• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2000년도 추계학술발표대회 및 bio-venture fair
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• pp.56-59
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• 2000

Thermophilic bacterium, Bacillus stearothermophilus NUB3621, was engineered to produce ethanol from glucose by introducing cloned thermostable pyruvate decarboxylase and alcohol dehydrogenase genes. A novel promoter sequence was screened and used for the enhancement of these two enzymes. Successful redirection of metabolic flux into ethanol was obtained. In addition, gene expression profiling using Bacillus subtilis DNA microarray was analyzed to overcome the intrinsic low glucose utilization of B.stearothermophilus. Many known and unknown genes were identified to be up or down regulated under glucose-containing media.

• 한국식품과학회지
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• 제48권6호
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• pp.542-547
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• 2016

The thermal properties of ramie leaf ${\beta}$-amylase (RBA) were examined to develop a novel process for enzyme purification. The thermostability of RBA extract prepared from ramie leaf powder was examined at various temperatures. RBA activity decreased slightly, whereas other carbohydrate-active enzymes, such as $\small{D}$-enzyme, were rapidly inactivated during 30 min incubation at $60^{\circ}C$. When the heat-treated extract was incubated with various substrates, maltose was produced exclusively as the major product, whereas the untreated crude extract produced maltose and other maltooligosaccharides. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, fewer protein bands were observed for the heat-treated extract than the untreated extract, indicating that the thermostable RBA was partially purified and other thermolabile enzymes were eliminated. Thus, the treatment of the RBA extract at $60^{\circ}C$ for 30 min resulted in 5.4-fold purification with a recovery yield of 90%.

• Journal of Microbiology and Biotechnology
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• 제31권3호
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• pp.483-491
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• 2021

Two putative genes, lip29 and est29, encoding lipolytic enzymes from the thermophilic bacterium Geobacillus thermocatenulatus KCTC 3921 were cloned and overexpressed in Escherichia coli. The recombinant Lip29 and Est29 were purified 67.3-fold to homogeneity with specific activity of 2.27 U/mg and recovery of 5.8% and 14.4-fold with specific activity of 0.92 U/mg and recovery of 1.3%, respectively. The molecular mass of each purified enzyme was estimated to be 29 kDa by SDS-PAGE. The alignment analysis of amino acid sequences revealed that both enzymes belonged to GDSL lipase/esterase family including conserved blocks with SGNH catalytic residues which was mainly identified in plants before. While Est29 showed high specificity toward short-chain fatty acids (C4-C8), Lip29 showed strong lipolytic activity to long-chain fatty acids (C12-C16). The optimal activity of Lip29 toward p-nitrophenyl palmitate as a substrate was observed at 50℃ and pH 9.5, respectively, and its activity was maintained more than 24 h at optimal temperatures, indicating that Lip29 was thermostable. Lip29 exhibited high tolerance against detergents and metal ions. The homology modeling and substrate docking revealed that the long-chain substrates showed the greatest binding affinity toward enzyme. Based on the biochemical and insilico analyses, we present for the first time a GDSL-type lipase in the thermophilic bacteria group.

• 미생물과산업
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• 제19권2호
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• pp.34-41
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• 1993

Industrial strain improvement is concerned with developing or modifying microorganisms used in production of commercially important fermentation products. The aim is to reduce the production cost by improving productivity of a strain and manipulating specific characteristics such as the ability to utilize cheaper raw materials or resist bacteriophages. The traditional empirical approach to strain improvement is mutation combined with selection and breeding techniques. It is still used by us to improve the productivity of organisms in amino acids, organic acids and enzymes production. The breeding of high L-lysine-producing strain Au112 is one of the outstanding examples of this approach. It is a homoserine auxotroph with AEC, TA double metabolic analogue resistant markers. The yield reaches 100 g/l. Besides, the citric acid-producing organism Aspergillus niger, Co827, its productivity reaches the advanced level in the world, is also the result of a series mutations especially with $^60Co{\gamma}$-radiation. The thermostable .alpha.-amylase producing strain A 4041 is the third example. By combining physical and chemical mutations, the strain A 4041 becomes an asporogenous, catabolite derepressed mutant with rifamycin resistant and methionine, arginine auxotroph markers. The .alpha.-amylase activity reaches 200 units/ml. The fourth successful example of mutation in strain improvement is the glucoamylase-producing strain Aspergillus niger SP56, its enzyme activity is 20,000 units/ml, 4 times of that of the parental strain UV-11. Recently, recombinant DNA approach provides a worthwhile alternative strategy to industrial strain improvement. This technique had been used by us to increase the thermostable .alpha.-amylase production and on some genetic researches.

