• Proceedings of the Korean Society for Applied Microbiology Conference
• /
• 1986.12a
• /
• pp.517.1-517
• /
• 1986

고온 호알카리성 Bacillus K-17의 Protease gene의 구조해명과 성질을 알기 위해서, E. coli HB101에 pER 322를 Vector로 하여 Protease gene을 Cloning하여 형질전환 된 균주를 선정하였다. 선정균주의 pretense activity를 Bacillus K-17의 상대활성도와 매우 유사하였으며 균체외에 보다 많은 효소 활성도를 지니고 있었다. 제한효소 Hind III로 절단하면 약 1.8kb와 0.4kb의 2개의 fragments 가 생성되었으며 Southern hybridization 결과 Cloning 된 gene이 Bacillus K-17에서 유래된 것임이 확인되었다.

• 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.37 no.5
• /
• pp.574-581
• /
• 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.

• Korean Journal of Microbiology
• /
• v.40 no.1
• /
• pp.12-16
• /
• 2004

Acid proteinase has been discovered in Aspergillus niger (acid protease A) and Cryphonectria parasitica (acid proteinase EapC) and it plays major roles in cheese formation from milk. In this study, a partial gene encoding acid proteinase in Penicillium oxalicum HCLF-34 was cloned by using PCR with degenerate primers corresponding to highly conserved regions of the acid proteinase. The partial acid proteinase gene in P. oxalicum HCLF-34 contains an open reading frame of 438 base pairs and encodes an acid proteinase protein of 146 amino aicds. The predicted amino acid sequences showed 71 % homology with acid protease A and 67% homology with EapC.

• 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.

• Korean Journal of Microbiology
• /
• v.41 no.1
• /
• pp.87-92
• /
• 2005

Pronase, a protease produced for commercial purpose by Streptomyces griseus, was composed of serine protease, alkaline protease, aminopeptidase and carboxypeptidase complex, and it has been widely used as anti-inflammatory drugs for human therapy. In this study, we developed a new integration vector, pHJ101 derived from pSET152, containing strong promoter, ermE, to overexpress a certain protease gene. Specific PCR primers for cloning of sprA (a gene for S. griseus protease A) and sprB (a gene for S. griseus protease B) genes were designed from the basis of nucleotide sequence in databases and amplified by PCR. Plasmid pHJ201 and pHJ202 were constructed by inserting of amplified each gene in a vector pHJ101. S. griseus HA and S. griseus HB were respectively obtained by conjugal process of a parent strain, S. griseus IFO 13350 with the recombinant Escherichia coli harboring plasmid pHJ201 or pHJ202. When protease activity was measured in flask cultivation, produced protease levels of S. griseus HA and S. griseus HB increased about 5.3 times and 5 times, respectively, more than that of parent strain. And, the constructed integrating plasmid pHJ101 was applicable for overexpression of a certain gene in Streptomyces sp.

• Journal of Microbiology
• /
• v.34 no.1
• /
• pp.68-73
• /
• 1996

An nprX gene of Bacillus subtilis NS15-4 encoding a neutral protease was cloned and its molecular characteristics were analyzed. The complete nucleotide sequence indicated that there is an open reading frame (0RF) possibly encoding 521 amino acid polypeptide. The ORF used all codons expected two cysteine and a proline having a codon bias index (CBI) of 0.09 in Escherichia coli. There were homologous sequences to the consensus sequence of -35 and -10 regions of E. coli promoters and to a Shine-Dalgarno (SD) sequence located 25 bp downstream of a mojor transcription initiation site. Moreover, there were also five minor transcription initiation sites at 6. 7. 8. 14 and 15 nt downstream of the major site. Northern blot analysis revealed the presence of about 1.8 kb mRNA transcript in E. coli having the nprX gene. The nucleotide sequence was identified in GenBank to be a gene for a neutral protease of B. sutilis with six nucleotide difference in the ORF region. The flanking regions of the NprX ORF showed much more differences form those of other neutral protease genes except the nprE gene of B. subtilis, which has the most homology to the nprX gene, and of which the flanking regions were identical to those of the nprX gene.

• Journal of Microbiology and Biotechnology
• /
• v.34 no.2
• /
• pp.436-456
• /
• 2024

Several thermostable proteases have been identified, yet only a handful have undergone the processes of cloning, comprehensive characterization, and full exploitation in various industrial applications. Our primary aim in this study was to clone a thermostable alkaline protease from a thermophilic bacterium and assess its potential for use in various industries. The research involved the amplification of the SpSKF4 protease gene, a thermostable alkaline serine protease obtained from the Geobacillus thermoglucosidasius SKF4 bacterium through polymerase chain reaction (PCR). The purified recombinant SpSKF4 protease was characterized, followed by evaluation of its possible industrial applications. The analysis of the gene sequence revealed an open reading frame (ORF) consisting of 1,206 bp, coding for a protein containing 401 amino acids. The cloned gene was expressed in Escherichia coli. The molecular weight of the enzyme was measured at 28 kDa using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The partially purified enzyme has its highest activity at a pH of 10 and a temperature of 80℃. In addition, the enzyme showed a half-life of 15 h at 80℃, and there was a 60% increase in its activity at 10 mM Ca²⁺ concentration. The activity of the protease was completely inhibited (100%) by phenylmethylsulfonyl fluoride (PMSF); however, the addition of sodium dodecyl sulfate (SDS) resulted in a 20% increase in activity. The enzyme was also stable in various organic solvents and in certain commercial detergents. Furthermore, the enzyme exhibited strong potential for industrial use, particularly as a detergent additive and for facilitating the recovery of silver from X-ray film.

• Korean Journal of Microbiology
• /
• v.30 no.6
• /
• pp.507-513
• /
• 1992

Serratia marecescens RH1 isolated from soil samples produced large amount of extracellular proteases. One of the genes encoding an extracellular protease form S. marcescens RH1 was cloned in Escherichia coli by shot gun cloning method. The cloned protease, SSP, was stably expressed by its own promoter and excreted into the extracellular medium from E. coli host (ORF) of 3.135 nucleotides corresponding to 1.045 amino acids (112 kDa). The nucleotide and deduced amino acid sequence of SSP showed high overall homology (88%) to one of the S. marcescens protease (27), but low homology to other serine protease families. The optimal pH and temperature of the enzyme were pH 9.0 and 45.deg.C respectively. The activity of protease was inhibited by phenylmethylsulfonyl fluoride (PMSF), which suggests that the enzyme is a serine protease.

• 한국생물공학회:학술대회논문집
• /
• 2000.04a
• /
• pp.527-530
• /
• 2000

A kiwi fruit ,called as the Chinese gooseberry, is originated from the Yangtze River Valley of Northern China and Zhejiang Province on the cost of Eastern China. Around 1950, a large mass production began at New Zealand with an Improved breeding. Plant origin actinidin from kiwi fruit belongs to the papain family of cysteine proteinase, which in plants includes papain from papaya, bromelain from pineapple, Cl4 protease from tomato and aleurain from barley. Actinidin is involved in the ripening-related gene family. In this study, protease gene of chinese wild kiwi fruit was isolated and characterized. 1.2kb PCR-amplified fragment was obtained from the total RNA using RT-PCR. pWACT-1 was obtained by subcloning of amplified fragment into pGEM-T Easy cloning vector and analyzed nucleotide sequence by DNA sequencing and amino acid sequence. In Result, high levels of homology between wild kiwi and New Zealand cultured-kiwi was obtained.