• Journal of Microbiology and Biotechnology
• /
• v.2 no.4
• /
• pp.231-236
• /
• 1992

A 5.8 kb chromosomal DNA fragment of Serratia marcescens ATCC 27117 including an extracellular serine protease gene was cloned in Escherichia coli. The cloned gene(pSMP18) caused specific excretion of the protease into the extracellular medium through the outer membrane of E. coli host cells. The protease purified from E. coli harboring pSMP18 was inactivated not by 100 mM EDTA but by 10 mM phenyl methyl sulfonyl flouride (PMSF). The molecular weight of the purified serine protease was about 66, 000 in the SDS-PAGE and the isoelectric point was approximately 5.7 in IEFㆍGel electrophoresis. The optimal pH and temperature for reaction of the purified serine protease were 9.5 and $45^\circ{C}$, respectively.

• The Korean Journal of Mycology
• /
• v.51 no.4
• /
• pp.265-276
• /
• 2023

This study aimed to isolate and characterize fungi from freshwater environments in South Korea and evaluate their extracellular enzyme activities. Fungal strains were collected from various freshwater sources and identified using phylogenetic analysis. The isolated fungi included known aquatic hyphomycetes and previously unreported species. Extracellular enzyme, including those of protease, amylase, lipase, cellulase, laccase, and chitinase, activities were evaluated. Among the isolated strains, several showed high enzyme activity, suggesting their potential role in organic matter decomposition in freshwater ecosystems. This research expands our knowledge of the diversity and enzyme activities of the fungi in freshwater environments, contributing to our understanding of their ecological roles.

• Journal of Life Science
• /
• v.11 no.1
• /
• pp.42-46
• /
• 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.

• Korean Journal of Microbiology
• /
• v.49 no.1
• /
• pp.78-82
• /
• 2013

A novel Chryseobacterium sp. JK1 strain isolated from soil had been reported that this isolate produced large amount of extracellular protease at mesophilic temperature in previous study. The optimal temperature and pH of extracellular protease were $40^{\circ}C$ and 7.0, respectively, showing narrow range of optimal temperature and relatively broad activity from pH 6.0 to 9.0. In addition, the protease showed greatest activity against skim milk and lowest against bovine serum albumin (BSA). The protease strongly inhibited by ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA) or phenylmethylsulfonyl fluoride (PMSF), and addition of cation $Ag^+$ or $Cu^{2+}$, and slightly inhibited by $Al^{3+}$. No significant inhibition was found with pepstatin, and addition of cation, $K^+$, $Ca^{2+}$, $Na^+$, $Fe^{2+}$ or $Mg^{2+}$. On the contrary, protease was enhanced by addition of divalent cation $Mn^{2+}$ (5 mM). Zymography analysis of concentrated culture supernatant revealed two major bands at 67 and 145 kDa. These results suggest that Chryseobacterium sp. JK1 strain produced extracellular neutral serine proteases which could apply in food industry.

• Applied Biological Chemistry
• /
• v.44 no.4
• /
• pp.246-250
• /
• 2001

In order to produce alkaline protease, psychrotrophic Bacterium which have high enzyme activity, was isolated by using enrichment culture from soil samples and identified as genus Bacillus sp. The optimal pH and temperature for the enzyme activity were pH 6 and $40^{\circ}C$. The temperature range of high enzyme activity was $20{\sim}40^{\circ}C$. The optimal initial pH of culture condition for enzyme was pH 6. The most favorable carbon and nitrogen sources for the production of protease by Bacillus sp. WRD-2 were 3% maltose and 4% yeast extract, respectively.

• Korean Journal of Microbiology
• /
• v.30 no.6
• /
• pp.501-506
• /
• 1992

An alkalophilic Vibrio sp. RH530 showing high proteolytic activity was isolated form soil samples by enrichment culture. The activity staining using gelatin SDS- polyacrylamide gel electrophoresis (PAGE ) revealed that the strain produced an alkaline major protease (Apr B) with a size of 27 kDa, and at least six minor proteases. The apparent sizes of four of the minor proteases were approximately 45, 28, 22 and 19 kDa. Apr B and five of the minor proteases were inhibited by serine protease inhibitors including PMSF and DFP, suggesting that they are serine proteases. One of the minor proteases was inhibited by metalloprotease inhibitors, not by serine protease inhibitors, indicating it to be a metalloprotease. Furthermore, the activities of Apr B and Prt 3 were not inhibited by SDS in the reaction mixture. The production of Apr B and some of the minor proteases was specifically affected by culture temperature (30 to 37.deg.C) and pH (7 to 10). The production of Apr B. Prt 2, Prt 5 and Prt 6 was mainly affected by culture temperature, while Prt 4 by culture pH. Prt 1 and Prt 3 were not affected by neither of these factors.

