• Microbiology and Biotechnology Letters
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• v.8 no.2
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• pp.77-85
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• 1980

This experiment was conducted to investigate the conditions for production and the characteristics of pretenses. Aspergillus oryzae KC-15, which is selected as a superior strain for the production of the protease, was used in this study. The results obtained were as follows: 1. The optimum culture time for the production of acid, neutral and alkaline protease on wheat bran medium were about 48, 48 and 72hr, respectively. The protease-produced by the strain were mainly alkaline and neutral one, but the production of acid protease was feeble extremely. 2. The addition of NaH$_2$PO$_4$, Na$_2$HPO$_4$, glucose, rice powder and Na-glutamate respectively to wheat bran media were effective for the production of alkaline and neutral protease, and the addition of (NH$_4$)$_2$HPO$_4$, glucose and rice powder respectively were effective for the production of acid protease. 3. Characteristics of professes(equation omitted) 4. As a heat resistance agent, NaH$_2$PO$_4$was the most effective one. The optimum amount of NaH$_2$PO$_4$was 10mg for alkaline and neutral protease, and 5mg for acid protease. 5. The heat resistance of the Protease by NaH$_2$PO$_4$was not recognized mostly above 6$0^{\circ}C$. 6. After the treatment of enzyme solution with 10mg of NaH$_2$PO$_4$for 30 minutes at 55$^{\circ}C$, the residual activities measured for alkaline, neutral and acid protease were 58, 57 and 55％ respectively.

• Korean Journal of Microbiology
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• v.18 no.2
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• pp.51-58
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• 1980

Mutational experiments were performed to improved to improve the protease productivity of Aspergillus flavus KU 153, which is selected among the wild strains. A UV-induced mutant strain having high protease productivity was obtained by the use of the clear zone method as a simple criterion for a primary screening test. Neutral and alkaline protease activities of hte mutant strain were higher than 1.8 times, comopared with those of the parental strain, respectively, while in the case of acid protease, it was 2.7 times. The mutant strain selected was more powerful in the production of cellulase and amylase, as well s protease in wheat bran, compared with those of the parental strain. protease production of the parental strain has reached maximum level at 3 days culture, while alkaline nad neutral protease production of the mutantstrain has reached at 2 days culture. On the other hand, the mutant strain formed the spore slowly, compared with the parental strain. Column chromatography of the neutral protease on DEAE-Sephadex A-50 showed that the mutant strain was not induced the formation of another neutral protease isozyme, but induced the variation in the function of regulatory gene.

• Microbiology and Biotechnology Letters
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• v.19 no.2
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• pp.116-121
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• 1991

A bacterial strain KN, which highly produced a protease, was isolated from several soil samples and identified to to belong to the genus Bacillus. We selected mutant strain Bacillus sp. KN103N, which was hyperproducer of protease and was resistant to D-cyclowerine, from the strain KN by several steps of mutagenesis. Neutral protease productivity of mutant strain KN103N was about 55 times as much as that of the original strain KN. The optimum pH and temperature for the enzyme activity were 7.0 and 50$^{\circ}C$, respectively and the enzyme was relatively stable at pH6.0~8.0 and below 40$^{\circ}C$. The enzyme was inactivated by EDTA, but not by DFP. These results indicate that the enzyme from Bacillus sp. KN103N was a neutral (metallo-) protease.

• Microbiology and Biotechnology Letters
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• v.30 no.4
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• pp.346-351
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• 2002

A bacterium producing the neutral pretense was isolated from soil, and was identified as Bacillus sp. DS-1 by 16S rRNA sequence comparison and biochemical determinations. The production of protease from Bacillus sp. DS-1 was increased 20% and 30% by the additions of 1% glucose and 1% yeast extract, respectively. The optimum pH and temperature for the protease activity were pH 7.0 and 55$^{\circ}C$. Bacillus sp. DS-1 produced a metalloprotease as a major protease in culture medium, since the pretense activity in culture supernatant was inhibited by the presence of 1 mM EDTA significantly.

