• 한국식품영양학회지
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• 제13권2호
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• pp.176-180
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• 2000

Microbial protease has been interesting due to the biological roles in the producing microorganism. A thermophilic Actinomyces produing protease was isolated from soil. The optimal medium composition and culture conditions for maximum protease production was as follows 0.5% soluble starch, 0.5% yeast extract. 0.1% K2HPO4, 0.05% CaCl2, initial pH 8.0 at 50$^{\circ}C$ for 48hours. The protease was purified by the procedure of ammonium sulfate precipitation, anion exchange chromatography(LC), DEAE high performance liquid chromatography and GPC HPLC. The purification fold of the purified enzyme was increased about 22.6. The optimal pH and temperature for reaction of the purified enzyme were 7.5 and 60$^{\circ}C$. The purified enzyme was stable for the pH range from 6.0 to 8.5, but was unstable when treated at 80$^{\circ}C$ for 10 minutes. The activity of the enzyme was inhibited by Ag+ and Cu2+.

• Applied Biological Chemistry
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• 제41권1호
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• pp.6-12
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• 1998

폐단백질을 활용하는 방도의 하나로 폐단백질 자원으로 부터 불용성 단백질의 분리 효율성을 높이고 기능성을 개선하기 위하여 protease를 생산하는 Aspergillus sp. MS-18 균주를 토양으로 부터 분리하고 이 균주가 생산하는 효소를 정제하여 특성을 살펴보았다. 효소 생산을 위한 최적 배양조건은 3% arabinose, 0.5% polypepton, 0.1% ammonium sulfate, 0.1% magnesium chloride 첨가로 3 일 배양이었다. 효소는 ion exchange chromatography, gel filtration 등으로 16.9 배 정제할 수 있었으며 비활성역가는 340.4 unit/mg이었다. 정제효소는 polyacryl amide gel 전기영동상 단일 밴드로 나타났으며, 분자량은 30,000 정도로 추정되었고 결정구조는 모서리가 둥그스럼한 막대 모양이었다. 정제 효소의 최적작용 pH와 온도는 9.0, $60^{\circ}C$였으며, pH 7.0-12.0까지 $50^{\circ}C$에서 안정하였다. 금속이온중 $Na^+$, $Mg^{2+}$, $Mn^{2+}$등에 의해 활성이 증대 되었으나, $Hg^{2+}$, $Cu^{2+}$, $Zn^{2+}$, $Pb^{2+}$에 의해 효소 활성이 저해되었고 저해제중 ethylenediaminetetra acetic acid와 phenyl methanesulfonyl fluoride에 의한 활성 저해가 관찰되어 금속 이온이 효소 활성에 관여하는 serine protease로 추정되었으며 정제효소의 Km, Vmax는 $29.33\;{\mu}mole/L$, $5.13\;{\mu}g/min$이었다.

• BMB Reports
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• 제37권5호
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• pp.574-581
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• 2004

The full-length cDNA of the lumbrokinase fraction 6 (F6) protease gene of Lumbricus rubellus was amplified using an mRNA template, sequenced and expressed in E. coli cells. The F6 protease gene consisted of pro- and mature sequences by gene sequence analysis, and the protease was translated and modified into active mature polypeptide by N-terminal amino acid sequence analysis of the F6 protease. The pro-region of F6 protease consisted of the 44 residues from methionine-1 to lysine-44, and the mature polypeptide sequence (239 amino acid residues and one stop codon; 720 bp) started from isoleucine-45 and continued to the terminal residue. F6 protease gene clones having pro-mature sequence and mature sequence produced inclusion bodies in E. coli cells. When inclusion bodies were orally administrated rats, generated thrombus weight in the rat' venous was reduced by approximately 60% versus controls. When the inclusion bodies were solubilized in pepsin and/or trypsin solutions, the solubilized enzymes showed hemolytic activity in vitro. It was concluded the F6 protease has hemolytic activity, and that it is composed of pro- and mature regions.

• 대한환경공학회지
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• 제36권4호
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• pp.265-270
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• 2014

영양 성분을 함유하고 있는 유기성 폐기물은 미생물에 의해 처리되어, 유용한 물질로 전환될 수 있다. 이러한 생물학적 공정에서 미생물 세포와 효소는 원료 물질인 기질과 함께 중요하다. 대규모화 공정에서도 미생물 세포와 효소는 공정 최적화에서 필수적인 요소이다. 본 연구에서는 이러한 생물학적 공정의 효율성을 높이는 목적으로 다량의 아미노산과 단백질을 함유하고 있는 많은 종류의 부패가 진전된 유기성 폐기물과 발효 식품에서 단백질 분해효소를 생산하는 미생물을 분리하였다. 단백질 분해 효소의 활성, 온도와 산도등 활성 조건과 활성 정도를 확인하여 선택된 균주들을 동정하였다. 산업적으로 저온에서 단백질을 분해하는 효소는 유기성 폐기물을 저온에서 처리할 수 있다. 저온에서 처리가 가능하다는 것은 폐기물의 처리 온도를 낮은 상태로 유지할 수 있어 그 만큼의 열(steam)비용을 줄일 수 있다. 또한 이 단백질 분해효소를 이용하여 단백질을 분해 후 다량의 아미노산을 생산할 수 있으므로 아미노산 생산 공정에도 적용이 가능하다. 이렇게 유기 폐기물을 처리하여 다양한 용도로 사용할 수 있으므로, 폐기물의 가치를 높일 수 있다. 다양한 활성 조건에서 단백질 분해효소를 다량으로 생산하는 균주를 분리하여 동정하고, 균주 배양 조건, 효소 생산의 최적 조건에 대한 연구를 수행하였다.

