• Korean Journal of Microbiology
• /
• v.28 no.2
• /
• pp.162-168
• /
• 1990

The aims of the present study were to evaluate the effects of culture conditions on the biosynthesis of extra-cellular alkaline protease in Streptomyces sp. The formation of aerial mycelia and spores were compared with the protease production in order to know the relations between the alkaline protease and the cell differentiation. As results, it was found that substrate concentration was very critical to regulate the formation of the protease, aerial mycelia, and spores, which were resulted from the changes of culture pH to acid. When the culture pH was adjusted with phosphate buffer from pH 6 to pH 9, the alkaline protease production was increased as the culture pH increased whereas aerial mycelia and spore formation were reversely related to the culture pH. Therefore, it was thought that the culture pH was an important factor to regulate the alkaline protease synthesis.

• Korean Journal of Microbiology
• /
• v.24 no.1
• /
• pp.32-37
• /
• 1986

In fermentation studies it revealed that Streptomyces sp. SMF 3001 started to synthesize extracellular alkaline protease from early exponential phase of cell growth. The biosynthesis of the alkaline protease was greatly induced by skim milk as a sola nitrogen source and further stimulation was observed under inorganic sulphur limited culture. However, it was found that the biosynthesis was apparently repressed by $NH_4^+$ and free amino acids, specially by cysteine. It was considered that the strain SMF 301 of Streptomyces sp. would produce the alkaline protease for the uptake of sulphur compounds from protein contained in the culture broth.

• Journal of Microbiology
• /
• v.33 no.2
• /
• pp.115-119
• /
• 1995

The alkaline protease of Xanthomonas sp. YL-37 has been purified, and the properties of the enzyme investigated. The alkaline protease of Xanthomonas sp. YL-37 was purified form crude enzyme by ammonium sulfate fractionation, CM-cellulose ion exchange chromatography, and Sephadex G-100 gel filtration. Through the series of chromatographies, the enzyme was purified to homogenecity with specific activity of 4.23 fold higher than that of the crude broth. The molecular weight of the purified protease has been estimated to be 62 KDa on SDS-polyacrylamide gel electrophoresis. The optimal pH and temperature for alkaline protease activity were 11.0 and 50.deg.C, respectively. The enzyme was stable between pH 5.0 and 10.0 and up to 50.deg.C. Enzyme activity was lost up to 50% on heating at 70.deg.C for 30 minutes. The activity of alkaline protease was inhibited by Cu$\^$2+/, Zn$\^$2+/, Hg$\^$2+/, PMSF, and activated by Mn$\^$2+/ and Ca$\^$2+/. The $K_{m}$ value for casein as a substrate was 4.0 mg/ml.

• Journal of Microbiology and Biotechnology
• /
• v.33 no.2
• /
• pp.195-202
• /
• 2023

Protease is a widely used enzyme particularly in the detergent industry. In this research, we aimed to isolate alkaline protease-producing bacteria for characterization as a laundry detergent additive. The screening of alkaline protease production was investigated on basal medium agar plus 1% skim milk at pH 11, with incubation at 30℃. The highest alkaline protease-producing bacterium was 6BS15-4 strain, identified as Bacillus gibsonii by 16S rRNA gene sequencing. While the optimum pH was 12.0, the strain was stable at pH range 7.0-12.0 when incubated at 45℃ for 60 min. The alkaline protease produced by B. gibsonii 6BS15-4 using dairy effluent was characterized. The optimum temperature was 60℃ and the enzyme was stable at 55℃ when incubated at pH 11.0 for 60 min. Metal ions K⁺, Mg²⁺, Cu²⁺, Na⁺, and Zn²⁺ exhibited a slightly stimulatory effect on enzyme activity. The enzyme retained over 80% of its activity in the presence of Ca²⁺, Ba²⁺, and Mn²⁺. Thiol reagent and ethylenediaminetetraacetic acid did not inhibit the enzyme activity, whereas phenylmethylsulfonyl fluoride significantly inhibited the protease activity. The alkaline protease from B. gibsonii 6BS15-4 demonstrated efficiency in blood stain removal and could therefore be used as a detergent additive, with potential for various other industrial applications.

• Journal of Microbiology and Biotechnology
• /
• v.21 no.12
• /
• pp.1250-1256
• /
• 2011

In this study, Bacillus licheniformis cells were immobilized by entrapment in calcium alginate beads and were used for production of alkaline protease by repeated batch process. In order to increase the stability of the beads, the immobilization procedure was optimized by statistical full factorial method, by which three factors including alginate type, calcium chloride concentration, and agitation speed were studied. Optimization of the enzyme production medium, by the Taguchi method, was also studied. The obtained results showed that optimization of the cell immobilization procedure and medium constituents significantly enhanced the production of alkaline protease. In comparison with the free-cell culture in pre-optimized medium, about 7.3-fold higher productivity was resulted after optimization of the overall procedure. Repeated batch mode of operation, using optimized conditions, resulted in continuous production of the alkaline protease for 13 batches in 19 days.

