• Journal of Microbiology
• /
• 제43권3호
• /
• pp.237-243
• /
• 2005

The protease purified from Bacillus cereus JH108 has the function of leucine specific endopeptidase. When measured by hydrolysis of synthetic substrate (N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide), the enzyme activity exhibited optimal activity at pH 9.0, $60^{\circ}C$. The endopeptidase activity was stimulated by $Ca^{++},\;Co^{++},\;Mn^{++},\;Mg^{++},\;and\;Ni^{++}$, and was inhibited by metal chelating agents such as EDTA, 1,10-phenanthroline, and EGTA. Addition of serine protease inhibitor, PMSF, resulted in the elimination of the activity. The endopeptidase activity was fully recovered from the inhibition of EDTA by the addition of 1 mM $Ca^{++}$, and was partially restored by $Co^{++}\;and\;Mn^{++}$, indicating that the enzyme was stabilized and activated by divalent cations and has a serine residue at the active site. Addition of $Ca^{++}$ increased the pH and heat stability of endopeptidase activity. These results show that endopeptidase requires calcium ions for activity and/or stability. A Lineweaver-Burk plot analysis indicated that the $K_m$ value of endopeptidase is 0.315 mM and $V_{max}$ is 0.222 ) is $0.222\;{\mu}mol$ of N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide per min. Bestatin was shown to act as a competitive inhibitor to the endopeptidase activity.

• Journal of Pharmaceutical Investigation
• /
• 제18권3호
• /
• pp.125-131
• /
• 1988

This study was undertaken to investigate the proteolytic enzyme from Neungee mushroom [Sarcodon aspratus (Berk) S. Ito]. The proteolytic activity of Neungee was higher than other several edible mushrooms under various pHs. The potency of proteolytic enzyme of Neungee was same as the digestive drugs containing protease. So the proteolytic activity of the enzyme was increased in neutral or weak alkaline pH, whose characteristics would be alkaline protease. The specific activity of the purified enzyme obtained by using Tris acryl CM-cellulose ion exchange increased 20 times as compared with that of the crude extract. The proteolytic enzyme was stable at room temperature, but decomposition was fast when incubated at higher temperature more than $40^{\circ}C$. The half life of the enzyme was longest in neutral pH and rate constant was increased in acidic or alkaline solution.

• Journal of Microbiology and Biotechnology
• /
• 제21권2호
• /
• pp.183-186
• /
• 2011

Biochemical methods were selected to evaluate the role of exopolymeric substances in the stability of biofilms used in bioremediation processes. Biofilms of Thiomonas arsenivorans formed on pozzolana were thus treated with pronase (protein target), lectins (Con A or PNA), calcofluor or periodic acid (polysaccharides target), DNase (DNA target), and lipase (triglycerides target). Neither protease nor DNase treatments had any effect on bacterial adhesion. Lectins and calcofluor treatments mainly affected young biofilms. Lipase treatment had a noticeable effect on biofilm stability whatever the biofilm age. Results suggest that it would be an increased resistance of mature biofilms that protects them from external attacks.

• 미생물학회지
• /
• 제47권3호
• /
• pp.255-262
• /
• 2011

무당벌레 소화기관으로부터 단백질 분해 우수세균 6균주를 분리하였다. 단백질 분해세균의 16S rRNA 유전자 염기서열을 해석하여 계통학적 특성을 검토한 결과, Staphylococcus sciuri subsp. sciuri (3균주), Bacillus subtilis (1균주), Bacillus thuringiensis (2균주)로 확인되었다. 이들 균주 중 pH 5.0 배지에서 58.5 U/ml의 높은 효소 활성을 나타내는 Staphylococcus sp. CB2-3을 최종 선발하였다. 효소의 특성을 조사한 결과, pH 4.0-6.0에서 높은 활성을 나타내어 산성 단백질 분해효소임이 확인되었다. 효소의 최적 반응 온도는 $40^{\circ}C$ 이었으며, $30-50^{\circ}C$의 범위에서 80% 이상의 효소 활성을 유지하였다. Staphylococcus sp. CB2-3 균주의 생육과 효소 활성을 위한 최적의 배지성분을 조사하였다. 탄소원으로 0.5% 솔비톨을 첨가하였을 때 효소 활성이 2배로 증가되었으며, 질소원으로 0.5% 탈지유를 첨가한 경우 효소 활성이 2.5배 증가되는 것으로 나타났다. 무기염류로는 KCl, $K_2HPO_4$, $FeSO_4$를 첨가하였을 때 효소 활성이 가장 높은 반면에 2가 금속이온인 $Co^{2+}$, $Zn^{2+}$, $Mn^{2+}$, $Cu^{2+}$를 첨가하였을 때는 균의 성장과 효소 활성이 심하게 저해되는 것으로 나타났다.

