• Journal of Microbiology and Biotechnology
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• 제18권5호
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• pp.933-941
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• 2008

An extracellular $\beta$-glucosidase produced by Monascus purpureus NRRL1992 in submerged cultivation was purified by acetone precipitation, gel filtration, and hydrophobic interaction chromatography, resulting in a purification factor of 92-fold. A $2^2$ central-composite design (CCD) was performed to find the best temperature and pH conditions for enzyme activity. Maximum activity was observed in a wide range of temperature and pH values, with optimal conditions set at $50^{\circ}C$ and pH 5.5. The $\beta$-glucosidase showed moderate thermostability, was inhibited by $HgCl_2$, $K_2Cr_O_4$, and $K_2Cr_2O_7$, whereas other reagents including $\beta$-mercaptoethanol, SDS, and EDTA showed no effect. Activity was slightly stimulated by low concentrations of ethanol and methanol. Hydrolysis of p-nitrophenyl-$\beta$-D-glucopyranoside (pNPG), cellobiose, salicin, n-octyl-$\beta$-D-glucopyranoside, and maltose indicates that the $\beta$-glucosidase has broad substrate specificity. Apparently, glucosyl residues were removed from the nonreducing end of p-nitrophenyl-$\beta$-D-cellobiose. $\beta$-Glucosidase affinity and hydrolytic efficiency were higher for pNPG, followed by maltose and cellobiose. Glucose and cellobiose competitively inhibited pNPG hydrolysis.

• Journal of Microbiology and Biotechnology
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• 제23권1호
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• pp.7-14
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• 2013

In the Bacillus amyloliquefaciens ${\alpha}$-amylase (BAA), the loop (residues 176-185; region I) that is the part of the calcium-binding site (CaI, II) has two more amino acid residues than the ${\alpha}$-amylase from Bacillus licheniformis (BLA). Arg176 in this region makes an ionic interaction with Glu126 from region II (residues 118-130), but this interaction is lost in BLA owing to substitution of R176Q and E126V. The goal of the present work was to quantitatively estimate the effect of ionic interaction on the overall stability of the enzyme. To clarify the functional and structural significance of the corresponding salt bridge, Glu126 was deleted (${\Delta}$E126) and converted to Val (E126V), Asp (E126D), and Lys (E126K) by site-directed mutagenesis. Kinetic constants, thermodynamic parameters, and structural changes were examined for the wild-type and mutated forms using UV-visible, atomic absoption, and fluorescence emission spectroscopy. Wild-type exhibited higher $k_{cat}$ and $K_m$ but lower catalytic efficiency than the mutant enzymes. A decreased thermostability and an increased flexibility were also found in all of the mutant enzymes when compared with the wild-type. Additionally, the calcium content of the wild-type was more than ${\Delta}E126$. Thus, it may be suggested that ionic interaction could decrease the mobility of the discussed region, prevent the diffusion of cations, and improve the thermostability of the whole enzyme. Based on these observations, the contribution of loop destabilization may be compensated by the formation of a salt bridge that has been used as an evolutionary mechanism or structural adaptation by the mesophilic enzyme.

• Journal of Microbiology and Biotechnology
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• 제32권6호
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• pp.800-807
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• 2022

Four aprE genes encoding alkaline serine proteases from B. subtilis strains were used as template genes for family gene shuffling. Shuffled genes obtained by DNase I digestion followed by consecutive primerless and regular PCR reactions were ligated with pHY300PLK, an E. coli-Bacillus shuttle vector. The ligation mixture was introduced into B. subtilis WB600 and one transformant (FSM4) showed higher fibrinolytic activity. DNA sequencing confirmed that the shuffled gene (aprEFSM4) consisted of DNA mostly originated from either aprEJS2 or aprE176 in addition to some DNA from either aprE3-5 or aprESJ4. Mature AprEFSM4 (275 amino acids) was different from mature AprEJS2 in 4 amino acids and mature AprE176 in 2 amino acids. aprEFSM4 was overexpressed in E. coli BL21 (DE3) by using pET26b(+) and recombinant AprEFSM4 was purified. The optimal temperature and pH of AprEFSM4 were similar to those of parental enzymes. However, AprEFM4 showed better thermostability and fibrinogen hydrolytic activity than the parental enzymes. The results indicated that DNA shuffling could be used to improve fibrinolytic enzymes from Bacillus sp. for industrial applications.