• Korean Journal of Microbiology
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• v.9 no.4
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• pp.163-168
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• 1971

Neutral protease which was obtained from a genus of Aspergilli as the crystal form were investigated for their purification and properties. The results of biochemical and enzymatic studies for their purification and properties in this enzyme were as follows. 1) On the wheat media containing 70%-water and $CaCo_{3}$, Aspergilus oryzae S.H.W. 131 is satisfactorily grown under the basic optimum conditions temperature $27^{\circ}C$- $30^{\circ}C$at relative humidity 100% for three days. 2) The enzyme solution extracted with water is successively purified through the passing on column of Asmti-177N for decolorization of it. And ion exchanger such as DEAAE Sphadex A-50 or Shepadex G-100 and fraction collector is necessary for the sepearte treatments of this enzyme. After washing it with organic solvents as aceton-EtOH, etc., it should be dried on the vacuum dryer at $40^{\circ}C$) The protease activity is determined by the amounts of amino acids, tyrosine. 4) The optimum pH of neutral protease is 6.0-8.0. 5) In effectively decomposing with this neutral protease, the optimum temperature is $35^{\circ}C$. 6) It is interesting that the amounts of metal ion affects the activity of neutral protease. For examples, if it were treated with manganic ion, its activity would be more effective than any other that.

• Journal of Microbiology and Biotechnology
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• v.10 no.1
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• pp.103-106
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• 2000

Bacillus cereus KCTC 3674 excretes at least two kinds of extracellular proteases into the growth medium. Two major bands of the protease activity with molecular weights of approximately 100 and 38 kDa were obtained after gelatin-SDS-PAGE. The protease with a molecular weight of 38kDa was identified as an extracellular neutral (metallo-) protease. The neutral protease was quite thermostabile but labile to alkaline pH. On the contrary, the 100-kDa protease was thermolabile but stable to alkaline pH. The production of 38-kDa neutral protease was strongly affected by temperature, oxygen, carbonylcyanied m-chlorophenylhydrazone(CCCP) that was defined as a protonophofre, and cerulenin which inhibited lipid synthesis and caused changes in the membrane composition. On the other hand, the production of the 100-kDa protease was strongly affected by only temperature and cerulenin. Quinacrine (0.2 mM), which inhibits the penicillinase-releasing proteases of Bacillus licheniformis, had no effect, whatsoever, on the production of extracellular proteases in B.cereus KCTC 3674.

• Korean Journal of Microbiology
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• v.10 no.2
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• pp.69-72
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• 1972

The studies of neutral protease which was obtained by passing through Sephadex A-50 had been reported not long ago. Since that time the author also conducted the research to be investigated the physical properties of acid protease absorbed by Sephadex A-50. The results are summarized as follows : 1) Cultivating Aspergillus oryza SHW-131 on a wheat bran medium, the acid protease including neutral protease is very sensitive for temperature. 3) Activity of acid protease is very sensitive for temeprature. 3) This enzyme was proved, what is called, to be a sort of weak acid protease. It's optimum pH was lied in about 4.5. 4) A range of pH for stability is far more narrow than any other protease. 5) The acid protease is dropped by EDTA solution in its activity.

• The Korean Journal of Zoology
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• v.32 no.1
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• pp.40-47
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• 1989

Alteration of intracellular calcium ion Concentration by adding of either calcium ionophore A23187 or EGTA in culture medium at 24 hr after cell plating resulted in remarkable changes in the progression of differentiation of chick embryo myoblast. When separated myoblast proteins using two-dimensional gel electrophoresis, synthesis patterns of several proteins changed upon the addition of either A23187 or EGTA. Treatment of A23187 and calciumactivated neutral protease at 24 hr after initial plating caused an increase in the rate of fusion compared to control culture. However, EGTA inhibited the myoblast fusion to a marked degree. A23187 treated at 24hr also increased the activity of protein kinase C during the fusionprogressed period. It seems that intracellular calcium ion plays an important role in the myoblast differentiation in vitro together with the protein kinase C and calcium-activated neutral protease.

• Fisheries and Aquatic Sciences
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• v.3 no.3_4
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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.

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