• KSBB Journal
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• 제16권2호
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• pp.212-215
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• 2001

The effect of various additives on the thermostability of Bacillus sp. cyclodextrin glucanotransferase (CGTase) was investigated. CaCl$_2$, starch, and glycerol had a positive effect on the thermostability of the CGTase, which was very stable for 6 months with added starch (5%, w/v) and CaCl$_2$(0.05 M) at 30$^{\circ}C$.

• 한국미생물·생명공학회지
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• 제19권4호
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• pp.368-371
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• 1991

Ethylene glycol, glycerol, sorbitol 그리고 sucrose가 Bacillus stearothermophilus가 생산하는 cyclodextrin glucanotransferase(CGYase)의 열안정성에 미치는 영향을 조사하였다. Glycerol, sorbitol 그리고 sucrose가 CGTase의 열안정성에 효과가 있었다. 이 효과는 첨가물의 농도와 밀접한 관계가 있었다. n-Butanol, 1,4-dioxane그리고 n-octane과 같은 유기 용매에서 CGTase의 열안정성을 조사하였다. 1,4-dioxane 과 n-octane은 CGTase의 열안정성을 증가시켰다. 특히, n-octane에서 CGTase를 $75^{\circ}C$에서 90분간 정치시킨 후에도 원래 효소활성의 81를 유지하였다.

• 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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• 제34권8호
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• pp.1660-1670
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• 2024

The aim of this study was to modify phytase YiAPPA via protein surficial residue mutation to obtain phytase mutants with improved thermostability and activity, enhancing its application potential in the food industry. First, homology modeling of YiAPPA was performed. By adopting the strategy of protein surficial residue mutation, the lysine (Lys) and glycine (Gly) residues on the protein surface were selected for site-directed mutagenesis to construct single-site mutants. Thermostability screening was performed to obtain mutants (K189R and K216R) with significantly elevated thermostability. The combined mutant K189R/K216R was constructed via beneficial mutation site stacking and characterized. Compared with those of YiAPPA, the half-life of K189R/K216R at 80℃ was extended from 14.81 min to 23.35 min, half-inactivation temperature (T₅₀³⁰) was increased from 55.12℃ to 62.44℃, and T_m value was increased from 48.36℃ to 53.18℃. Meanwhile, the specific activity of K189R/K216R at 37℃ and pH 4.5 increased from 3960.81 to 4469.13 U/mg. Molecular structure modeling analysis and molecular dynamics simulation showed that new hydrogen bonds were introduced into K189R/K216R, improving the stability of certain structural units of the phytase and its thermostability. The enhanced activity was primarily attributed to reduced enzyme-substrate binding energy and shorter nucleophilic attack distance between the catalytic residue His28 and the phytate substrate. Additionally, the K189R/K216R mutant increased the hydrolysis efficiency of phytate in food ingredients by 1.73-2.36 times. This study established an effective method for the molecular modification of phytase thermostability and activity, providing the food industry with an efficient phytase for hydrolyzing phytate in food ingredients.

• 한국식품영양학회지
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• 제10권2호
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• pp.160-165
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• 1997

오징어의 actomyosin과 myosin은 3$0^{\circ}C$에서 최대 Vmax를 나타냈고 대합의 actomyosin과 myosin은 35$^{\circ}C$에서, HMM은 $25^{\circ}C$에서 최대 Vmax를 보였다. 또한 근원섬유단백질의 열 안정성은 염농도의 차이에 따라 크게 변하여 염농도가 높을수록 변성의 정도가 컸으며 대합의 근원섬유단백질이 오징어의 근원섬유단백질보다 높은 열 안정성을 나타냈다. 근원섬유단백질에 3%의 에탄올을 첨가하여 가온하면 변성은 가속화되었으며 변성속도에 있어서도 동물간에 차이가 있는 것으로 나타났다.

• 한국식품영양과학회지
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• 제28권1호
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• pp.81-86
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• 1999

The factors affecting thermostability and reactivation of peroxidase from soybean sprouts(Glycine max L.) were investigated. The enzyme was the most stable at pH 7.0 and below 60oC. Thermostability of the enzyme was reduced by addition of sodium chloride and saccharides. The partially inactivated enzyme by heat treatment at 75oC for 10 min was reactivated up to 211.5% at the optimal reactivation condition. The optimal pH and temperature for reactivation of the enzyme were pH 9.0 and 40oC, respectively. The reactivation was completely inhibited by addition of sulfhydryl reagent such as L cysteine.

• 한국식품영양과학회지
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• 제30권5호
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• pp.844-847
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• 2001

감자로부터 polyphenol oxidase를 추출하여 열안정성에 영향을 주는 요인을 식품의 제조공정에 유용한 정보를 제공하기 위해 연구를 하였다. 감자의 PPO는 pH 7.0에서 가장 안정하였고, 이 효소는 8$0^{\circ}C$에서 1분간의 열처리로 효소 활성이 70% 저해되었다. 감자 PPO의 열안정성은 sodium chloride의 첨가로 감소되었으며, 2-mercaptoethanol과 dithiothreitol과 같은 환원제의 첨가로 역시 효소활성이 저해되었다.

