• Fisheries and Aquatic Sciences
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• v.2 no.2
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• pp.218-225
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• 1999

A protease without tryptic and chymotryptic activities was purified from the hepatopancreas of shrimp, Penaeus orientalis, using Q-Sepharose ionic exchange, benzamidine Sepharose-6B affinity, Mono-Q, and gel chromatography. Molecular weight (M.W.) of the protease was estimated to be 27kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS­PAGE). The amino acid composition of the protease was different from that of protease from P. japonicus or trypsin from P. orientalis. The protease was completely inhibited by benzamidine, $N\alpha-p-tosyl-L-lysine$ chloromethyl ketone (TLCK), and phenylmethylsulfonyl fluoride (PMSF) and was not affected by leupeptin, pepstatin, N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), iodoacetate, and ethylenediamine tetra acetate (EDTA). The enzyme did not have any activity against Na-benzoyl-DL-arginine p-nitroanilide (BAPNA) or N-benzoyl-L-tyrosine ethyl ester (BTEE) which are specific substrates of trypsin and chymotrypsin, respectively. However, the protease showed hydrolytic activity for a carboxyl terminal of Tyr, Trp, Phe, Glu, and Cys.

• Fisheries and Aquatic Sciences
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• v.13 no.1
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• pp.84-87
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• 2010

A peptide that inhibits HIV-1 protease was isolated from a hydrolysate of oyster (Crassostrea gigas) proteins digested with thermolysin. The peptide was using membrane filtration, gel permeation chromatography, ion exchange chromatography, and reverse-phase high performance liquid chromatography. Amino acid sequence of the peptide was determined to be Val-Phe-Glu-Leu. Chemically synthesized Val-Phe-Glu-Leu showed an $IC_{50}$ value of 106 ${\mu}M$.

• Journal of Microbiology
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• v.45 no.2
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• pp.158-167
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• 2007

In this study, we have cloned a novel cDNA encoding for a papain-family cysteine protease from the Uni-ZAP XR cDNA library of the polychaete, Periserrula leucophryna. This gene was expressed in Escherichia coli using the T7 promoter system, and the protease was characterized after partial purification. First, the partial DNA fragment (498 bp) was amplified from the total RNA via RT-PCR using degenerated primers derived from the conserved region of cysteine protease. The full-length cDNA of cysteine protease (PLCP) was prepared via the screening of the Uni-ZAP XR cDNA library using the $^{32}P-labeled$ partial DNA fragment. As a result, the PLCP gene was determined to consist of a 2591 bp nucleotide sequence (CDS: 173-1024 bp) which encodes for a 283-amino acid polypeptide, which is itself composed of an 59-residue signal sequence, a 6-residue propeptide, a 218-residue mature protein, and a long 3'-noncoding region encompassing 1564 bp. The predicted molecular weights of the preproprotein and the mature protein were calculated as 31.8 kDa and 25 kDa, respectively. The results of sequence analysis and alignment revealed a significant degree of sequence similarity with other eukaryotic cysteine proteases, including the conserved catalytic triad of the $Cys^{90},\;His^{226},\;and\;Asn^{250}$ residues which characterize the C1 family of papain-like cysteine protease. The nucleotide and amino acid sequences of the novel gene were deposited into the GenBank database under the accession numbers, AY390282 and AAR27011, respectively. The results of Northern blot analysis revealed the 2.5 kb size of the transcript and ubiquitous expression throughout the entirety of the body, head, gut, and skin, which suggested that the PLCP may be grouped within the cathepsin F-like proteases. The region encoding for the mature form of the protease was then subcloned into the pT7-7 expression vector following PCR amplification using the designed primers, including the initiation and termination codons. The recombinant cysteine proteases were generated in a range of 6.3 % to 12.5 % of the total cell proteins in the E. coli BL21(DE3) strain for 8 transformants. The results of SDS-PAGE and Western blot analysis indicated that a cysteine protease of approximately 25 kDa (mature form) was generated. The optimal pH and temperature of the enzyme were determined to be approximately 9.5 and $35^{\circ}C$, respectively, thereby indicating that the cysteine protease is a member of the alkaline protease group. The evaluation of substrate specificity indicated that the purified protease was more active towards Arg-X or Lys-X and did not efficiently cleave the substrates with non-polar amino acids at the P1 site. The PLCP evidenced fibrinolytic activity on the plasminogen-free fibrin plate test.