• Journal of Chest Surgery
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• 제42권2호
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• pp.165-174
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• 2009

배경: 심장 수술의 발전과 함께 자기 조직이 아닌 다른 인공 보철편의 필요가 늘어나 그에 따른 다양한 대체제가 연구 개발, 이용되고 있다. 본 연구는 더 나은 인공 조직보철편의 개발을 위해 이종이식 판막(돼지)과 심낭(돼지, 소)을 고정액에 따른 조직의 물리적 성질의 변화와 장력의 관계, 탄력도 변화, 열성 안정성을 알아보고자 하였다. 대상 및 방법: 이종이식 판막과 심낭을 처리하지 않은 그룹(fresh), glutaraldehyde (GA)로 고정한 그룹, glutaraldehyde (GA)에 ethanol 등과 같은 용매(solvent)를 첨가하여 고정한 그룹 세 그룹으로 나누어 검사하였다. 1) 각각의 군을 물리적 활성도 검사를 시행하였다. 2) 각 군별로 이종이식편의 일방향성 장력과 탄력도를 검사 비교하였다. 3) 소 심낭과 돼지 심낭을 각군 간에 열성 안정성 검사를 시행하였다. 결과: 1) 물리적 활성도 검사에서 촉진시 유의한 차이가 없었고, 봉합과, 신장도면에서 저농도 GA에 비해 GA+용매 처리군이 좀더 단단하게 느껴졌다. 2) 일반적으로 돼지의 대동맥, 폐동맥 판막의 원주방향 일방향성 장력과 탄력도는 아무것도 처리하지 않은 것보다 GA 혹은 GA+용매 처리 시 나아졌고, GA 혹은 GA+용매로 처리한 것 간에는 유의한 차이가 없었다(p>0.05). 소와 돼지의 심낭의 경우에도 GA 혹은 GA+용매로 처리한 것 간에는 유의한 차이가 없었다(p>0.05). 3) 각 실험과 군간에 GA 농도별로 비교 시 모든 군에서 그렇지는 않았지만, 고농도로 GA로 처리한 군(돼지의 폐동맥 판막, 돼지 심낭)에서 탄력도가 더 증가하는 경향이었다. 4) 소와 돼지 심낭 절편의 열 안정성 검사에서 GA 처리군과 GA+용매로 처리군간에 급격 수축 시점이 $80^{\circ}C$로 서로 차이가 없었다(소 심낭: p=0.057, 돼지 심낭: p=0.227). 결론: 이종 이식 보철편의 GA 고정 시 용매의 첨가는 압력-장력, 압력-탄력도, 열성 안정성 등 물리적 손실은 가져오지 않는 것으로 생각되며, 계속해서 더 나은 이식 보철 편 개발을 위한 여러 기능성 용매의 사용이나 세정 액의 연구개발이 필요하다.

• 생명과학회지
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• 제23권1호
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• pp.15-23
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• 2013

Bacillus amyloliquefaciens CH51은 분자량 27 kDa 크기의 subtilisin 타입의 혈전용해능을 지니는 단백질분해효소인 AprE51을 생산하였다. 이전연구에서 더 우수한 혈전용해 활성을 갖는 AprE51-6이 세포외 돌연변이법으로 생산되었으며, 본 연구에서는 이 개선된 효소인 AprE51-6의 열안정성을 증진시킬 목적으로 B. subtilis subtilisin E의 아미노산과의 상동성 분석을 통하여 두 아미노산인 Gly-166과 Asn-218이 치환되었다. 그 결과 G166R과 N218S 돌연변이체는 혈전용해능을 보이는 용해능 배지에서 원 효소보다 각각 1.8배와 4.5배 높은 혈전용해능을 보였다. 정제된 두 돌연변이효소인 AprE51-7과 AprE51-8는 원효소인 AprE51-6에 비하여 1.9 그리고 2.5배 높은 $k_{cat}$값을 나타내었고, 2.1과 1.9배 낮은 기질친화력을 나타내는 $K_m$값을 보여주었다. 특히 AprE51-8는 나토키나아제에 비하여 알칼리 pH 영역에서 높은활성을 유지하였고, $60^{\circ}C$에서 더 우수한 열안정성을 보여주었다. 열안정성의 정도를 나타내는 척도인 반감기 값에서도 AprE51-7과 AprE51-8는 $50^{\circ}C$에서 21.5분과 27.3분으로 기존의 AprE51보다 2배 그리고 2.6배 더 긴 반감기를 보였다.