• Journal of Microbiology and Biotechnology
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• 제19권12호
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• pp.1536-1541
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• 2009

The DNA shuffling technique has been used to generate libraries of evolved enzymes in thermostability. We have shuffled two thermostable cytidine deaminases (CDAs) from Bacillus caldolyticus DSM405 (T53) and B. stearothermophilus IFO12550 (T101). The shuffled CDA library (SH1067 and SH1077 from the first round and SH2426 and SH2429 from the second round) showed various patterns in thermostability. The CDAs of SH1067 and SH1077 were more thermostable than that of T53. SH2426 showed 150% increased halftime than that of T53 at $70^{\circ}C$. The CDA of SH2429 showed about 200% decreased thermostability than that of T53 at $70^{\circ}C$. A single amino acid residue replacement that presented between SH1077 and SH2429 contributed to dramatic changes in specific activity and thermostability. On SDS-PAGE, the purified CDA of SH1077 tetramerized, whereas that of SH2429 denatured and became almost monomeric at $80^{\circ}C$. A simulated three-dimensional structure for the mutant CDA was used to interpret the mutational effect.

• Journal of Microbiology and Biotechnology
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• 제25권1호
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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.

• 한국미생물·생명공학회지
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• 제3권2호
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• pp.73-82
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• 1975

In the course of studies on the production of thermostable amylases by thermophilic actinomycetes isolated from soils the investigation was carried out on the production of $\alpha$-amylase by G-1011 strain which had presented the most remarkable $\alpha$-amylase formation ability among 128 amylolytic isolates. The results were as follows ： 1. Characteristics of G-1011 strain were compared with those descriptions of thermophilic actinomycetes given in Bergey's Manual. The strain was identical to these species of actinomycetes. The details of physiological properties of the strain luould he published in near future. 2. The optimum temperature for incubation of the cell growth of G-1011 strain and $\alpha$-amylase production by the strain was revealed to 5$0^{\circ}C$. 3. The effective medium for $\alpha$-amylase formation by the strain was consisted of 3.0%, soluble starch, 1.0%, peptone, 0.5%, yeast extract, 0.5%, NaCl, 0.1%, MgSO$_4$ㆍ7$H_2O$ 0.02%, $K_2$MPO$_4$and 0.002% FeSO$_4$ㆍ7$H_2O$. The pH of the medium was ajusted to 7.0 with phosphate buffer solution. 4. The maximum production of $\alpha$-amylase (3420 D. U/ml) by G-1011 strain resulted when it was grown for 16 hours with the culture of reciprocal shaking.

• 생명과학회지
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• 제7권1호
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• pp.30-38
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• 1997

내열성 {\alpha}$-amulase를 생산하는 미생물을 분리하기 위하여 각종 분리원으로 시료를 채취하여 55$^{\circ}C$ 이상에서 생육하고 가장 내열성 {\alpha}$-amulase 생산능이 우수한 균주를 분리, 동정한 결과 thermophilic Bacilus 속으로 추정되었다. 균체생육과 효소생산의 최적온도는 60~$65^{\circ}C$였고, 초발 pH8.0에서 가장 높은 효소생산능을 보였다. {\alpha}$-amulase 생산에 가장 양호한 탄소원은 soluble starch, dextrin, prtato starch, corn starch 등의 다당류이었으며 glucose, fructose 등의 단당류에서는 효소생산이 미약하거나 억제를 받았다. {\alpha}$-amulase 생산에 가장 중요한 질소원은 yeast extract이었따. 0.1% $CaCl_2{\cdot}2H_2O$, 0.001%의 Tween-80의 첨가는 {\alpha}$-amulase 생산성을 증가시켰다. 본 공사균이 생산한 조효소액의 특성을 검토해 본 결과, 8$0^{\circ}C$에서 최적 효소활성을 보였으며, 10$0^{\circ}C$에서도 23%의 잔존활성을 나타내었다. 최적 pH는 5.0이었다. $Ca^{2+}$은 효소활성 증진에는 영향을 미치지 않았다. 공시균으로부터 분리한 genomic DNA를 중온성 BAcillus subtilis KCTC 1024에 형질전환시킨 결과, transformant는 wild type에 비하여 약 2배의 효소활성이 증가되었다.

