soluble form. The extracted protein was purified using Ni-NT agarose chromatography. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of purified StmHex resolved at a molecular weight of 83.6 kDa, which corresponds to the expected molecular weight of StmHex (Fig. 1c). Purified StmHex was further used for characterization.

image

Figure 1. Amplification, cloning and expression of StmHex. (a) The Hex gene was amplified using Stm genomic DNA as template with gene specific primers. Lane 1: DNA ladder mix, lane 2: 2.26-kb amplicon of StmHex. (b) Cloning of StmHex in NcoI and HindIII sites of pET22b+ (Novagen) was confirmed by restriction digestion.:ane 1: DNA ladder mix, lane 2: NcoI and HindIII digest released 2.26-kb StmHex. (c) StmHex was eluted from the Ni-NTA column using elution buffer containing 250 mM imidazole. The molecular mass of the standards is indicated in kDa to the left of lane 1. Lane 1: prestained protein marker, lane 2: Ni-NTA purified StmHex (25 μg).

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Sequence analysis

The amino acid sequence of StmHex was blasted at the NCBI database to search for homologues and a phylogenetic tree was constructed using mega 5. StmHex shared 41% homology with NagC from Streptomyces thermoviolaceus (BAC76622), 36% with Nag2 from V. harveyii 650, 34% with GlcNAcase A from Pseudoalteromonas piscicida (BAB17855) and 26% with chitobiase from Serratia marcescens.

Kinetic analysis

The kinetic parameters of the hydrolytic activity of StmHex were determined with chitohexaose (DP6) and chitobiose (DP2) as substrates (Fig. 2). Velocity measurements with increasing substrate concentration, fitted to Michaelis–Menten kinetics, revealed that Vmax, Km and (kcat/Km) values for DP6 were 10.55 nkat (mg protein)−1, 271.1 μM and 0.246 s−1 mM−1, respectively, and for DP2 were 30.65 nkat (mg protein)−1, 2365 μM and 0.0820 s−1 mM−1, respectively.

image

Figure 2. Kinetic parameters of StmHex towards DP6 and DP2. A kinetic study was carried out using 2000–1800 μM of DP6 and DP2, and 0.5 μg of StmHex in 50 mM sodium acetate buffer, pH 5.0. The reaction products were quantified using a reducing group assay. The average of the triplicate data was fitted to the Michaelis–Menten equation by a nonlinear regression function of graphpad Prism version 6.0.

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Optimum temperature and pH

StmHex was incubated with DP6 at 20–100 °C to determine the optimum temperature. StmHex exhibited highest specific activity at 40 °C (Fig. 3a). After 4 h of incubation at 40 °C StmHex showed only a marginal decrease in activity (data not shown). Preincubation of StmHex at different temperatures, followed by reducing end assay, revealed that activity of StmHex was unaffected at 30 and 40 °C, but decreased by c. 50% at 50 °C. A gradual decrease in activity was evident up to 100 °C (Fig. 3b). StmHex showed highest specific activity in sodium acetate buffer, pH 5.0. Enzyme activity decreased at extreme high and low pH, and had > 60% activity between pH 5.0 and 11.0 (Fig. 4).

image

Figure 3. Effect of temperature on StmHex activity. (a) The optimum temperature for activity of StmHex was determined using 300 μM of DP6 and 5 μg of StmHex for 1 h in 50 mM sodium acetate buffer, pH 5.0, under standard assay conditions. The average of triplicate data was used for analysis. (b) StmHex was preincubated at 30–100 °C for 1 h followed by addition of 300 μM of DP6 and incubated at 37 °C for 1 h. The enzyme assay was performed under standard assay conditions. The average of triplicate data was used for analysis.

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image

Figure 4. Effect of pH on StmHex activity. The optimum pH for activity of StmHex was determined using 300 μM of DP6 and 5 μg of StmHex for 1 h at 40 °C in different pH buffers of 50 mM ranging from pH 2.0 to 12.0 under standard assay conditions. The average of triplicate data was used for analysis. Sodium citrate (image_n/fml12237-gra-0001.png), Sodium acetate (image_n/fml12237-gra-0002.png), Sodium phosphate (image_n/fml12237-gra-0003.png), Glycine-NaOH (image_n/fml12237-gra-0004.png), Sodium phosphate -NaOH (image_n/fml12237-gra-0005.png).

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HPLC of hydrolysis products from chitooligosaccharides

The time course of hydrolysis of chitooligosaccharides by StmHex was followed using isocratic HPLC, suggesting a substrate-size dependence of the activity that had a tendency to decrease with increase in the DP (Fig. 5). Among the test chitooligosaccharides, DP2 was hydrolysed rapidly to DP1 within 2 min, while DP5 and DP6 substrates required up to 60 min for complete hydrolysis to GlcNAc. The rate of hydrolysis of chitooligosaccharides was in the order: DP2 > DP3 > DP4 > DP5 ≅ DP6 (Fig. 5). At initial time points, the substrates DP3–DP6 were hydrolysed to DPn−1 + DP1 (where n is the substrate used) and released intermediate products that were one unit shorter than the starting substrates. During the time course of the reaction, these intermediate products were further degraded to the final product DP1.

