• 생명과학회지
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• 제31권8호
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• pp.761-770
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• 2021

본 연구에서는 반응표면분석법을 이용하여 전통발효식품인 막걸리로부터 분리한 Bacillus amyloliquefaciens SRCM115785 균주에 대하여 protease 생산량을 증가시키기 위한 배지의 최적 농도를 확립하고자 하였다. 선정한 11개의 배지 성분 중 각 성분이 protease 생산에 미치는 영향에 대한 분석을 위해 Plackett-Burman design (PBD)를 설계하여 통계분석한 결과 glucose, yeast extract, beef extract를 protease 생산 향상을 위한 요인으로 최종 선별하였다. 선별된 3개의 성분에 대해 protease 생산을 위한 각 성분별 최적 농도를 결정하기 위해 central composite design (CCD)분석을 설계하여 protease 최대 생산을 위한 각 배지조성별 농도는 glucose 6.75 g/l, yeast extract 12.42 g/l, beef extract 17.48 g/l로 예측되었다. ANOVA 분석을 통해 실험모델의 적합성을 증명하였고, 설계한 최적배지에서 반복실험을 진행하여 protease 생산량을 측정한 결과 예측값과 매우 유사한 값을 나타냄을 확인하였다. 최종적으로 일반 배지에 비해 137% 환이 증가하였으며, 추가로 정량 분석 결과 기존 25.72 U/ml 대비 59.28 U/ml로 230.47% 증가함을 확인하였다. 본 연구를 통해 protease 생산량 증가를 위한 배지 성분의 최적화를 확립하였고, 이를 바탕으로 산업용 효소로서 protease의 효율적인 활용방안에 대한 기초자료로서 활용될 수 있을 것으로 기대된다.

• Journal of Microbiology and Biotechnology
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• 제9권4호
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• pp.469-474
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• 1999

Thermoactinomyces sp. E79 produced two types of thermostable alkaline proteases extracellularly. A minor protease was separated from a major protease by using DEAE-column chromatography. This enzyme was purified to homogeneity by ammonium sulfate and DEAE-Sepharose ion-exchange chromatography. The purified minor protease showed different biochemical properties compared to the major protease. The molecular mass of the purified enzyme was estimated by SDS-PAGE to be 36 kDa. Its optimum temperature and pH for proteolytic activity against Hammarsten casein were $70^{\circ}C$ and 9.0, respectively. The enzyme was stable up to$75^{\circ}C$ and in an alkaline pH range of 9.0-11.0. The enzyme was inhibited by phenylmethylsulfonyl fluoride (PMSF) and $Hg^{2+}, indicating that the enzyme may be a cysteine-dependent serine protease. In addition, the enzyme cleaved the endoproteinase substrate, succinyl-Ala-Ala-Pro-Phe-p- nitroanilide, and the $K_m$ value for the substrate was 1.2 mM.

• 한국미생물·생명공학회지
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• 제48권3호
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• pp.358-365
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• 2020

An organic solvent- and bleach-stable protease-producing strain was isolated from a polluted river water sample and identified as Aeromonas veronii OB3 on the basis of biochemical properties (API 20E) and 16S rRNA sequence analysis. The strain was found to hyper-produce alkaline protease when cultivated on fish waste powder-based medium (HVSP, 4080 U/ml). The biochemical properties and compatibility of OB3 with several detergents and additives were studied. Maximum activity was observed at pH 9.0 and 60℃. The crude protease displayed outstanding stability to the investigated surfactants and oxidants, such as Tween 80, Triton X-100, and H₂O₂, and almost 36% residual activity when incubated with 1% SDS. Remarkably, the enzyme demonstrated considerable compatibility with commercial detergents, retaining more than 100% of its activity with Ariel and Tide (1 h, 40℃). Moreover, washing performance of Tide significantly improved by the supplementation of small amounts of OB3 crude protease. These properties suggest the potential use of this alkaline protease as a bio-additive in the detergent industry and other biotechnological processes such as peptide synthesis.

• Journal of Microbiology
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• 제40권1호
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• pp.26-32
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• 2002

From the culture supernatant of the psychrotrophic strain of Bacillus amyloliquefaciens an extracellular serine protease was purified to apparent homogeneity by successive purification steps using QAE-Sephadex, SP-Sephadex and Sephacryl S-100 column chromatography. The pretense is monomeric, with a relative molecular mass of 23,000. It is inhibited by the serine protease inhibitor phenylmethylsulfonyl fluoride, but not by EDTA. The enzyme is most active at pH 9-10 and at $45^{\circ}C$, although it is unstable at $60^{\circ}C$.

