• Mycobiology
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• 제33권1호
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• pp.23-29
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• 2005

The effects of different concentrations of three amino acids as carbon and or nitrogen sources on mycelial dry weights, changes in pH values of synthetic medium, ammonia secretion and extracellular protease activity by three zoosporic fungi, pathogens of fish and shellfish, were studied. As compared with the control, the addition of isoleucine and aspartic acid as nitrogen sources were generally stimulative for mycelial dry weight production whereas phenylalanine was inhibitory irrespective to the tested fungal species. When amino acids served as carbon and nitrogen sources, the mycelial dry weights of the three fungi were increased (mostly non-significantly) relative to untreated control but weights were decreased as the concentrations of the three amino acids raised. The addition of individual amino acids as carbon and nitrogen sources to the medium significantly increased pH values of the medium comparable to the control. The addition of each of the three amino acids as carbon and nitrogen sources to the medium significantly induced ammonia secretion by the three species of zoosporic fungi. Ammonia secretion in synthetic medium amended with amino acids as nitrogen source raised by the three zoosporic fungi relative to untreated control except in case of Achlya racemosa treated with isoleucine. Extracellular protease activity was almost promoted in case of Achlya proliferoides and Saprolegnia furcata cultures treated with isoleucine and aspartic acid individually in presence of glucose and vice versa in case of phenylalanine. However, extracellular protease activity of A. racemosa decreased compared with the control at various concentrations of isoleucine and both phenylalanine and aspartic acid assumed inconsistent effects. Extracellular protease activity of the three zoosporic fungi in the medium devoid of glucose varied depending upon zoosporic fungal species, the tested amino acid and the applied concentrations. The values of protease activity were approximately less two folds than that obtained in presence of glucose.

• Journal of Life Science
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• 제11권1호
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• pp.42-46
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• 2001

Subtilisin YaB, produced by alkalophilic Bacillus strain YaB, is an extracellular alkaline serine protease having 55% homology to subtilisin BPN'. It is synthesized as a 378-amino acid preproenzyme and secreted into the culture medium as a 265-amino acid mature protease. To examine the role of pro-sequence for the secretion of subtilisin YaB, we have studied the expression, in Bacillus subtilis, of a mutant preprosubtilisin YaB in which active site Ser214 is substituted with Cys. The use of a six protease-deficient strain, WB600, was required for its efficient production. The prosubtilisin YaB, thus produced, was indeed secreted into the culture medium and was processed to its mature form upon treatment with exogenously added active subtilisin YaB. From these results, we have concluded that the processing of pro-sequence is not essential for the secretion of the enzyme.

• Journal of Microbiology and Biotechnology
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• 제8권6호
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• pp.639-644
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• 1998

Extracellular serine proteases were isolated from a soil bacterium, alkalophilic coryneform bacterium TU-19, which have been grown in a liquid medium optimized at 3$0^{\circ}C$ and pH 10.0. Three different sizes, 120 kDa (protease I), 80 kDa (protease II), and 45 kDa (protease III), of serine pro teases were purified using Sephadex G-150 and QAE-Sephadex chromatography (Kang et al. 1995. Agric. Chem Biotech. 38: 534-540). SDS-PAGE showed that the 120 kDa protease was degraded into the 80 kDa protease in 20 mM Tris-HCI (pH 8.0) buffer solution. This degradation was enhanced in the presence of 0.5 M NaCl and 5 mM EDTA, but was inhibited in the presence of 5 mM $CaCl_2$. These results indicated that the $Ca^{2＋}$ ion seems to stabilize the 120 kDa protease like other proteases derived from Bacillus species. The $NH_2$-terminal amino acid sequences of the 10 residues of both proteases were completely identical: Met-Asn-Thr-Gln-Asn-Ser-Phe-Leu-Ile-Lys. In contrast to this, the 80 kDa protease has 1.5 times higher specific activity than the 120 kDa protease does (Kang et al. 1995. Agric. Chern. Biotech. 38: 534-540). Therefore the C-terminal of the 120 kDa protease seems to be autolyzed to the 80 kDa protease but this autolysis did not decrease the protease activity. Optimum pH and temperature of both 80 kDa and 120 kDa proteases were pH 10.5 and $45^{\circ}C$, respectively, and pH and thermal stability were almost identical. Several divalent ions except the $Fe^{2＋}$ ion showed similar effects on activities of both proteases, which are similarly resistant to three different detergents.

