• Korean Journal of Fisheries and Aquatic Sciences
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• v.19 no.6
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• pp.537-546
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• 1986

In the previous paper(Kim et al, 1986), the alkaline proteinase from the pyloric caeca of mackerel was shown relatively strong activity in the alkaline pH range. Therefore purification of the enzyme has been undertaken to identify the proteolytic enzyme and three alkaline proteinases were isolated by ammonium sulfate fractionation, DEAE-Sephadex A-50 column chromatography and Sephadex G-100 gel filtration. One percent sodium chloride solution was the most effective for the extraction of alkaline proteinase from the pyloric caeca of mackerel. Three alkaline proteinases temporarily designated Enz. A, B and C were isolated from the pyloric caeca of mackerel, and identified to be homogeneous with electrophoresis. The specific activity of the purified Enz. A, B and C was increased to 34, 53 and 37-fold over the crude enzyme solution, respectively. Yield of them was 1.6, 2.1 and $1.5\%$, respectively, and a combined yield was $5.2\%$.

• Microbiology and Biotechnology Letters
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• v.17 no.6
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• pp.545-551
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• 1989

Thermostable alkaline protease of alkalophilic Bacillus sp. No. 8-16 has been purified, and the properties of the enzyme investigated. The characteristic point of the organism used is especially good growth in alkaline and thermal condition. The alkaline protease of the strain No. 8-16 was purified from crude enzyme by acetone precipitation, CM-cellulose ion exchange chromatography, Sephadex G-100 and Sephadex G-75 gel filtration. Through the series of chromatograpies, the enzyme was purified to homogeneity with specific activity of 37 fold higher than that of the crude broth. Characteristics of the purified enzyme were as follow; $K_m$ value for the enzyme was 1.3 mg/ml, the alkaline protease showed a maximal activity at 7$0^{\circ}C$ and from the pH 6.0 through 12.0, and stable for 1 hr. at 6$0^{\circ}C$. The moleclar weight of the enzyme was estimated to be 33,000 by Sephadex G-100 gel filtration. The activity of the alkaline protease was inhibited by iodoacetic acid and Ag$^+$, Hg$^+$, PMSF (phenylmethylsulfonyl fluoride), and activated by $Ca^{2+}$ and Mn$^{2+}$.

• Korean Journal of Microbiology
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• v.46 no.4
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• pp.383-388
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• 2010

Alkaline protease-overproducing strains of Vibrio metschnikovii were developed by using the molecular evolution from the classical mutants V. metschnikovii L12-23, N4-8, and KS1. Each vapK (Vibrio alkaline protease K) was obtained from the genomic DNAs of mutants by PCR to carry out the DNA shuffling. The modified vapK-1 obtained by DNA shuffling was used again as a template for the error-prone PCR to make the vapK-2. Both genes were cloned in the plasmid pKF3 to construct the recombinant plasmids which have one or two copies of the modified genes. The recombinant plasmids were back-transformed to V. metschnikovii KS1 to construct recombinant V. metschnikovii that expresses the alkaline protease. About 3.9-fold more protease activity was measured in the strain which has the plasmid containing two copies of vapK-2 when compared to strain KS1. When compared to wild type V. metschnikovii RH530, 43-fold more activity was achieved. Comparison of amino acids among vapK, vapK-1, and vapK-2 revealed that the active sites was highly conserved and not changed. However, many amino acids except the active sites were changed. These results suggested that the changes in amino acids might play an important role in the increase of protease activity by allowing the easy access of substrate to active sites of the protease. The fermentation of alkaline protease from the V. metschnikovii KS1 harboring the plasmid that contains two copies of vapK-1 showed the possibility of this strain to be used as industrial producer.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.19 no.6
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• pp.521-528
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• 1986