• Textile Coloration and Finishing
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• v.5 no.3
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• pp.206-215
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• 1993

In the prior study, wool gabardines were treated with alkaline proteases which were some kinds of enzyme to decompose protein, and their tensile strengths were determined, and the surface of the fibers were also observed using a scanning electron microscope. Enzylon ASN 30 and Alkalase 2.5L DX did not show much effect on the weight loss of wool, however, the weight loss of wool increased considerably with treating Esperase 8.0L. Pretreatment of wool with dichloloisocyanuric acid before protease-treatment increased the weight loss of wool to a great extent. In this study, the enzyme treated wools dyeing behaviors with acid dye, Milling Cyanine 5R, were mainly investigated. The protease-treatment remarkably increased not only the rate of dyeing but also the saturation dye uptake. From these results, it seemed likely that the structural relaxation of adhesive filler of interscale or intercellular cement facilitated the dye penetration into the fibers, at the same time, the change in the inner structure of the wool fibers by the protease made the fixation of the dyes more efficient.

• The Korean Journal of Food And Nutrition
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• v.25 no.1
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• pp.188-195
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• 2012

To produce rapidly the traditional Kanjang soy sauce-like product with rich flavors, lactic acid bacteria of Enterococcus spp. isolated from Chungkukjang was used as one of starter cultures. Among 119 Enterococcus spp., eight strains were selected by protease-secreting activities and identified as four E. faecium, three E. faecalis, and one E. gallinarium. The strains showed low resistances toward eight antibiotics and had no resistant genes to the vancomycin. Especially, E. faecium O24 was cultivated well on 5% NaCl medium that was selected for further study as the starter. E. faecium O24 grew well on the steamed soybean and the counts increased by ten times overnight, which produced mostly 80 mg% glutamic acid and aspartic acid as the seasoning amino acids on the product. Various organic acids including principal lactic acid were also produced. Flavors of maltol and guaiacol, typical soy-sauce flavor, were produced in the mixed cultures of Zygosaccharomyces rouxii and Candida versatilis. Therefore, E. faecium O24 could be a starter of soybean fermentation for soy sauce-like product with rich flavors rapidly.

• Microbiology and Biotechnology Letters
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• v.25 no.4
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• pp.379-385
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• 1997

Several protease-deficient mutants of Aspergillus niger have been isolated by halo-screening on skim milk plate after UV irradiation of conidiospores. The extracellular proteolytic activities of the mutant strains grown in an optimized medium varied from 3% to 85% of that of the parental strain. Especially, two mutant strains named as ANPD-129 and ANPD-153, which had 3% and 49% of acid protease activity of the parental strain, respectively, were further characterized both physiologically and genetically. The growth rates of the mutants, ANPD-129 and ANPD-153, were similar to that of the parental strain, unlike other protease-deficient mutants. The diploid formed between the two mutants restored protease activity to a similar level of that of the parental strain. This result revealed that ANPD-129 and ANPD-153 had mutations at different loci. Using master strains with marked chromosomes these loci were assigned to linkage groups. The mutation locus (prt129) in ANPD-129 was assigned to linkage group VI and the locus (prt153) in ANPD-153 to linkage group III.

• Microbiology and Biotechnology Letters
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• v.23 no.3
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• pp.322-328
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• 1995

A thermophilic bacteria showing proteolytic activity against defatted soybean was isolated from soil. It was identified as Bacillus amyloliquefaciens based on its morphological and physiological characteristics. The Bacillus amyloliquefaciens NS 15-4 was cultivated at 50$\circ$C by rotary shaking in a medium containing defatted soybean. An extracellular protease from this strain was purified to homogeneity by ammonium sulfate precipitation, ion exchange, and hydrophobic interaction chromatographies. The molecular weight of the enzyme was estimated to be approximately 30,000 by SDS-PAGE and the N-terminal amino acid sequence of the enzyme was turned out to be AQSVPYGISQIKAPA. The optimum temperature and pH for the enzyme reaction were 60$\circ$C and 11, respectively, and its thermostability was increased by the addition of calcium ion. The enzyme was inactivated by phenylmethylsulfonylfluoride, suggesting it be a serine protease. Comparing with other commercial proteases, the enzyme showed relatively high proteolytic activity against defatted soybean, a water-insoluble protein substrate.

