• 한국미생물·생명공학회지
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• 제21권5호
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• pp.468-472
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• 1993

For the production of Casein Phosphopeptide(CPP) inhibiting the insolubility of calcium, 10% sodium caseinate was treated with 1.5% of protease from Streptococcus sp.. Optimal conditions and productivity for the CPP production, and properties of the CPP were compared with tryptic hydrolysates of sodium caseinate. Optimum conditions of pH, temperature and reaction time were 8.0, 50C, 4 hrs, respectively. Under these conditions the productivity of CPP was 23% and Molecular weight of CPP was ranged from 3, 000 to 17, 000. The results also showed that the insolubility of calcium was completely inhibited by using 1.5 times of CPP for the amount of calcium.

• 미생물학회지
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• 제48권4호
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• pp.298-304
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• 2012

가정에서 제조된 된장 16점으로부터 $60^{\circ}C$에서 성장하는 Bacillus속 균주 63주를 분리하였으며 이로부터 내열성 protease를 생산하는 1개 균주를 선발하였다. 분리균의 형태적, 생화학적 특성과 16S rRNA 유전자서열을 조사한 결과 Bacillus licheniformis로 확인되었다. 분리균의 배양상등액은 반응온도 $60-65^{\circ}C$와 pH11에서 최대 protease 활성을 보였으며, $60^{\circ}C$에서 30분간 열처리한 후에도 87%의 protease 활성을 유지하였다. 질소원, 탄소원, 금속이온, 인 등의 배지성분이 protease 생산성에 미치는 영향을 조사한 결과 유당과 soytone peptone이 분리균의 효소 생산을 증가시키는 탄소원과 질소원으로 확인되었다. 분리균은 말기대수기 이후부터 protease를 생산하기 시작하였으며, 유당 (3%), soytone peptone (1.5%), $MgSO_4$ (0.1%), $K_2HPO_4$ (0.03%)와 $KH_2PO_4$ (0.03%)로 구성된 최적화 배지에서 배양시간이 28시간일 때 protease의 생산성이 최대 550 U/ml에 이르렀다.

• 한국미생물·생명공학회지
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• 제19권5호
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• pp.450-455
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• 1991

Serratia marcescens ATCC 21074로부터 돌연변이 유도에 의해 protease 생산능이 높은 ampicillin 耐性의 변이주 Serratia sp. SMNT-1을 분리하였다. 이 균주는 原 균주에 비하여 約 11배 정도 높은 protease 생산성을 나타내었다. 이 효소는 pH 9.0, $40^{\circ}C$에서 최대 활성을 나타내었으며, 저온하에서는 pH 6.0-10.0 범위에서 안정하였으나, $37^{\circ}C$, 알카리 조건하에서 불안정하였다. 이 효소는 $60^{\circ}C$에서 10분간 열처리시 실활되었다.

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

• Journal of Microbiology
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• 제39권4호
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• pp.305-313
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• 2001

The spr D gene encoding Strptomyces griseus protease D(SGPD); a chymotrypsin-like proteae, was cloned from Strptomyces griseus IFO13350 and sequence. Most of the amino-acid sequence deduced from the nucleotide sequence is idential to that Strptomyces griseus IMRU3499 except that one amino acid has been deleted and Trp 369 has been substituted into Cys369 in the SGPD from S. griseus IFO13350 without affecting the protease activity. The spr D gene was overexpressed in Streptomyce liv-idans TK24 as a heterologous host. Various media with different compositions were also used to max-imize the productivity of SGPD inthe heterologous host. The SGPD productivity was best when the transformant S. lividans TK24 was cultivated in R2YE medium. The relative chymotrypsin activity of the culture broth measured with an artificial chromogenic substrate, N-scuccinyl-ala-ala-pro-phe-p-nitroanilide, was 16 units/ml. A high level of SGPD was also produced in YEME and SAAM medial but it was relatively lower that in R2YE medium and negligible amounts of SGPD were produced in GYE, GAE and Benedict media. The growth of S. lividans reacted the maximum level of cell mass at days 3 and 4 of the culture, but SGPD production started in the stationary phase of cell growth and kept increase in till the 10$^{th}$ day of culture in R2YE and YEME medium, but in GYE media the productivity reached maximum level at 8days of cultivation. The introduction of the spr D gene into S. lividans TK24 triggered biosyntheis of the pigmented antibiotic , actinorhodin, which implies some protease may paly a very improtant role in secondary-metabolite formation in sStreptomyces.