In this paper, proteolytic activity of the tissue extracts from the internal organs such as alimentary canal, pancreas, pyloric caeca, stomach, liver and spleen of mackerel, Scomber japonicus, and sardine, Sardinops melanosticta, was compared with each other under the optimum reaction condition. The proteinases distributed in alimentary canal, pancreas, pyloric caeca and spleen were active in alkaline pH range, but those in stomach were shown the activity in acid pH range, furthermore those in liver were exhibited the activity in acid, neutral and alkaline pH range. The proteinases distributed in the internal organs of both fish were stable at the heat treatment of $45^{\circ}C$ for 5 minutes. The proteinases from stomach and pyloric caeca of the two fish and those from pancreas of sardine were less stable than those from any other internal organs of both fish. Whereas the proteinases from spleen and neutral proteinases from liver were shown to be stable by the heat treatment at $55^{\circ}C$ for 5 minutes. The proteinases from pyloric caeca of both fish, and stomach, pancreas and spleen of mackerel were stable during the whole storage days at $5^{\circ}C$, but the other proteinases were slowly inactivated after 14 days of storage. The enzymes were seemed to be more stable in the storage at $-15^{\circ}C$ than at $5^{\circ}C$.

• Korean Journal of Microbiology
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• v.51 no.1
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• pp.75-80
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• 2015

Pseudoalteromonas donghaensis HJ51, isolated from the East Sea, has been reported as a novel strain to produce extracellular protease. Crude supernatant was used to determine optimal activity and optimal production conditions for the enzyme. It was found that the optimal temperature and pH of the protease were $40^{\circ}C$ and pH 7.5-10.5, respectively. The enzyme activity was kept to 88% at the pH 11. In metal requirement analysis, the enzyme exhibited the highest activity when 10 mM $Fe^{3+}$ was supplied. While supplementation of additional carbon sources used in study showed no positive effect on cell growth and enzyme activity, the addition of beef extract, tryptone, or casamino acids instead of peptone of PY-ASW containing 1% glucose increased enzyme production to 21, 7, 4%, respectively. Taken together these properties, the enzyme produced from P. donghaensis HJ51 can be applied to the industries that require protease activity under alkaline pH and low temperature.

• The Korean Journal of Food And Nutrition
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• v.19 no.4
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• pp.496-501
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• 2006

This study was to isolate a bacterium producing a alkaline protease from mud flats of the west seaside of Korea and to investigate the biochemical analysis of the alkaline protease producing from the isolate. The isolate was named as Gelidibacter sp. HK-1 based on 16S rRNA sequence, Gram staining and the photograph of electron microsceope. Optimum temperature for growth and pretense production of the isolate was $25^{\circ}C$. Growth of the isolate was reached at stationary phase after 10hrs followed by inoculation. Maximum activity of protease produced from the isolate was shown after 14hrs. Optimum temperature and pH for the protease activity were $45^{\circ}C$ and pH 9, respectively. Molecular weight of the pretense was about 50KD and the partial amino acid sequence of the pretense was Ala-Try-Ala-Leu-Asn-Thr-Ser-Val-Thr-Glu-Thr-Phe-Ala-Lys. The partial amino acid sequences of the protease showed significant homology with a pretense produced from Streptomyces avermitilis.

• Journal of Life Science
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• v.20 no.11
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• pp.1656-1661
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• 2010

Alkaline protease for the hydrolysis of egg yolk protein was immobilized on five carriers - Duolite A568, Celite R640, Dowex-1, Dowex 50W and Silica gel R60. Duolite A568 showed a maximum immobilization yield of 24.7%. Optimum pH for the free and immobilized enzyme was pH 8 and 9, respectively. However, no change was observed in optimum temperature ($50^{\circ}C$). Thermal stability was observed in immobilized enzymes compared to free enzymes. The immobilized enzyme retained 86% activity after 10 cycle operations in a repeated batch process. The effect of flow rate on the stability of enzyme activity in continuous packed-bed reactor was investigated. Lowering flow rate increased the stability of the immobilized enzyme. After 96 hr of continuous operation in a packed-bed reactor, the immobilized enzyme retained 83 and 61% activity when casein and egg yolk were used as a raw materials, respectively.