• Journal of Microbiology and Biotechnology
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• 제18권5호
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• pp.852-858
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• 2008

An extracellular protease (Mc1) was isolated from the nematode-trapping fungus Monacrosporium cystosporium by gel filtration, anion-exchange, and hydrophobic interaction chromatographies. This protease had a molecular mass of approximately 38 kDa and displayed an optimal activity at pH 7-9 and $56^{\circ}C$ (over 30 min). Its proteolytic activity was highly sensitive to the serine protease inhibitor PMSF (phenylmethylsulfonylfluoride, 0.1 mM), indicating that it belonged to the serine-type peptidase group. The Michaelis constant ($K_m$) and $V_max$ for substrate N-Suc-Ala-Ala-Pro-Phe-pNA were $1.67{\times}10^{-4}\;M$ and 0.6071 $OD_{410}$ per 30 s, respectively. This protease could degrade a broad range of substrates including casein, gelatin, BSA (bovine serum albumin), and nematode cuticle. Moreover, the enzyme could immobilize the free-living nematode Panagrellus redivivus and the pine wood nematode Bursaphelenchus xylophilus, suggesting that it might playa role in infection against nematodes. The encoding gene of Mc1 was composed of one intron and two exons, coding for a polypeptide of 405 amino acid residues. The deduced amino acid sequence of Mcl showed 61.4-91.9% identity to serine proteases from other nematode-trapping fungi. Our results identified that Mcl possessed biochemical properties including optimal reaction condition and substrate preference that are different from previously identified serine proteases.

• 한국식품과학회지
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• 제21권3호
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• pp.379-386
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• 1989

본 연구에서는 농축대두단백으로 두유를 만들고 미생물 pretense와 papain을 단독 또는 혼합 사용하여 대두단백질을 가수분해한 후, 단백질이 일부 가수분해 된 두유에서 L. acidophilus의 생육과 산생성을 관찰하고 제조된 젖산균음료의 관능성을 조사하였다. Pretense 처리로 젖산균의 산생성이 촉진되었는데 미생물 pretense가 papain보다 효과적이었으며 2종의 효소를 혼합 사용했을 때는 상승효과가 보였다. 그러나 pretense 처리로 pH와 생균수는 큰 변화가 없었다. 미생물 pretense의 경우는 가수분해시간 15분까지, papain의 경우는 가수분해시간 45분까지 산생성 촉진효과가 현저했으나 그 후에는 3시간이 경과하여도 큰 변화가 없었다. 미생물 pretense 0.2% 또는 papain 0.2% 처리로 대두젖산균음료의 전체적인 기호도와 맛이 다소 개선되었다. Protease 처리 15분에 비단백태질소의 함량이 현저하게 증가하였고 후 3시간까지 서서히 증가하였다.

• Journal of Microbiology and Biotechnology
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• 제22권1호
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• pp.34-45
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• 2012

A thermophilic Bacillus stearothermophilus F1 produces an extremely thermostable serine protease. The F1 protease sequence was used to predict its three-dimensional (3D) structure to provide better insights into the relationship between the protein structure and biological function and to identify opportunities for protein engineering. The final model was evaluated to ensure its accuracy using three independent methods: Procheck, Verify3D, and Errat. The predicted 3D structure of F1 protease was compared with the crystal structure of serine proteases from mesophilic bacteria and archaea, and led to the identification of features that were related to protein stabilization. Higher thermostability correlated with an increased number of residues that were involved in ion pairs or networks of ion pairs. Therefore, the mutants W200R and D58S were designed using site-directed mutagenesis to investigate F1 protease stability. The effects of addition and disruption of ion pair networks on the activity and various stabilities of mutant F1 proteases were compared with those of the wild-type F1 protease.

• Fibers and Polymers
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• 제7권2호
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• pp.180-185
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• 2006

A pineapple protease, bromelain, was used to improve the dyeing properties of protein fibers such as wool and silk. The optimal condition for the activity of the pineapple protease was about $60^{\circ}C$ at pH 7. The wool and silk were treated with the protease extracted from a pineapple and the K/S values of the dyed wool and silk were measured using a spectrophotometer in order to compare the dye uptake. The protease treatment enhanced the dyeing properties of protein fibers without severe changes in mechanical properties. The surface appearances of protease-treated fibers were observed by microscopy.

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

• 한국식품영양학회지
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• 제13권2호
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• pp.171-175
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• 2000

Microbial proteases have certain unique characteristics, and are now widely used in food, leather, detergent, and pharmaceutical industries. Thermophilic Actinomyces producing the protease was isolated from soil in Wonju city. This strain was able to grow and produce protease at the culture temperature of 50$^{\circ}C$. The maximum protease production was obtained when 0.5% soluble starch and 0.4% yeast extract were used as carbon and nitrogen source, respectively. The other culture condition for the maximal productivity of the protease was 0.1% K2HPO4, and 0.05% CaCl2 at initial pH 8.0 for 48 hours.