• Journal of Microbiology and Biotechnology
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• 제21권12호
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• pp.1250-1256
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• 2011

In this study, Bacillus licheniformis cells were immobilized by entrapment in calcium alginate beads and were used for production of alkaline protease by repeated batch process. In order to increase the stability of the beads, the immobilization procedure was optimized by statistical full factorial method, by which three factors including alginate type, calcium chloride concentration, and agitation speed were studied. Optimization of the enzyme production medium, by the Taguchi method, was also studied. The obtained results showed that optimization of the cell immobilization procedure and medium constituents significantly enhanced the production of alkaline protease. In comparison with the free-cell culture in pre-optimized medium, about 7.3-fold higher productivity was resulted after optimization of the overall procedure. Repeated batch mode of operation, using optimized conditions, resulted in continuous production of the alkaline protease for 13 batches in 19 days.

• 미생물학회지
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• 제38권4호
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• pp.235-240
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• 2002

식품폐수처리장의 활성슬러지와 논, 밭의 토양에서 우수한 protease 생성 균주을 분리 선별하였다. 이 중 효소활성이 우수한 PB16은 그람 음성, 간균이며 protease activity는 6.49 U/ml이었다. 생리, 생화학적 특성 및 16S rRNA 염기서열분석을 실시한 결과, Aeromonas hydrophila (99.0%) 인 것으로 확인 되었다. Bioscreen C를 사용한 최적 성장조건 평가는 합성폐수에 vitamin과mineral을 첨가한 배지의 중식속도(0.26 $h^{-1}$)가 무 첨가배지(0.21 $h^{-1}$)보다 높았으며, 분리균주의 합성폐수 유기물 제거능 실험에서는 초기 soluble-CODcr 2,472 mg/l인 고농도 합성폐수를 1일, 3일 반응 후 각각 59, 87%의 제거율을 나타내었다.

• 한국식품영양학회지
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• 제6권2호
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• pp.89-95
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• 1993

A Nuluk, a Korean traditional Koji for brewing, was made with wheat flour and Aspergillus oryzae L2 which had a good aroma and strong abilities In producing saccharogenic and dextrogenic enzymes. The cultural conditions for the production of saccharogenic and proteolytic enzymes were tested. The productivity of dextrogenic enzyme was improved when Nuluk was made with unsteamed wheat flour as compared with steamed one, but that of proteolytic enzyme was reduced. The addition of water containing 0.5% hydrochloric acid was unfavorable for the production of those two enzymes. The optimum ratio of water added to wheat flour for the production of those two enzymes was 28$^{\circ}C$ on the basis of wheat flour, The productivity of saccharogenic enzyme was enhanced when the Nuluk was molded after 20 hours of precultivation, but that of proteolytic enzyme was reduced as compared with no molding. The optimum temperatures for the production of saccharogenic enzyme and proteolytic enzyme were 36$^{\circ}C$ and 28$^{\circ}C$, respectively.

• KSBB Journal
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• 제22권4호
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• pp.185-190
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• 2007

Productivity of secreted recombinant protein depends largely on its stability in the extracellular environment with protease. Most hGM-CSF produced by transgenic tobacco cell cultures and secreted to the medium was confirmed to be rapidly degraded by protease in medium. To increase the productivity, therefore, various protein stabilizers such as gelling agents such as carrageenan and alginate, polymers, polyols, and amino acids have been tested. The stability of hGM-CSF in spent medium without cells was improved by the presence of gelling agents. However, the reason for the enhanced production by the addition of gelling agents may be due to the increased expression level and permeability rather than stability. The addition of DMSO inhibited the cell growth, but improved specific yield. The others were not effective for stability as well as hGM-CSF production.

• 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.