image

Figure 5. Time-course of hydrolysis of chitooligosaccharides by StmHex as analysed by HPLC. The reaction mixture containing 2.5 mM chitooligosaccharides (DP6–DP2) in 50 mM sodium acetate buffer, pH 5.0, was incubated with 5 μg StmHex for different time periods starting from 0 to 60 min at 40 °C. At each time point, 50 μL of the reaction mixture was withdrawn and an equal amount of 70% acetonitrile was added to stop the reaction. The reaction solution (20 μL) was analysed by isocratic HPLC using a ShodexAsahipack NH2P-50 4E column and eluted chitooligosaccharides were monitored by recording absorption at 210 nm. The top profile shows a standard mixture of chitooligosaccharides ranging from DP1 to DP6, while other profiles below are at different incubation periods as indicated. Control represents the substrate without enzyme treatment. Alternate letters (a–j) represent product profile and concentration of chitooligosaccharides released by StmHex from DP2, DP3, DP4, DP5 and DP6 substrates, respectively. Products were quantified from respective peak areas by using standard calibration curves of chitooligosaccharides ranging from DP1 to DP6.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Chitinolytic enzymes produced from various sources are responsible for biological conversion of chitin, and their enzymatic properties have been extensively investigated (Purushotham et al., 2012; Kolstad et al., 2013; Suma & Podile, 2013). Thus, the production of inexpensive chitinolytic enzymes has received attention for the production of biochemicals from chitin and its oligomers. In this study, we cloned, heterologously expressed and purified StmHex using Ni-NTA column chromatography. The purified StmHex was characterized for its kinetics, optimum temperature, optimum pH and mode of hydrolysis on chitooligosaccharides.

Michaelis–Menten kinetics of StmHex with DP6 as substrate revealed higher overall catalytic efficiency over Nag2 of V. harveyii 650 (kcat/Km 1.66 × 10–4 s−1 M1; Suginta et al., 2010; Table 1). According to Keyhani & Roseman (1996), Koga et al. (1996) and Yang et al. (2008) N-acetyl-β-hexosaminidases with GH20 domain exhibited highest catalytic efficiency for chitobiose and no activity against long-chain polymers. The StmHex with GH20 domain did not show activity on chitin polymers, but active on chitooligomers including chitobiose.

Table 1. Comparison of overall catalytic efficiency of hexosaminidases from different organisms
 OrganismSubstratekcat/Km (s−1 mM−1)Reference
  1. a

    Values are the means of triplicate determinations.

StmHexa Stenotrophomonas maltophilia (GlcNAc)20.0820This study
StmHexa S. maltophilia (GlcNAc)60.246This study
OfHex1 Ostrinia furnacalis (GlcNAc)23428.0Liu et al. (2010)
VfExo I Vibrio furnissii (GlcNAc)2Keyhani & Roseman (1996)
VfExo I V. furnissii (GlcNAc)6Keyhani & Roseman (1996)
VhNag 2 V. harveyi (GlcNAc)60.166Suginta et al. (2010)
VhNag 2 V. harveyi (GlcNAc)20.055Suginta et al.(2010)
PfNag A Pseudomonas fluorescens pNP-GlcNAcPark et al. (2010)
LeHex20A Lentinula edodes (GlcNAc)6992.0Konno et al. (2012)
LeHex20A L. edodes (GlcNAc)2576.0Konno et al. (2012)

StmHex was active at 40 °C (Fig. 3a), similar to N-acetyl-β-glucosaminidase from Alteromonas sp. strain 0-7 (Tsujibo et al., 1995) and N-acetyl-β-glucosaminidase from V. furnissii (Keyhani & Roseman, 1996). Enzyme assay in different pH range buffers revealed that StmHex was optimally active at pH 5.0, similar to NagC from S. thermoviolaceus (Kubota et al., 2004) and chitobiase from V. parahemolyticus (Zhu et al., 1992); StmHex exhibited activity over a broad pH range.

StmHex reaction products were examined by HPLC using chitooligosaccharide (DP2–DP6) substrates. Nag2 from V. harveyii 650 required 25 h for complete hydrolysis of DP6 (Suginta et al., 2010), while StmHex required 1 h. N-acetyl-β-glucosaminidase from Pseudomonas fluorescens JK-0412 (Park et al., 2010) and Aeromonas hydrophila SUWA-9 (Lan et al., 2004) attained complete DP5 hydrolysis by 24 and 7 h, respectively, and StmHex completed the same in 1 h. NagC from S. thermoviolaceus exhibited activity only on DP2–DP5 substrates and not on DP6 substrate (Kubota et al., 2004). When colloidal chitin and α-chitin were used as substrates, StmHex did not release any products even after prolonged reaction (data not shown), indicating that the enzyme was not active on polymeric chitin, similar to hexosaminidases reported from V. furnissii and Penaeus japonicus (Keyhani & Roseman, 1996; Koga et al., 1996).

StmHex is an exo-acting enzyme that could be efficiently used for bioconversion of chitooligosaccharides into GlcNAc, in association with other chitinases, for large-scale applications.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We thank DST-FIST, UGC-SAP and DBT-CREBB for support to the Department of Plant Sciences, University of Hyderabad, and the European Union for a research grant within the project ‘NanoBioSaccharides’. K.S. thanks the Council of Scientific and Industrial Research (CSIR), Government of India, for a Senior Research Fellowship. The authors declare no conflict of interest.

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  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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