• Microbiology and Biotechnology Letters
• /
• v.23 no.2
• /
• pp.145-149
• /
• 1995

For the purpose of developing a new biodegradable detergent, we have isolated a gene encoding wide-range temperature applicable alkaline protease from Xanthomonas sp. YL-37 (Lee et al., 1994, Kor. J. Appl. Microbiol. Biotechnol.). An alkaline protease gene was isolated from the gene bank that was prepared from the chromosomal DNA of Xanthomonas sp. YL-37. From the results of agarose gel electrophoresis and a restriction enzyme mapping, a 2.7 kb DNA fragment containing the alkaline protease gene was inserted in the plasmid pUC9. Extracellular activity of a clone having alkaline protease gene was detected on SDS-polyacrylamide gel with activity staining assay. The molecular weight of alkaline protease was determined to be about 64 kDa from 11% SDS-PAGE analysis. Alkaline protease activity, produced from E. coli which harboring the plasmid, showed no difference at reaction temperature 20, 30 and 40$\circ$C, respectively. This result showed that alkaline protease produced from E. coli harboring the plasmid was apparently the same as that of Xanthomonas sp. YL-37.

• BMB Reports
• /
• v.41 no.2
• /
• pp.102-107
• /
• 2008

Extracellular proteases play an important role in a wide range of host physiological events, such as food digestion, extracellular matrix degradation, coagulation and immunity. Among the large extracellular protease family, serine proteases that contain a "paper clip"-like domain and are therefore referred to as CLIP-domain serine protease (clip-SP), have been found to be involved in unique biological processes, such as immunity and development. Despite the increasing amount of biochemical information available regarding the structure and function of clip-SPs, their in vivo physiological significance is not well known due to a lack of genetic studies. Recently, Drosophila has been shown to be a powerful genetic model system for the dissection of biological functions of the clip-SPs at the organism level. Here, the current knowledge regarding Drosophila clip-SPs has been summarized and future research directions to evaluate the role that clip-SPs play in Drosophila immunity are discussed.

• Journal of Applied Biological Chemistry
• /
• v.43 no.3
• /
• pp.147-151
• /
• 2000

A gene, nprM, from Bacillus megaterium ATCC 14945 was obtained by PCR using primers synthesized based on two nprM sequences from two different strains, and cloned into Escherichia coli. The gene nprM encoded an extracellular neutral protease, and the molecular mass of the expressed enzyme was estimated to be approximately 36kDa on a denaturating gel. The enzyme was activated by $Ca^{2+}$, and the optimum concentration of $Ca^{2+}$ was 5 mM. The enzyme was inhibited by EDTA but not by PMSF. The optimal pH and temperature of the cloned enzyme were $50^{\circ}C and pH 7.5-8.0, respectively, and were similar to those of the enzyme from the gene gonor cell. The cloned NprM caused internal cleavage of the native endoglucanase of B. subtilis BSE616 as a model foregin protein, and resulted in a small truncated but still active endoglucanase.

• Journal of fish pathology
• /
• v.19 no.2
• /
• pp.119-126
• /
• 2006

The characteristics of the extracellular products (ECPs) of Vibrio harveyi grown under various conditions were studied. The extracellular products (ECPs) of Vibrio harveyi is well known as an important pathogenic factor. The optimal isolation condition of the V. harveyi ECPs was incubation with 1.5% NaCl added TSA medium at 25℃ for 24 h. The buffer system for ECPs isolation controlled as pH 8.0 displayed optimal condition. The major protein of ECPs isolated from the five V. harveyi strains originated from Korea (FF8, FF10, FR1, FR2 and FT1 strain) were revealed to serine protease.