• 한국미생물·생명공학회지
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• 제20권3호
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• pp.295-301
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• 1992

토양으로부터 alkaline protease 생성능이 강한 Streptomyces griseus HC-1141을 분리하였으며, 정제 효소의 최적작용 pH 와 온도는 8.0, 60'C 였으며, pH 7.0-9.0의 범위와 $60^{\circ}C$이하에서 안정하였다. 금속 이온중 $Mn^{2+}$, $Ca^{2+}$, 등에 의해 활성이 증대되었으나 $Hg^{2+}$, $Cu^{2+}$, $Zn^{2+}$, $Ba^{2+}$, $Fe^{2+}$ 등에 의해 효소 활성이 저해되었고, 효소활성 저해제 중 Epsilon-aminocaproic acid, 2,4-dinitrophenol, iodine 등에 의해서는 현전한 효소활성저해가 관찰되지 않았으나 ethylenediaminetetraacetic acid와 p-chloromercuribenzoic acid에 의해 활성저해가 관찰되어 효소분자 중 SH기가 활성에 어느정도 관여하는 metallo enzyme으로 추정되었다. 정제효소의 $K_m$, $V_{max}$ 및 활성화에너지는 $2.229{\times}10^{-4}$M, $46.08 {\mu}$g/min, 3.643 kcal/mol 이었으며 hemoglobin과 egg albumin보다 casein을 더 잘 가수분해하였다. 또한 여러 가지 detergent에 대하여 강한 저항성을 가지고 있었다.

• Bulletin of the Korean Chemical Society
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• 제33권7호
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• pp.2213-2218
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• 2012

Efficient synthetic routes were developed to prepare a sizable amount (4-15 grams) of the chiral epoxides 4-6 as versatile intermediates for the synthesis of aspartyl protease inhibitors of therapeutic interest such as HIV protease and ${\beta}$-secretase. Oxidative cleavage of the C(2)-C(3) double bond of L-ascorbic acid followed by functional group manipulation led to the preparation of the epoxide 10, which was opened with an azide to yield a common aziridine intermediate 12. Through opening of the aziridine ring of 12 with either a carbon or a sulfur nucleophile, chiral epoxide precursors 4-6 could be prepared for various HIV protease inhibitors. Except for the final low melting epoxides 5 and 6, all intermediates were obtained as crystalline solids, thus the synthetic pathway can be easily applied to a large-scale synthesis of the chiral epoxides.

• 산업식품공학
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• 제15권2호
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• pp.182-187
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• 2011

김치 및 젓갈 등의 150여 전통 발효 식품을 시료로 하여 protease 활성을 갖는 유산균을 분리한 결과, 24 U/mgcrude protein의 높은 활성을 갖는 젖산균 BV-26 균주을 분리하였다. API 50CHL kit를 이용하여 BV-26 균주의 당 이용성을 분석하고 16S rRNA 염기서열(99.9% 상동성)을 비교한 결과, 분리된 균주를 L. plantarum BV-26으로 표기 하였다. L. plantarum BV-26의 생장과 protease 활성 변화를 MRS 배지를 이용하여 측정한 결과, L. plantarum BV-26의 생장은 배양 6시간 이후 활발하게 진행되어 18시간에 최고의 균체 농도를 보였으며, protease 활성은 배양 후 12시간부터 생성되기 시작하여 16시간에서 최고의 활성을 나타내는 것으로 확인되었다. 따라서 본 연구에서 분리된 L. plantarum BV-26을 동물사료의 발효용 스타터로 이용할 경우 유산균이 갖는 유익한 장점 및 안전성을 확보할 수 있을 뿐만 아니라, 특히 대두박의 발효시 사료의 영양적 가치를 높일 수 있을 것으로 기대된다.

• 대한본초학회지
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• 제26권4호
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• pp.89-94
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• 2011

Objective : Kyenegum has been frequently used for characterizing digestive symptoms in the traditional and oriental medicines. This study was conducted to investigate the characteristics of extracts from velvet antlers using the 4 different kinds of extracting methods. Methods : The extracts of velvet antlers were extracted using a $65^{\circ}C$ DW (9hrs), a Kyenegum crude enzyme, a $121^{\circ}C$ DW (2hrs), and a Kyenegum protease. To evaluate the characteristic of velvet antler extracts, we examined the brix, soluble solid, amino acid, mineral composition, and collagen protein. Results : As a result of the comparisons of velvet antlers extracted by the traditional extraction and the crude enzyme of kyenegum, the brix and soluble solid showed the higher contents for kyenegum enzymes. Also, mineral contents of the extracted velvet antlers were higher, particularly in Ca and P for those. The contents of collagen protein, hydroxyproline and hydroxylysine, were found to be more than twice in kyenegum protease compared with other extracting methods. Conclusion : These results indicated that the Kyenegum crude enzyme and protease are very effective to extract of velvet antlers.

