• Bulletin of the Korean Chemical Society
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• v.27 no.6
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• pp.921-924
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• 2006

• Journal of Microbiology and Biotechnology
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• v.24 no.7
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• pp.925-935
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• 2014

A cold-adapted carbohydrate esterase, CEST, belonging to the carbohydrate esterase family 6, was cloned from Microbulbifer thermotolerans DAU221. CEST was composed of 307 amino acids with the first 22 serving as a secretion signal peptide. The calculated molecular mass and isoelectric point of the mature enzyme were 31,244 Da and pH 5.89, respectively. The catalytic triad consisted of residues Ser37, Glu192, and His281 in the conserved regions: GQSNMXG, QGEX(D/N), and DXXH. The three-dimensional structure of CEST revealed that CEST belongs to the ${\alpha}/{\beta}$-class of protein consisted of a central six-stranded ${\beta}$-sheet flanked by eight ${\alpha}$-helices. The recombinant CEST was purified by His-tag affinity chromatography and the characterization showed its optimal temperature and pH were $15^{\circ}C$ and 8.0, respectively. Specifically, CEST maintained up to 70% of its enzyme activity when preincubated at $50^{\circ}C$ or $60^{\circ}C$ for 6 h, and 89% of its enzyme activity when preincubated at $70^{\circ}C$ for 1 h. The results suggest CEST belongs to group 3 of the cold-adapted enzymes. The enzyme activity was increased by $Na^+$ and $Mg^{2+}$ ions but was strongly inhibited by $Cu^+$ and $Hg^{2+}$ ions, at all ion concentrations. Using p-nitrophenyl acetate as a substrate, the enzyme had a $K_m$ of 0.278 mM and a $k_{cat}$ of $1.9s^{-1}$. Site-directed mutagenesis indicated that the catalytic triad (Ser37, Glu192, and His281) and Asp278 were essential for the enzyme activity.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2004.06a
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• pp.215-218
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• 2004

Dextrans make up a class of polysaccharides that are D-glucans of various structures with contiguous $\alpha$-1longrightarrow6 ~6 glycosidic linkages in the main chains and $\alpha$-1longrightarrow2, $\alpha$-1longrightarrow3, or $\alpha$-1longrightarrow4 branch glycosidic linkages, depending on the specificity of the particular dextransucrase. Glucansucrases that catalyze glucans synthesis from sucrose. When other carbohydrates, in addition to sucrose, are present in the enzyme digest, the enzyme transfers glucose to the carbohydrate acceptors in the secondary reaction that diverts some of the glucose from incorporation into glucan. Many carbohydrate acceptors have been recognized and the products that result are dependent on the particular enzyme and the structure of the particular acceptor. Because of these unique catalytic characteristics, various dextransucrases have many important industrial and medical uses. To improve the understanding of their action mode and extend their applications, this study describes mechanism of glucan synthesis and potential industrial uses of dextransucrases, and our recent findings on the structural, functional organization and directed evolution of the glucansucrases to offer for designing glucansucrases with improved properties.

• Journal of Microbiology and Biotechnology
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• v.13 no.6
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• pp.969-975
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• 2003

Two conserved amino acid residues in the extra sugar-binding space near the catalytic site of Thermus maltogenic amylase (ThMA) were analyzed for their role in the hydrolysis and transglycosylation activity of the enzyme. Site-directed mutagenesis was carried out by replacing N33l with a lysine (N331K), E332 with a histidine (E332H), or by replacing both residues at the same time (N331K/E332H). The measured $K_m$ values indicated that affinities toward all substrates tested, including starch, pullulan, ${\beta}-cyclomaltodextrin$, and acarbose, were lower in all the mutants compared to that of wild-type ThMA, leading to reduced hydrolysis activity. In addition, the lower ratio of transglycosylation to hydrolysis in the mutants compared to that in the wild-type ThMA indicated that these mutants preferred hydrolysis to the transglycosylation reaction. These results demonstrated that the conserved dipeptide at 331 and 332 of ThMA is directly involved in the formation and accumulation of transfer products by accommodating acceptor sugar molecules.

• Journal of Microbiology and Biotechnology
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• v.24 no.4
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• pp.440-446
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• 2014

The carbohydrate-binding module (CBM) is an important domain of most cellulases that plays a key role in the hydrolysis of cellulose. The neutral endoglucanase (EG1) gene was reconstructed. A redesigned endoglucanase, named EG2, was constructed with a CBM containing a linker from Corticium rolfsii (GenBank Accession No. D49448). The redesigned EG genes were expressed in Escherichia coli, and their characteristics are discussed. Results showed that the degradation of cellulose by EG2 was about double that by EG1. The specific activities of EG1 and EG2 were tested under optimal conditions, and EG2 had higher activity ($169.1{\pm}2.74$ U/mg) toward CMC-Na than did EG1 ($84.0{\pm}1.98$) in the process of cellulose degradation. The optimal pH and temperature, pH stability, and heat stability of EG1 and EG2 were similar. Results indicated that the CBM plays an essential role in the hydrolysis of cellulose. We can improve EG's catalytic power by adding the CBM from Corticium rolfsii.