• Journal of Microbiology and Biotechnology
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• 제27권4호
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• pp.649-659
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• 2017

Extremophilic microorganisms have established a diversity of molecular strategies in order to survive in extreme conditions. Biocatalysts isolated by these organisms are termed extremozymes, and possess extraordinary properties of salt allowance, thermostability, and cold adaptivity. Extremozymes are very resistant to extreme conditions owing to their great solidity, and they pose new opportunities for biocatalysis and biotransformations, as well as for the development of the economy and new line of research, through their application. Thermophilic proteins, piezophilic proteins, acidophilic proteins, and halophilic proteins have been studied during the last few years. Amylases, proteases, lipases, pullulanases, cellulases, chitinases, xylanases, pectinases, isomerases, esterases, and dehydrogenases have great potential application for biotechnology, such as in agricultural, chemical, biomedical, and biotechnological processes. The study of extremozymes and their main applications have emerged during recent years.

• E2M - 전기 전자와 첨단 소재
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• 제7권6호
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• pp.527-533
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• 1994

In this study, it was studied on dielectric breakdown strength and thennostability properties due to the structure variation of matrix resin and treatment of coupling agent of epoxy insulating materials. The interpenetrating network structure was formed by simultaneous heating curing the epoxy resin with single network structure and the methacrylic acid resin. Also inner structure was observed and the glass transition temperature was measured on these three type specimens. Dielectric breakdown properties were investigated by applying DC, AC and impulse voltage. As a result, the glass transition temperature and the dielectric breakdown strength of specimen with interpenetrating network structure was more higher than another two type specimens.

• 한국미생물·생명공학회지
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• 제25권1호
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• pp.37-43
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• 1997

Triphenylmethane was decolorized rapidly by enterbacter cloacae MG 82 at initial reaction time. The spheroplast showed higher activity of triphenylmentane decolorization than that of intact cell suspension. The outer part of the bacterial cell envelope and the peptidoglycan are important for the function of transport barrier of triphenylmethane. In intact cell, decolorization activity was higher at 37$\circ $C than at $\circ $C, indicating that triphenylmethane decolorization is due to the enzyme reaction. Culture filtrate showed no decolorization activity, while cell-free extract appeared high activity of 1.45 units, clearly showing that decolorization activity was due to the cell-free extract. Comparing decolorization activities of cell fractions, it was found that decolorization activity was located at the compartment of cytoplasmic membrane. The enzyme activity was also shown to be Mg$^{++}$-dependent. The optimum pH and temperature of enzyme activity were 7.0 and 50$\circ $C, respectively. The thermostability of this enzyme at 35$\circ $C was kept to 58% for 3 hours.

• 한국세라믹학회지
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• 제12권2호
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• pp.15-20
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• 1975

Ceramic resistors to be stable at high temperature were manufactured from using MgO, SiO2, SnO2, Bi2O3, and CeO2 by sintering in air at 125$0^{\circ}C$. Electrical resistivity with elevated temperatures was studied for the various system of the above oxides. The resistor, 1.0 MgO-1.0 SiO2-0.575 SnO2-0.005 Sb2O3-0.025 Bi2O3-0.013 CeO2 has the resistivity, (14.55$\pm$0.3)$\times$103 ohm in a temperature range from $25^{\circ}C$. to 80$0^{\circ}C$. It is concluded that the ceramics prepared by a dielectric compound and metal oxide semi-conductor has a good thermostability for electrical appliciations.

• KSBB Journal
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• 제11권2호
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• pp.186-192
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• 1996

The effects of chemical modification on the enzymes' stability in polar organic solvents were studied with subtilisin Carlsberg in dimethylformamide-water mixtures as a model system. Three out of nine lysine residues of subtilisin Carlsberg were coupled to either trimellilic or pyromellitic anhydrides thereby, for each lysine residue modified, resulting in the net replacement of one basic amino group by two or three acidic carboxyl groups, respectively. In water at 60$^{\circ}C$, both trimellitic and pyromellitic anhydride-modified subtilisin Carlsberg showed increased thermostability by 2.6 times and 1.6 times, respectively, as compared to that of unmodified enzyme. In 70% dimethylformamide at 25$^{\circ}C$, however, only pyromellitic acrid was shown to enhance the stability of subtilisin Carlsberg by 5.5 times increasing the half life time of irreversible inactivation from 4.9hr for unmodified enzyme to 27.8hr for modified enzyme.