• Advances in environmental research
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• 제3권4호
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• pp.307-319
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• 2014

Four chickpea cultivars viz. kabuli (Pusa 1088 and Pusa 1053) and desi (Pusa 1103 and Pusa 547) differing in sensitivity to high temperature conditions were analyzed in earthern pot (30 cm) at different stages of growth and development in the year of 2010 and 2011. Pusa-1053 (kabuli type) showed maximum photosynthetic rate and least by Pusa-547 (desi type), whereas maximum cell membrane thermostability were recorded in Pusa-1103 and minimum in Pusa-1088. Among the treatments, the plants grown under elevated temperature conditions had produced 13.01% more significant data in comparison to plants grown under continuous natural conditions. Stomatal conductance were reduced 44.25% under elevated temperature conditions than natural conditions, whereas 35.56%, when plants grown under initially natural conditions upto 30DAS, then 30-60DAS elevated temperature and finally shifted to natural conditions till harvest. In case of Pusa-1103, stomatal conductance was maximum as compared to rest of 2.7% from Pusa-1053, 8.9% from Pusa-1088, and 10.3% in Pusa-547 throughout the study. Plants grown under continuous elevated temperature conditions had produced 15.30% and 15.32% more significant membrane thermostability index in comparison to continuous natural conditions at vegetative stage and 19.40% and 18.44% at flowering stage, while the better response was recorded at pod formation stage. Pusa-1053 had given 2.8% more membrane thermostability index than Pusa-1088 and Pusa-1103 had given 1.6% more membrane thermostability index than Pusa-547 in the present study. The membrane disruption caused by high temperature may alter water ion and inorganic solutes movement, photosynthesis and respiration. Thus, thermostability of the cell membrane depends on the degree of the electrolyte leakage.

• 식품기술
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• 제8권4호
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• pp.179-191
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• 1995

Thermoanaerobacter ethanolicus유래 thermophilic amylopullulanase의 thermophilicity와 thermostability의 기작을 규명하기 위하여 N-말단과 C-말단으로부터 nested deletion mutatnt와 sitedirected mutagenesis등에 의한 변이효소를 제조, 분석하였다. 이러한 까다로운 변이효소를 제조하여 amylopullulanase의 특정부위가 효소의 thermophilicity와 thermostability에 관여하고 있는 것을 확인했다. N-말단의 start amino acid에서 194와 324 아미노산잔기에 이르는 부위 (TPR)가 이 효소의 높은 최적반응온도의 유지에 관련되어 있고 1102와 1224잔기에 이르는 부위 (TSR)는 thermodenaturation이 잘 일어나지 않도록 하고 있었다. 야생형 amylopullulanase (Apu), 변이효소중 ApuN342와 ApuN106/C379는 비슷한 효소비활성과 Km값을 가지고 있었다. TPR부위의 site-directed mutagenesis에 의한 변이효소중 P240A (proline$\rightarrow$alanine), P244A, P240A-P244A는 야생형의 최적반응온도 $80^{\circ}C$와 똑같았지만 효소의 열안전성(반감기)는 $85^{\circ}C$에서 21, 105, 128분을 보여 주었다. TSR에서의 변이효소중 P1159A, P1202A는 열안전성은 비슷하였지만 반응최적온도는 $85^{\circ}C$와 $90^{\circ}C$로 야생형 Apu보다 오히려 높아졌다. 따라서 proline은 분자내에서 thermophilicity와 thermostability를 항상 증가시키는 쪽으로 영향을 주지 않는 것으로 보인다. Proline은 그 위치와 주위의 다른 아미노산잔기와 같이 종합적으로 분자의 구조에 영향을 미치고 있는 것으로 판단된다.

• Journal of Microbiology and Biotechnology
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• 제26권7호
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• pp.1163-1172
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• 2016

The Ω-loop is a nonregular and flexible structure that plays an important role in molecular recognition, protein folding, and thermostability. In the present study, molecular dynamics simulation was carried out to assess the molecular stability and flexibility profile of the porcine trypsin structures. Two Ω-Loops (fragment 57-67 and fragment 78-91) were confirmed to represent the flexible region. Subsequently, glycosylation site-directed mutations (A73S, N84S, and R104S) were introduced within the Ω-loop region and its wing chain based on its potential N-glycosylation sites (Asn-Xaa-Ser/Thr consensus sequences) and structure information to improve the thermostability of trypsin. The result demonstrated that the half-life of the N84S mutant at 50℃ increased by 177.89 min when compared with that of the wild-type enzyme. Furthermore, the significant increase in the thermal stability of the N84S mutant has also been proven by an increase in the T_m values determined by circular dichroism. Additionally, the optimum temperatures of the wild-type enzyme and the N84S mutant were 75℃ and 80℃, respectively. In conclusion, we obtained the thermostability-improved enzyme N84S mutant, and the strategy used to design this mutant based on its structural information and N-linked glycosylation modification could be applied to engineer other enzymes to meet the needs of the biotechnological industry.