• Korean journal of food and cookery science
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• v.11 no.1
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• pp.26-32
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• 1995

This study was carried out to investigate the effect of protease on the functionality of soymilk protein. The protease from Bacillus polymyxa was selected because of the least production of bitter taste and calcium-aggregation. The results are summarized as follows: 1. Solubility of SMP(soymilk protein) and SPI(soyprotein isolate) were lowest at pH 4.7 and increased as the pH value reached closer to either ends. PT-SMP(pretense treated soymilk protein) showed higher solubility at all pH range, especially at pH 4.7 than SMP, SPI. 2. Emulsion activity of three samples was lowest at pH 4.7 and significantly increased as pH approched higher acidic or alkaline regions. PT-SMP showed similar activity to other samples, but less stability. 3. Foam capacity of PT-SMP was lowest at pH 8 and increased in acidic, alkaline pH. PT-SMP showed higher foam capacity at all pH range, but lower foam stability than SMP and SPI. 4. PT-SMP showed higher heat coagulability than other samples at all pH range except pH 4.7.

• Journal of agriculture & life science
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• v.45 no.6
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• pp.201-212
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• 2011

The alkaline protease gene was cloned from a halo-tolerant alkalophilic Bacillus clausii I-52 isolated from the heavily polluted tidal mud flat of West Sea in Inchon Korea, which produced a strong extracellular alkaline protease (BCAP). Based on the full genome sequence of Bacillus subtilis, PCR primers were designed to allow for the amplification and cloning of the intact pro-BCAP gene including promoter region. The full-length gene consists of 1,143 bp and encodes 381 amino acids, which includes 29 residues of a putative signal peptide and an additional 77-amino-acid propeptide at its N-terminus. The mature BCAP deduced from the nucleotide sequence consists of 275 amino acids with a N-terminal amino acid of Ala, and a relative molecular weight and pI value was 27698.7 Da and 6.3, respectively. The amino acid sequence shares the highest similarity (99%) to the nattokinase precursor from B. subtilis and subtilisin E precursor from B. subtilis BSn5. The substrate specificity indicated that the recombinant BCAP could hydrolyze efficiently the synthetic substrate, N-Suc-Ala-Ala-Pro-Phe-pNA,and did not hydrolyze the substrates with basic amino acids at the P1 site. The recombinant BCAP was strongly inhibited by typical serine protease inhibitor, PMSF, indicating that BCAP is a member of the serine proteases.

• Journal of Life Science
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• v.23 no.12
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• pp.1486-1494
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• 2013

A high protease-producing strain was isolated and identified from the digestive tract of octopus vulgaris by detecting a hydrolysis circle of protease and its activity. The strain was identified by morphology observation, biochemical experiments, and 16S rRNA sequence analysis. The protease obtained from the strain was purified by a three-step process involving ammonium sulfate precipitation, carboxy methyl-cellulose (CM-52) cation-exchange chromatography, and DEAE-Sephadex A50 anion-exchange chromatography. The properties of protease were characterized as well. The strain Bacillus sp. QDV-3, which produced the highest activity of protease, was isolated. On the basis of the phenotypic and biochemical characterization and 16S rRNA gene-sequencing studies, the isolate was identified as follows: domain: Bacteria; phylum: Firmicutes; class: Bacilli; order: Bacillales; family: Bacillaceae; and genus: Bacillus. The isolate was shown to have a 99.2% similarity with Bacillus flexus. A high active protease designated as QDV-E, with a molecular weight of 61.6 kDa, was obtained. The enzyme was found to be active in the pH range of 9.0-9.5 and its optimum temperature was $40^{\circ}C$. The protease activity retained more than 96% at the temperature of $50^{\circ}C$ for 60 min. Phenylmethylsulfonyl fluoride (PMSF) inhibited the enzyme activity, thus confirming that this protease isolated from Bacillus sp. QDV-3 is an alkaline serine protease. Metal ions, $Mn^{2+}$ and $Mg^{2+}$, were determined to enhance the protease activity, whereas $Ba^{2+}$, $Zn^{2+}$, and $Cu^{2+}$ were found to inactivate the enzyme.