• Applied Biological Chemistry
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• v.31 no.3
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• pp.274-283
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• 1988

Aqualysin I is an alkaline serine protease which is secretet into the culture medium by Thermus aquaticus YT-1, an extreme thermophile. Aqualysin I was purified, and its partial amino acid sequence was determined. The gene encoding aqualysin I was cloned into E. coli using synthetic oligodeoxyribonucleotides as hybridization probes. The nucleotide sequence of the cloned DNA was determined. The primary structure of aqualysin I, deduced from the nucleotide sequenc, agreed with the determid amino acid sequences, including the $NH_2-$ and COOH terminal sequence of the tryptides derived from aqualysin I. Aqualysin I comprised 281 amino acid residues and its molecular mass was determined to be 28350. On alignment of the whole amino acid sequence, aqualysin I showed high sequence homology with the subtilisin type serine protease, and 43% identity with proteinase K, 37-30% with subtilisins and 34% with thermitase. Extremely high sequence identity was observed in the regions containing the active-site residues, corresponding to Asp32, His64 and Ser221 of subtilisin BPN'. Aqualysin I contains two disulfide bonds, Cys67-Cys99 and Cys163-Cys194, and these disulfide bonds seem to contribute to the heat stability of the enzyme. The determined positions of the twe disulfide bonds of aqualysin I agreed with those predicted previously on the basis of computer graphics of the crystallographic data for subtilisin BPN'. Therefore, these findings sugests that the three-dimensional structure of aqualysin I is similar to that of subtilisin BPN' Aqualysin I is produced as a lage precursor, which contains $NH_2-$ and COOH- terminal portions besides the mature protease sequence.

• Preventive Nutrition and Food Science
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• v.2 no.2
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• pp.109-120
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• 1997

In order to elucidate roles of lactic acid bacteria(LAB) for the antibiosis occurring in th fermenting environment of Kimchi, 2.052 strains of LAB were isolated from Kimchi. Fifty tow strains which showed antagonistic effect against 4 indicator strains were finally selected and investigated. Based upon responses to protease treatment, antibiosis of the 52 strains of LAB were classified into 3 types. Type A antibiosis resulted from action of antibiotic-like substances which were not affected by protease treatment and which had broad action spectra against even natural inhabitants of Kimchi. Type B antibiosis was due to bacteriocin-like substances which were very sensitive to treatment of protease and more effective against foreign bacteria than original inhabitant microflora. Type C antibiosis was owing to proteinaceous compounds which were activated or induced by the presence of protease and then exerted antibacterial activities. Therefore, lactic acid bacteria appeared to contribute to antibiosis of Kimchi by the concerted action of these three different types of antibacterial compounds. As one of model system for type B bacteriocin, the antagonistic compound produced by LAB31-9 as well as th producer strain itself was further charaacterized. Strain LAB31-9 was identified as L. casei. Bacteriocin produced by LAB31-9 was proteinaceous and stable over wide range of pH and to various solvents, but very labile to heat treatment. Its mode of action was bactericidal. Based upon these data, bacteriocin produced by LAB31-9 was named as 'caseicin K319'. Genetic determinant for the bacteriocin production of LAB31-9 was located in the chromosome.

• Journal of Nutrition and Health
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• v.45 no.5
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• pp.429-436
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• 2012

This study was performed to examine the characteristics of protein of red crab (Chionoecetes japonicus) shell powder hydrolyzed by commercial proteases. Red crab shell was digested by commercial proteases, such as Protamex (P), Neutrase (N), Flavourzyme (F), Alcalase (A), Protease M (PM) and Protease A (PA). Protein yield analyzed by Biuret assay, absorbance at 280 nm and brix revealed that PA was the enzyme having the highest proteolytic activity. SDS PAGE showed that molecular weight of proteins produced by protease treatments was various and below 150 kDa. Combinational treatment of proteases (PA + P, PA + PM, PA + F, PA + A) was tried whether these increase protein hydrolysis from red crab shell powder compared to a PA single treatment. Soluble protein content was similar, but amino acid concentration by combinational treatments was higher than PA single treatment [PA + P 247.4 mg/g > PA + F (206.4 mg/g) > PA + A (133.4 mg/g) > PA + PM (59.1 mg/g) > PA (54.9 mg/g)]. Amino acid composition by combinational treatments was slightly different. Most abundant essential amino acids were phenylalanine, glycine, alanine, and leucine, whereas tyrosine and cystine were not detected.