• 농업생명과학연구
• /
• 제46권1호
• /
• pp.163-176
• /
• 2012

인천 연안 갯벌에서 분리한 호알카리성 Bacillus clausii I-52로부터 세포외 알카리성 단백질 분해효소(BCAP)의 발현 및 생산성을 증가시키기 위하여 BCAP promoter, ribosome 결합 서열, 신호서열, 전구체 서열 및 활성형 BCAP 유전자를 cloning한 재조합 plasmid pHPS9-fuBCAP을 penicillin-protoplast 법으로 B. clausii I-52의 염색체 DNA에 integration 하였고, 도입된 plasmid pHPS9-fuBCAP 유전자는 PCR에 의해 확인하였다. 가장 높은 단백질 분해효소 상대 활성을 보이는 선별된 transformant C5를 생산 최적화 배지(대두박 2%, 밀가루 1%, 구연산나트륨 0.5%, $K_2HPO_4$ 0.4%, $Na_2HPO_4$ 0.1%, NaCl 0.4%, $MgSO_47H_2O$ 0.01%, $FeSO_47H_2O$ 0.05%, 물엿 2.5%, 탄산나트륨 0.6%)에서 액침 배양법(배양온도, $37^{\circ}C$; 배양 시간, 48 h; 교반 속도, 650 rpm; 통기 속도, 1 vvm)으로 배양하여 단백질 분해효소를 발현 및 분비시켰을 때, BCAP 발현 양(134,670 U/ml)은 wild-type(83,960 U/ml)에 비하여 약 1.6 배 증가하였으며, 비활성도(91,611.5 U/mg 단백질)는 wild-type(71,760 U/mg 단백질)에 비하여 약 1.3 배 증가하였다. 또한, B. clausii I-52 염색체 DNA에 integration된 pHPS9-fuBCAP plasmid는 단백질 발현과 함께 8일간의 계대배양 동안에 안정하게 유지되고 있음을 확인하였다.

• Journal of Microbiology and Biotechnology
• /
• 제18권3호
• /
• pp.410-416
• /
• 2008

The gene SfXyn10, which encodes a protease-resistant xylanase, was isolated using colony PCR screening from a genomic library of a feather-degrading bacterial strain Streptomyces fradiae var. k11. The full-length gene consists of 1,437bp and encodes 479 amino acids, which includes 41 residues of a putative signal peptide at its N terminus. The amino acid sequence shares the highest similarity (80%) to the endo-1,4-${\beta}$-xylanase from Streptomyces coelicolor A3, which belongs to the glycoside hydrolase family 10. The gene fragment encoding the mature xylanase was expressed in Escherichia coli BL21 (DE3). The recombinant protein was purified to homogeneity by acetone precipitation and anion-exchange chromatography, and subsequently characterized. The optimal pH and temperature for the purified recombinant enzyme were 7.8 and $60^{\circ}C$, respectively. The enzyme showed stability over a pH range of 4.0-10.0. The kinetic values on oat spelt xylan and birchwood xylan substrates were also determined. The enzyme activity was enhanced by $Fe^{2+}$ and strongly inhibited by $Hg^{2+}$ and SDS. The enzyme also showed resistance to neutral and alkaline proteases. Therefore, these characteristics suggest that SfXyn10 could be an important candidate for protease-resistant mechanistic research and has potential applications in the food industry, cotton scouring, and improving animal nutrition.

• 미생물학회지
• /
• 제30권6호
• /
• pp.528-532
• /
• 1992

계면 활성제 특히 Triton X-100 의 첨가에 의해 ATP-의존성 효소의 subunit 인 Clp P 의 안정도를 높히고, 염의 첨가에 의한 효소 활성의 유지 정도와 Clp P 호소에 대한 생화학적 성질을 조사하였다. Clp A 가 가지고 있는 ATPase 활성을 나타내기 위하여서는 Clp P 의 단백질 분해 효소활성이 요구되며 Clp 효소의 다량체형성을 조사한 결과 ATP 가 존재하지 않을 경우에는 Clp A 와 Clp P 효소가 각각 용출하였으며 ATP 및 Mg 를 첨가하였을 때에는 complex 를 형성하여 분자량이 600,000 이상으로서 Clp P 효소는 Hexamer 로서 효소활성을 나타내며 Clp A 와 Clp P 의 다량체 형성이 요구되고 형성시에 ATP 가 관여하고 있는 것으로 나타났다.