• Microbiology and Biotechnology Letters
• /
• v.6 no.1
• /
• pp.9-16
• /
• 1978

Effects of the addition of Ginseng extract and it's incubation time on enzyme activity (${\alpha}$-amylase, ${\beta}$-amylase, protease) of Aspergillus app. was investigated. 1. ${\alpha}$-Amylase activity of Asp. oryzae was higher for 48 hours than control, however, the same after 48 hours. ${\beta}$-Amylase activity was stimulated at the concentration of 0.1 to 1.0％, and decreased at the concentration of 3.0 to 5.0％. The acid protease activity was higher than control for 72 hours and in the medium of 120 hours was decreased significantly. The alkaline protease activity was lower than control. However, alkaline protease activity was higher than acid protease activity. 2. ${\alpha}$-Amylase of Asp. niger was increased in proportion to increasing of the extract concentration. ${\beta}$-Amylase was increased at the concentration from 0.5 to 3.0％ and it's activity was depressed in proportion to increasing of the extract concentration for 48 hours and on the contrary it was improved in proportion to increasing of the extract concentration for 72 hours except for 5.0％ concentration with marked decreasing. Alkaline protease activity was increased at lower concentration (0.1∼0.5％) for 24 to 48 hours and it was depressed at higher concentration (1.0 to 5.0), however after 48 hours incubated, it's activity was decreased in preparation to increasing of the extract concentration.

• Journal of Microbiology and Biotechnology
• /
• v.11 no.4
• /
• pp.558-563
• /
• 2001

A new alkalophilic strain of Bacillus pumilus JB¬05 producing bleach-stable and halo-tolerant alkaline protease was isolated from cement industry effluents in Karnataka, India. The effects of carbon and nitrogen sources on protease production by this alkalophilic strain were observed after a 30-h incubation. A high level of alkaline protease activity was obtained in the presence of starch as the carbon and peptone as the nitrogen sources. The partially purified enzyme showed an optimum temperature and pH activity at $58^{\circ}C$ and 10.5, respectively. The enzyme was completely inhibited by PMSF (95.0%) indicating it as a serine protease. It is bleach-stable as it retained 35% original activity in the presence of 10% (v/v) hydrogen peroxide at $30^{\circ}$C after 2 h and is halo-tolerant as it retained 70% original activity in the presence of 2.5 M sodium chloride at $30^{\circ}C$ after 2 h incubation.

• Mycobiology
• /
• v.32 no.2
• /
• pp.74-78
• /
• 2004

An alkaline protease produced by Aspergillus niger C-15 was purified and characterized. The enzyme was purified 19.41-fold with a specific activity of 74150 U/mg and a recovery of 34.4% by gel filtration and ion exchange chromatography. The molecular weight of the enzyme was estimated to be 34 kDa. The optimum pH and temperature for the protease activity were pH 8.0 and $60^{\circ}C$, respectively. The enzyme activity inhibited by EDTA suggests that the preparation contains a metalloprotease. The enzyme activity of the metalloprotease was completely inhibited by 5 mM $HgCl_2$ and $FeCl_3$, while partially inhibited by $CuSO_4$, and $MnCl_2$. When polyols such as glycerol, mannitol, sorbitol and xylitol, were added to the reaction medium, most polyols tested enhanced protease activity. Especially, glycerol showed the highest effect. The alkaline metalloprotease was stable at high temperature and retained more than 90% of the initial activity at $60^{\circ}C$ and 86.4% under addition of glycerol.

• Microbiology and Biotechnology Letters
• /
• v.17 no.4
• /
• pp.358-364
• /
• 1989

A crystalline alkaline pretense- producing Streptomyce sp. YSA-130 was isolated from soil in alkaline medium(pH 10.5). The optimum culture condition of Streptomyce sp. YSA-130 for the production of alkaline protease was as follows; 2.0% soluble starch, 1.0% soytone, 0.3% $K_2$HPO$_4$, 0.02% MgSO$_4$.7$H_2O$, 0.8% Na$_2$CO$_3$, pH 10.5, 3$0^{\circ}C$, and 12 hr. The alkaline pretense from the culture broth of Streptomyce sp. YSA-130 was purified about 24 folds by ammonium sulfate precipitation , dialysis, DEAE-cellulose ion exchange chromatography, gel filtration on Sephadex G-15 and crystallization. Optimum temperature and pH of purified enzyme were 6$0^{\circ}C$, and 11.5. Temperature and pH stability of purified enzyme were 5$0^{\circ}C$, and 5.5-12.0. Calcium ion was effective to stabilize the enzyme at higher temperature. The molecular weight of the purified enzyme was approximately 30,000. The purified enzyme was inactivated by diisopropyl flurophosphate(DFP) but not affected by metal ion, EDTA, sulfhydryl reagent and stable detergent.

• Microbiology and Biotechnology Letters
• /
• v.6 no.1
• /
• pp.33-40
• /
• 1978

Immobilization of alkaline protease was investigated by absorbing the enzyme on adsorbents. Alkaline protease was adsorbed on silica gel selected as a carrier to immobilize the enzyme. In this study, properties of the immobilized enzyme were compared with those of the soluble enzyme. 1) The optimum pH (10.0) of the enzyme was not changed, but the activity was increased at alkaline pH by immobilization. 2) The optimum temperature of the immobilized enzyme was shifted from 50$^{\circ}C$ to 45$^{\circ}C$, while the temperature-activity Profile became broader than those of the soluble enzyme. 3) The pH stability of the immobilized enzyme was significantely increased at pH 4.0, althouth it did not change in the neutral and alkaline pH region. 4) The heat stability of the enzyme was enhanced in the temperature range of 55$^{\circ}C$∼65$^{\circ}C$ by the immobilization. 5) The immobilized enzyme retained 40％ of its original activity after repetitive use for 6 times. 6) The enzyme stability was greately improved for a prolonged storage at 4$^{\circ}C$.