• 한국식품영양과학회지
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• 제18권3호
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• pp.348-355
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• 1989

Alkaline protease 생성능이 강한 Aspergillus fumigatus 균주를 토양에서 분리하고, 생성효소를 정제하여 특성을 조사한 결과 최적 pH는 9.0, pH안정성은 $pH\;8.0{\sim}10.0$, 최적온도는 $50^{\circ}C$였으며, $50^{\circ}C$이하의 온도에서 안정하나 그 이상의 온도에서는 급격한 효소 불활성화를 보였고, 금속염 $Mn^{++},\;Cu^{++},\;Ba^{++},\;Mg^{++}$ 등에 의해서 활성이 다소 증대되나 $K^+,\;Fe^{+++},\;Ag^{++},\;Pb^{++},\;Na^+,\;Ca^{++},\;Hg^+,\;Zn^{++}$에 의해 저해를 받았다. 활성저해제인 EDTA, 2,4-DNP, ${\varepsilon}-amino$ caproic acid에는 큰 저해를 받지 않으나, PCMB에 많은 저해를 받는 것으로 미루어 활성 부위가 SH기인 cystein protease로 추정되었다. Km값은 $8.33{\times}10^{-4}mole/{\ell}$, Vmax는 $47.62{\mu}g/min$였으며, casein과 hemoglobin을 trypsin보다 더 잘 분해하고, casein을 hemoglobin보다 잘 분해하였다.

• Fisheries and Aquatic Sciences
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• 제3권3_4호
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• pp.188-194
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• 2000

A protease purified from hepatopancreas of shrimp, Penaeus japonicus, had maximum activity at $70^{\circ}C$ and in neutral and alkaline pH ranges. Specific activity at optimum reaction condition of the protease was estimated to be approximately 12 U/mg/min. The protease was stable in neutral and alkaline pH ranges and activity was retained after heat treatment at $50^{\circ}C$ for 30 min. Apparent $K_m$ and $V_{max}$ value against casein substrate were estimated to be $0.29\%$ and $7.8see^{-1}$, respectively, and those against N-CBZ-L-tyrosine p-nitropheny1 ester (CBZ­Tyr-NE) were 0.38 mM and $2,400 see^{-1}$, respectively. The N-termina1 sequence of the protease showed high homology to the trypsin from same species and the proteases from shrimp. Myosin heavy chain (MHC) from shrimp tail meat was the most susceptible to the protease and actin/tropomyosin were degraded progressively during 4 hr incubation, but to a lesser degree than MHC.

• 한국미생물·생명공학회지
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• 제28권3호
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• pp.167-171
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• 2000

두엄에서 분리한 Thermoactinomycese sp. E79는 탈지 대두박(defatted soycean meal)을 특이적이로 분해하는 내열성 alkaline 단백질 분해효소를 생산한다. 이 효소를 생산하기 위한 환경인자를 조사하였는데 배지의 초기 pH가 6에서 8까지는 유사한 균체농도를 얻을 수 있었고 pH10에서는 단백질분해효소의 발현이 되지 않았다. 탄소원은 수용성 전분을 이용할 경우 최적의 값을 보여 9.2U/mL의 효소역가를 얻었고 포도당을 탄소원으로 사용한 경우 단백질분해효소의 발현이 억제되었다 최적의 효소발현을 위해 tryptone을 세포성장에 soytone을 단백질 분해효소 생산에 가장 적합한 질소원으로 선택하였다. 호기성 세균인 Thermoactinomycese sp. E79의 산소요구성으 알아보기 위해 산소전달속도를 달리하여 발효인자를 결정하였고 volumetric oxygen transfer coefficient 가 1.93$\times$102 hr-1 일 때 균체농도 6.58 g/L 효소역자 43.0 U/mL 의최대값을 보였다 또한 효소역가를 증강시키기 위해 200mg/L의 humic acid를 첨가한 경우 비첨가 대조구에 비해 단백질 분해효소 역가는 1.64배 세포성장은 1.77배 증가하였다.