• KSBB Journal
• /
• 제31권4호
• /
• pp.284-290
• /
• 2016

In this research, recombinant human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) was produced by transgenic rice cells. RAmy3D promoter was used for overcome the limitation of low expression level in transgenic plant cells, and the secretion of target protein was accomplished by signal peptide. However, the RAmy3D promoter system which can be induced only by sugar starvation causes the decrease of cell viability. As a result, cell death promotes the release of protease which degrades the target proteins. The protein stability and productivity can be significantly influenced by proteolysis activity. Therefore, development of new strategies are necessary for the in situ recovery of target proteins from cell culture media. In this study, in situ recovery was performed by various strategies. Direct addition of Protein A resin with nylon bag leads to cell death by increased shear stress and decrease in production of hCTLA4Ig by protease. Medium exchange through modified flask could recover hCTLA4Ig with high cell viability and low protease activity, on the other hand, the productivity was lower than that of control. When in situ recovery was conducted at day 7 after induction in air-lift bioreactor, 1.94-fold of hCTLA4Ig could be recovered compared to control culture without in situ recovery. Consequently, in situ recovery of hCTLA4Ig from transgenic rice cell culture could enhance productivity significantly and prevent degradation of target proteins effectively.

• Applied Biological Chemistry
• /
• 제31권3호
• /
• pp.274-283
• /
• 1988

Aqualysin I is an alkaline serine protease which is secretet into the culture medium by Thermus aquaticus YT-1, an extreme thermophile. Aqualysin I was purified, and its partial amino acid sequence was determined. The gene encoding aqualysin I was cloned into E. coli using synthetic oligodeoxyribonucleotides as hybridization probes. The nucleotide sequence of the cloned DNA was determined. The primary structure of aqualysin I, deduced from the nucleotide sequenc, agreed with the determid amino acid sequences, including the $NH_2-$ and COOH terminal sequence of the tryptides derived from aqualysin I. Aqualysin I comprised 281 amino acid residues and its molecular mass was determined to be 28350. On alignment of the whole amino acid sequence, aqualysin I showed high sequence homology with the subtilisin type serine protease, and 43% identity with proteinase K, 37-30% with subtilisins and 34% with thermitase. Extremely high sequence identity was observed in the regions containing the active-site residues, corresponding to Asp32, His64 and Ser221 of subtilisin BPN'. Aqualysin I contains two disulfide bonds, Cys67-Cys99 and Cys163-Cys194, and these disulfide bonds seem to contribute to the heat stability of the enzyme. The determined positions of the twe disulfide bonds of aqualysin I agreed with those predicted previously on the basis of computer graphics of the crystallographic data for subtilisin BPN'. Therefore, these findings sugests that the three-dimensional structure of aqualysin I is similar to that of subtilisin BPN' Aqualysin I is produced as a lage precursor, which contains $NH_2-$ and COOH- terminal portions besides the mature protease sequence.

• 미생물학회지
• /
• 제18권4호
• /
• pp.161-171
• /
• 1980

A mutant strain having increased productivity of both enzymes, protease and amylase, was obtained from A. flavus KU 153, isolatd from South Korea for its high protease production by successive ultra-violet light irradiation, Two glucoamylases from the mutant strain selected were purified from wheat branculture by successive salting out, followed by dialysis and column chromatography, and their characteristics were compared with those of the wild strain. Glucoamylase production of the mutant selected was increased about 3.3 times compared with the wild strain, and 2.1 times compared with the parental strain, ${\alpha}-amylase$ activity of the mutant selected was about 2 times hugher than that of the wild strain or the parental strain. Protease and cellulase productivities of the muant selected were all alike compared with those of the highly proteolytic mutant, the parental strain. Therefore, it was considered that the back mutation on the protease production did not occurred in the formation process of the glucoamylase producing mutant. Total activities of glucoamylase I and II from the mutant selected were 2.86 and 3.65 times higher compared with those from the wild strain, respectively. Considering the optimal pH-thermal stability and Km-Vmax value of glucoamylase I and II from both strains, wild and mutant, it was deduced that the characteristics of glucoamylase I and II from the wild strain did not altered during the mutation process. Therefore, it was concluded that the selected mutant did not induce the formation of another glucoamylase isozyme, or the changes in the characteristics of the glucoamylase, but induce the productivity of the same glucoamylase I and II by the action of regulatory gene.