• Journal of Applied Biological Chemistry
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• 제44권3호
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• pp.113-118
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• 2001

Xylose (glucose) isomerase was purified to homogeneity from cell-extracts of Streptomyces chibaensis J-59 via ammonium sulfate precipitation followed by chromatography on DEAE-cellulose, and gel filtration on Sephacryl S-300. The purified enzyme is a homotetramer with a native molecular mass of 180 kDa and a subunit molecular mass of 44 kDa. The amino acid N-terminal sequence of glucose isomerase from S. chibaensis J-59 was determined to be Ser-Tyr-Gln-Pro-Thr-Pro-Glu-Asp-Arg-Phe-Thr-Phe-Gly-Leu. The first 14 amino acids of the N-terminal sequence of the enzyme showed strong analogies with N-terminal sequences of glucose isomerase produced by other Streptomyces spp. The optimum pH and temperature for activity were 7.5 and 85, respectively. The purified enzyme required $Mg^{2+}$, $Co^{2+}$, and $Mn^{2+}$ for the activity, $Mg^{2+}$ being the most effective. The enzyme was not inhibited by $Ca^{2+}$, but was inhibited by $Hg^{2+}$, $Ag^+$, and $Cu^{2+}$. The $K_m$, $V_{max}$, and $k_{cat}$ values of S. chibaensis J-59 isomerase for glucose were 83 mM, 40.9 U/mg, and $1,843min^{-1}$, respectively. In the presence of $Co^{2+}$, cell-free enzymes retained 100% without loss of activities by the heat-treatment at $70^{\circ}C$ for 7 days. The enzyme retained 50% residual activity after heating at $85^{\circ}C$ for 13.5 h, at $90^{\circ}C$ for 126 min. The enzyme is more thermostable than any other glucose isomerases of Streptomyces spp.

• 한국미생물·생명공학회지
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• 제16권6호
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• pp.484-488
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• 1988

토양분리균 Pseudomonas sp.가 생산하는 inulinase를 분리.정제하여 얻은 단일 단백질 효소 PI과 PII는 탄수화물 함량이 각각 15%와 2.4%인 당 단백질 형태의 endo-inulinase로서 두 효소가 모두 촉매활성에 필수적인 tryptophan 잔기를 가지고 있었다. 분자량은 PI 210,000, PII 170,000으로 측정되었다. 1mM pCMB 존재에 의해 두 효소가 약80% 정도의 활성저해를 보였으나 5mM cysteine 또는 1mM dithiothreitol을 첨가하면 효소활성이 거의 완전 회복되는 특성을 나타내었다. 최종 가수분해산물인 fructose(1mM)에 의해 PI, PII 효소가 각각 15% 정도의 활성저해를 받는 반면 Co$^{+2}$ 이온은 50~60%의 높은 활성화 효과를 보였다. 두 효소는 pH 4.0~7.5사이에서 매우 안정하였으며, 열에 대하여서도 비교적 안정하여 6$0^{\circ}C$, 120분 가열에 의해 PI이 약 27%, PII가 약 40%의 실활을 나타낼 뿐이다. 또한 60units의 효소를 사용, 2% inulin을 5$0^{\circ}C$에서 72시간 가수분해 시켰을 때 PI약 70%, PII약56%의 기질 분해율을 보였다.