• 한국균학회지
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• 제42권4호
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• pp.333-340
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• 2014

미생물을 이용한 생물학적 방제제의 개발을 위해 연작피해가 없는 순창군 토양으로부터 다양한 미생물 201종을 분리하였고, 이들의 생물학적 활성을 조사하였다. 201종의 분리 세균 중에서 다양한 식물병원성 곰팡이에 대하여 항균활성이 우수한 5종을 선별하였다. 5종의 분리주에 대하여 siderophore를 생산하며 cellulase, protease, amylase와 같은 곰팡이 세포벽 분해효소를 생산하는 능력이 가장 우수한 SCS3 균주를 최종 선별하였다. 최종 선별한 균주 SCS3의 형태학적, 생리학적 및 생화학적 특성을 조사하였고, 16S rRNA 염기서열의 분석에 의해 B. subtilis SCS3으로 명명하였으며, 염기서열 분석에 기반하여 계통수를 작성하였다. 이상의 결과로부터 B. subtilis SCS3은 식물병원성 곰팡이의 방제를 위한 생물학적 방제제로 유용하게 이용될 수 있을 것으로 생각한다.

• BMB Reports
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• 제34권6호
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• pp.509-516
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• 2001

A genomic DNA fragment that contains the gene, which codes for a novel extracellular serine protease in Burkholderia pseudomallei, was cloned by using pQE40 as a vector. It was maintained in Escherichia coli JM109. The expression of the gene(s) resulted in the production of a 52 kDa protease. The recombinant protease was purified from the culture filtrate via ammonium sulfate fractionation, gel filtration, and anion-exchange chromatography. The purified protease had an optimum pH and temperature of pH 8.9 and $38^{\circ}C$, respectively. The protease activity was inhibited by EGTA, EDTA, and PMSF, but not 1,10-phenanthroline. The first 11 amino acid residues from the N-terminus of the purified protease were identified as LAPNDPYYYGY. PNDPYY was found to show homology to the Bacillus cereus microbial serine protease and B. subtilis PD498 serine protease. These results indicate that the protease that was purified in this study is an extracellular calcium-dependent serine protease. The purified protease was able to digest the human serum 19A, IgG, albumin, and transferrin, as well as bovine muscle actin and myosin. Furthermore, it was able to promote or cause dermonecrosis in experimental rabbits. These results propose the possible role of a novel B. pseudomallei extracellular calcium-dependent serine protease in the virulence of the pathogen.

• 한국축산식품학회지
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• 제28권5호
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• pp.675-680
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• 2008

The physical quality and microbial safety of Korean beef jerky was evaluated at various steps during its preparation. Microbial counts in raw beef demonstrated mesophillic bacteria at 4.20 Log CFU/g, psychrotrophic bacteria at 3.85 Log CFU/g, anaerobic bacteria at 4.90 Log CFU/g, and yeast and molds at 1.92 Log CFU/g. Spore-forming bacteria and coliforms were not detected in raw beef samples. Spices and spiced meats showed similar trends in microbial counts, demonstrating minimal microbial contamination during these stages of preparation. The final beef jerky product exhibited counts of mesophillic bacteria at 1.15-1.66 Log CFU/g, psychrotrophic bacteria at 1.15-1.66 Log CFU/g, and anaerobic bacteria at 0.81-1.72 Log CFU/g. Spore-forming bacteria, yeast and molds, and coliforms were not detected in beef jerky. Significant differences from added ingredients occurred for instron textural profile analysis traits for hardness. In general, Korean beef jerky with humectant and tenderizer had lower hardness than control (without humectant and tenderizer). Also, the sample added with 0.01% protease from Streptomyces griseus had lower hardness than all samples. All samples had 0.7l to 0.72 water activities, and the color and pH were not shown in significant changes of all samples.

• 한국염색가공학회지
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• 제18권5호
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• pp.8-14
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• 2006

An earthworm protease, Lumbricus rubellus, was used to improve the dyeing properties of protein fibers such as wool and silk. The optimal condition for the activity of the earthworm pretense was about $40^{\circ}C$ at pH 7. The wool and silk were treated with the protease extracted from an earthworm and the K/S values of the dyed wool and silk were measured using a spectrophotometer in order to compare the dye uptake. The pretense treatment enhanced the dyeing properties of protein fibers without severe changes in mechanical properties. The surface appearances of pretense-treated fibers were observed by microscopy analysis.

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

• 한국토양비료학회지
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• 제43권6호
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• pp.987-994
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• 2010

바실러스와 그람음성균은 녹비시용구에서 높았지만, 미생물량은 비료+돈분퇴비 시용구에서 높았다. 인산효소와 유기물분해효소는 무처리구에 비해 유기물 시용구에서 모두 높은 값을 보였다. 영년 밭토양의 PLFA에 의한 군집분석은 볏짚퇴비 및 돈분퇴비시용구가 다른 처리와 상이한 특성을 보였다. 영년 밭 토양의 우점균은 Bacillus flexus, Bacillus subtilis 및 Bacillus megaterium 등이었으며, 토양에서 분리된 세균은 대부분 amylase, protease 및 lipase의 활성을 가지고 있었다.