• Journal of Microbiology and Biotechnology
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• v.25 no.6
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• pp.845-855
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• 2015

The present study describes the gene cloning and high-level expression of an alkaline and thermostable lipase gene from Trichosporon coremiiforme V3. Nucleotide analysis revealed that this lipase gene has an open reading frame of 1,692 bp without any introns, encoding a protein of 563 amino acid residues. The lipase gene without its signal sequence was cloned into plasmid pPICZαA and overexpressed in Pichia pastoris X33. The maximum lipase activity of recombinant lipase was 5,000 U/ml, which was obtained in fed-batch cultivation after 168 h induction with methanol in a 50 L bioreactor. The purified lipase showed high temperature tolerance, and being stable at 60℃ and kept 45% enzyme activity after 1 h incubation at 70℃. The stability, effects of metal ions and other reagents were also determined. The chain length specificity of the recombinant lipase showed high activity toward triolein (C18:1) and tripalmitin (C16:0).

• Journal of Life Science
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• v.12 no.6
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• pp.682-687
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• 2002

We analyzed improvement on the enzyme activity of CPase Taq by addition of various metal ions. The enzyme activity was increased more then four times by 1 mM cobalt ion and almost three times by 1 mM calcium ion. However, the active center metal zinc ion did not affect the enzyme activity. In order to investigate whether the active center metal affects the enzyme activity, zinc ion which is occupied the active center of the enzyme was replaced by cobalt ion which activates the enzyme activity very effectively. Since the cobalt ion in the active center of the cobalt-substituted CPase Taq did not affect the enzyme activity, it could act as the natal metal ion in the active center of the enzyme.

• 한국생물공학회:학술대회논문집
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• 2003.04a
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• pp.527-529
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• 2003

A fructosyltransferase from Leuconostoc mesenteroides TL1 was purified. The molecular mass of the enzyme was estimated to be 38 kDa based on the activity staining. The pH and temperature optima of the enzyme were 6.3 and $40^{\circ}C$, respectivly. Structural analysis of the polymer prepared from sucrose by the enzyme was determined by NMR Spectroscopy: It shows the heterogeneous linkages of levan-like fructan and dextran-like glucan.

• Microbiology and Biotechnology Letters
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• v.24 no.5
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• pp.560-566
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• 1996

A strain capable of producing thermostable chitinase suitable for chitooligosaccharide production was isolated from high temperature environment and identified as Bacillus licheniformis. The chitinase from Bacillus licheniformis KFB-Cl4 was only induced by addition of colloidal thitin into the basal medium as carbon source, showing the decrease of the chitinase production by supplernental addition of other carbon sources into the medium containing 1.0% colloidal chitin. Among organic and inorganic nitrogen sources, yeast extract was the most effective for the increase of total activity and specific activity, and had high affinity for the enzyme production. The optimum temperature of cell growth and thermostable chitinase production was 55$\circ$C. The optimum culture medium was composed of 1.2% colloidal chitin, 0.15% K$_{2}$HPO$_{4}$, 0.05% KH$_{2}$PO$_{4}$, 0.01% MgSO$_{4}$-7H$_{2}$O, 0.1% yeast extract (pH 6.5). Bacillus licheniformis KFB-C14 produced the thermostable chitinase of 3.89 units per ml culture fluid and 7.4 units per mg protein under rotary shaking at 150 rpm for 40 hr.

• Journal of Microbiology and Biotechnology
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• v.25 no.1
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• pp.44-49
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• 2015

Thermostable enzymes derived from Thermotoga maritima have attracted worldwide interest for their potential industrial applications. Structural analysis and docking studies were preformed on T. maritima β-glucosidase enzyme with cellobiose and pNP-linked substrates. The 3D structure of the thermostable β-glucosidase was downloaded from the Protein Data Bank database. Substrates were downloaded from the PubCehm database and were minimized using MOE software. Docking of BglA and substrates was carried out using MOE software. After analyzing docked enzyme/substrate complexes, it was found that Glu residues were mainly involved in the reaction, and other important residues such as Asn, Ser, Tyr, Trp, and His were involved in hydrogen bonding with pNP-linked substrates. By determining the substrate recognition pattern, a more suitable β-glucosidase enzyme could be developed, enhancing its industrial potential.

• Korean Journal of Food Science and Technology
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• v.26 no.4
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• pp.398-402
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• 1994

The participation of thermostable amylase in the decrease of viscosity of pine nut's porridge was investigated using the crude enzyme obtained from ammonium sulfate fractionation of pine nut extracts. The fraction precipitated at $35{\sim}55%$ saturation of ammonium sulfate had the highest specific activity of the enzyme. ${\alpha}-amylase$ activity was maximal at $75^{\circ}C$, pH 5.4. Amylograph data showed that addition of the enzyme to rice flour resulted in the significant decrease of its viscosity, suggesting the existence of thermostable ${\alpha}-amylase$ in pine nut.