• Journal of Microbiology and Biotechnology
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• v.14 no.5
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• pp.1075-1080
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• 2004

Dextransucrase (DSRB742) from Leuconostoc mesenteroides NRRL B-742CB is a glucosyltransferase that catalyzes the synthesis of dextran using sucrose, or the synthesis of oligosaccharides when acceptor molecules, like maltose, are present. The DSRB742 gene (dsrB742) was cloned and the properties were characterized. In order to identify critical amino acid residues, the DSRB742 amino acid sequence was aligned with glucosyltransferase sequences, and three amino acid residues reported as sucrose binding amino acids in Streptococcus glucosyltransferases were selected for site-directed mutagenesis experiments. Asp-533, Asp-536, and His-643 were independently replaced with Ala or Asn. D533A and D536A dextransucrases showed reduced dextran synthesis activities, 2.3% and 40.8% of DSRB742 dextransucrase, respectively, and D533N, D536N, H643A, end H643N dextransucrases showed complete suppression of dextran synthesis activities altogether. Additionally, D536N dextransucrase showed complete suppression of oligosaccharide synthesis activities. However, modifications at Asp-533 or at His-643 retained acceptor reaction activities in the range of 8.4% to 21.3% of DSRB742 acceptor reaction activity. Thus at least two carboxyl groups of Asp-533 and Asp-536, and His-643 as a proton donor, are essential for the catalysis process.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 1986.12a
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• pp.524.3-524
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• 1986

Carboxymethyl cellulase (CMCase) produced by cloned B. megaterium was found to contain 5.2% carbohydrate but no metal ion. The enzyme was isoelectric at pH 7.23 and was high is basic amino acids. The N-terminal of the enzyme was glutamic acid. The cellulolytic activity of this enzyme was extended to the small molecular substrates such as from cellotriose to cellopentaose. In additon, the enzyme showed transglycoslation activity. The pK values of the enzyme we estimated to be 4.4 and 6.7, andthat of the enzyme-substrate complex were 4.2 and 7.2, respectively. The enzyme was not affected by the treatment with iodoacetic acid, but the modification of enzyme with carbodiimide and diethyl pyrocarbonate resulted in a marked loss of the enzyme activity. These results suggest that the active site of enzyme essentially contains carboxylic and imidazole group of amino acid residues.

• Proceedings of the Korean Biophysical Society Conference
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• 1999.06a
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• pp.35-35
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• 1999

Amylases catalyze the hydrolysis of starch material and play central roles in carbohydrate metabolism. The structure and a size exclusion column chromatography proved that the enzyme is a dimer in solution. The N -terminal segment of the enzyme folds into a distinct domain and comprises the enzyme active site together with the central (${\alpha}$/ ${\beta}$)$\sub$8/ barrel of the adjacent subunit.(omitted)

• Journal of Microbiology and Biotechnology
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• v.19 no.10
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• pp.1077-1084
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• 2009

A genomic library was constructed to clone a xylanase gene (Mxyn10) from Demequina sp. JK4 isolated from a deep sea. Mxyn10 encoded a 471 residue protein with a calculated molecular mass of 49 kDa. This protein showed the highest sequence identity (70%) with the xylanase from Streptomyces lividans. Mxyn10 contains a catalytic domain that belongs to the glycoside hydrolase family 10 (GH10) and a carbohydrate-binding module (CBM) belonging to family 2. The optimum pH and temperature for enzymatic activity were pH 5.5 and $55^{\circ}C$, respectively. Mxyn10 exhibited good pH stability, remaining stable after treatment with buffers ranging from pH 3.5 to 10.0. The protein was not significantly affected by a variety of chemical reagents, including some compounds that usually inhibit the activity of other related enzymes. In addition, Mxyn10 showed activity on cellulose. These properties mark Mxyn10 as a potential enzyme for industrial application and saccharification processes essential for bioethanol production.

• Journal of Microbiology and Biotechnology
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• v.19 no.3
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• pp.277-285
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• 2009

The nucleotide sequence of the Paenibacillus curdlanolyticus B-6 xyn10A gene, encoding a xylanase Xyn10A, consists of 3,828 nucleotides encoding a protein of 1,276 amino acids with a predicted molecular mass of 142,726 Da. Sequence analysis indicated that Xyn10A is a multidomain enzyme comprising nine domains in the following order: three family 22 carbohydrate-binding modules (CBMs), a family 10 catalytic domain of glycosyl hydrolases (xylanase), a family 9 CBM, a glycine-rich region, and three surface layer homology (SLH) domains. Xyn10A was purified from a recombinant Escherichia coli by a single step of affinity purification on cellulose. It could effectively hydrolyze agricultural wastes and pure insoluble xylans, especially low substituted insoluble xylan. The hydrolysis products were a series of short-chain xylooligosaccharides, indicating that the purified enzyme was an endo-$\beta$-1,4-xylanase. Xyn10A bound to various insoluble polysaccharides including Avicel, $\alpha$-cellulose, insoluble birchwood and oat spelt xylans, chitin, and starches, and the cell wall fragments of P. curdlanolyticus B-6, indicating that both the CBM and the SLH domains are fully functioning in the Xyn10A. Removal of the CBMs from Xyn10A strongly reduced the ability of plant cell wall hydrolysis. These results suggested that the CBMs of Xyn10A play an important role in the hydrolysis of plant cell walls.