• Fisheries and Aquatic Sciences
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• v.13 no.1
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• pp.36-48
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• 2010

An agar-degrading Agarivorans sp. AG17 strain was isolated from the red seaweed Grateloupia filicina collected from Jeju Island. A beta-agarase gene from Agarivorans sp. AG17 was cloned and designated as agrA. agrA has a 2,985 bp coding region encoding 995 amino acids and was classified into the glycoside hydrolase family (GHF)-50. Predicted molecular mass of the mature protein was 105 kDa. His-tagged agrA was overexpressed in Escherichia coli and purified as a fusion protein. The enzyme showed 158.8 unit/mg specific activity (optimum temperature at $65^{\circ}C$ and pH 5.5 in acetate buffer) with unique biochemical properties (high thermal and pH stabilities). Enzyme produced neoagarohexaose, neoagarotetraose and neoagarobiose by degrading agar, and hydrolyzed neoagaro-oligosaccharides were biologically active. Hence the purified enzyme has potential for use in industrial applications such as the development of cosmetics and pharmaceuticals.

• Journal of Microbiology
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• v.43 no.1
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• pp.34-37
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• 2005

In the present study, the xylA gene encoding a thermostable xylose (glucose) isomerase was cloned from Streptomyces chibaensis J-59. The open reading frame of xylA (1167 bp) encoded a protein of 388 amino acids with a calculated molecular mass of about 43 kDa. The XylA showed high sequence homology (92% identity) with that of S. olivochromogenes. The xylose (glucose) isomerase was expressed in Escherichia coli and purified. The purified recombinant XylA had an apparent molecular mass of 45 kDa, which corresponds to the molecular mass calculated from the deduced amino acid and that of the purified wild-type enzyme. The N-terminal sequences (14 amino acid residues) of the purified protein revealed that the sequences were identical to that deduced from the DNA sequence of the xylA gene. The optimum temperature of the purified enzyme was $85^{\circ}C$ and the enzyme exhibited a high level of heat stability.

• BMB Reports
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• v.39 no.1
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• pp.105-110
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• 2006

We have screened 766 strains of fungi from the BIOTEC Culture Collection (BCC) for xylanases working in extreme pH and/or high temperature conditions, the so-called extreme xylanases. From a total number of 32 strains producing extreme xylanases, the strain BCC7928, identified by using the internal transcribed spacer (ITS) sequence of rRNA to be a Marasmius sp., was chosen for further characterization because of its high xylanolytic activity at temperature as high as $90^{\circ}C$. The crude enzyme possessed high thermostability and pH stability. Purification of this xylanase was carried out using an anion exchanger followed by hydrophobic interaction chromatography, yielding the enzyme with >90% homogeneity. The molecular mass of the enzyme was approximately 40 kDa. The purified enzyme retained broad working pH range of 4-8 and optimal temperature of $90^{\circ}C$. When using xylan from birchwood as substrate, it exhibits $K_m$ and $V_{max}$ values of $2.6{\pm}0.6\;mg/ml$ and $428{\pm}26\;U/mg$, respectively. The enzyme rapidly hydrolysed xylans from birchwood, beechwood, and exhibited lower activity on xylan from wheatbran, or celluloses from carboxymethylcellulose and Avicel. The purified enzyme was highly stable at temperature ranges from 50 to $70^{\circ}C$. It retained 84% of its maximal activity after incubation in standard buffer containing 1% xylan substrate at $70^{\circ}C$ for 3 h. This thermostable xylanase should therefore be useful for several industrial applications, such as agricultural, food and biofuel.

• Journal of Microbiology and Biotechnology
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• v.8 no.6
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• pp.560-567
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• 1998

We have found that Thermoactinomyces vulgaris KFB-Cl00 produces a chitinase. The optimum temperature and pH of the enzyme activity were $55^{\circ}C$ and 6.5. The enzyme was stable after heat treatment at $80^{\circ}C$ for 30 min and stable in acidic and basic conditions (PH 6.0~11.0). The thermostable endo-chitinase from Thermoactinomyces vulgaris KFB-C100 was cloned into the plasmid pBR322 by using E. coli DH5$\alpha$ as a host strain. The positive clone carrying a recombinant plasmid (PKCHI23) with a 4.1-kb fragment containing the chitinase gene was found. The recombinant plasmid was analyzed to determine the essential region for chitinase activity and obtained a 2.3-kb fragment, which was sub cloned into pTrc99A using the PstI and SalI sites to construct pTrc99A/pKCHI23-3. The resulting plasmid exerted high chitinase activity upon transformation of E. coli XL1-Blue cells. Chitinase was overproduced 14 times more in the clone cells than in the wild-type cells and the enzyme was purified to homogeneity. The purified enzyme showed the similar properties as the native chitinase from T. vulgaris in terms of molecular weight and substrate specificity. The catalytic action of the cloned enzyme was an endo type, producing chitobiose as a major reaction product.