Purification and characterization of a halotolerant serine proteinase from thermotolerant Bacillus licheniformis RKK-04 isolated from Thai fish sauce

Toyokawa, Yoichi; Takahara, Hiroaki; Reungsang, Alissara; Fukuta, Masakazu; Hachimine, Yuki; Tachibana, Sanro; Yasuda, Masaaki

doi:10.1007/s00253-009-2434-5

Cited by 49 publications

(28 citation statements)

References 30 publications

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“…Halotolerant proteases have been screened and isolated from various micro-organisms such as Bacillus clausii (Joo and Chang, 2005), B. subtilis strain FP-133 (Setyorini et al 2006), B. cereus MTCC 6840 (Joshi et al 2007), B. licheniformis BA17 (Nikerel et al 2008), B. aquimaris strain VITP4 (Shivanand and Jayaraman 2009) and B. licheniformis RKK-04 (Toyokawa et al 2010) in recent years.…”

Section: Discussionmentioning

confidence: 99%

Biosynthesis optimization and purification of a solvent stable alkaline serine protease from Halobacterium sp.

Vijayaraghavan

Jebamalar²,

Vincent

2011

Ann Microbiol

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Three halotolerant bacterial isolates that were isolated from the salt pan of coastal Tamilnadu, India, were screened on skimmed milk agar plates containing 1-25% (w/v) NaCl for protease secretion. Among the three positive isolates, Halobacterium sp. showed the maximum protease secretion (4.5 mm diameter zone) and was hence selected for this study. The enzyme was a serine protease as evidenced by complete inactivation of enzyme after treatment with a serine protease inhibitor (phenylmethylsulfonyl fluoride). Culture conditions which optimize protease production that included pH, fermentation period, NaCl in addition to different factors such as carbon and nitrogen sources, casein concentration were investigated. Optimum bacterial growth and proteolytic activity was achieved after 48 h of incubation at 37°C, pH 10.0 with 5% (w/v) NaCl concentration and 2.5% (w/v) casein. Initial experiments revealed that the culture medium containing 1.0% (w/v) lactose as the carbon source and 1.0% (w/v) ammonium chloride as the nitrogen source supported maximized enzyme production. Analysis carried out using SDS-PAGE revealed that the extracellular enzyme consisted of a single polypeptide chain of 43 kDa which was purified by a combination of ammonium sulphate fractionation, DEAE cellulose and Sephadex G-75 gel filtration chromatography. This protease was stable for a wide range of temperatures (40-60°C), NaCl concentrations (1-5%, w/v) and pH (8.0-10.0) with maximal activity observed at 60°C, 5% NaCl and pH 10.0. Proteolytic activity was enhanced by 1.0 mM of Ca 2+ (137%) and strongly inhibited by Mn 2+ (67%). Most importantly, the proteolytic activity remained stable in alkaline pH and was found to be active at higher temperatures. The broad range of temperature optima with respect to enzyme activity was indicative of this enzymes suitability in fish sauce fermentation, which was carried out at 35-45°C. This enzyme was stable in organic solvents such as toluene and hexane and the relative enzyme activity was 103 and 114%, respectively, which mainly finds use in the paint industry.

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Section: Discussionmentioning

confidence: 99%

Biosynthesis optimization and purification of a solvent stable alkaline serine protease from Halobacterium sp.

Vijayaraghavan

Jebamalar²,

Vincent

2011

Ann Microbiol

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“…A similar result was reported with B. subtilis RTSBA6.00 and Bacillus sp. (Toyokawa et al, 2010;Haile and Gessesse, 2012 (Vijayaraghavan et al, 2012). The alkaline protease digested the gelatine layer of the used X-ray film and removed the silver ions from the X-ray film ( Fig.…”

Section: Optimization and Validationmentioning

confidence: 99%

Statistical Approach for the Production and Partial Characterization of Alkaline Stable Protease from a Newly Isolated Bacillus sp. IND6 for Silver Recovery

Vijayaragh¹,

Kalaiyaras²,

Prakash³

2015

Research J. of Microbiology

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A low-cost culture medium for the production of bacterial alkaline protease was developed after response surface methodological approach. Central composite design was applied to optimize the concentration of the three significant factors, namely pH, sucrose and yeast extract. A second-order model equation was obtained and then validated experimentally. The model adequacy was highly satisfactory as the coefficient of determination was 0.98. The maximum enzyme production was 2213 U mLG 1 after 72 h of incubation, which showed about fivefold increase in enzyme production than unoptimized medium. The partially purified enzyme showed optimum activity at 50°C at pH 9.0. Crude enzyme exhibited a significant stability with most of the tested commercial laundry detergents. This enzyme removed the gelatin layer of used X-ray film effectively. Considering its promising properties, Bacillus cereus IND6 enzymatic preparations may be considered a potential candidate for detergent applications and silver recovery.

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“…그러므로 혈전분해 를 위한 연구가 다양한 방법으로 진행되고 있으며 특히, 미생 물 유래 혈전분해효소를 이용한 혈전분해특성을 규명하는 연 구가 지속적으로 수행되고 있다 [4,[7][8][9]21]. 미생물 유래 효소를 이용한 연구에 있어서 미생물에 의한 효율적인 효소 생산은 무엇보다도 중요한 연구분야라고 할 수 있으며 미생물 유래 혈전용해효소에 관한 연구에 있어서는 효소특성 규명에 관한 연구는 많은 보고가 이루어지고 있지만 효율적인 효소 생산을 위한 연구는 반드시 선행되어야 할 중요한 연구분야임에도 불구하고 현재까지 연구 보고가 매우 미흡한 실정이다.…”

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Effect of Tryptic Soy Broth (TSB) and Luria-Bertani (LB) Medium on Production of Subtilisin CP-1 from Bacillus sp. CP-1 and Characterization of Subtilisin CP-1

Park

2012

Journal of Life Science

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A bacterial strain producing a fibrinolytic enzyme, subtilisin CP-1, was isolated from Doen-Jang, a Korean traditional fermentation food. Based on the analysis of gene sequence of 16S rRNA and biochemical analysis, the strain was identified as Bacillus sp. and named as Bacillus sp. CP-1. To investigate the effect of the medium on the production of fibrinolytic enzyme from Bacillus sp. CP-1, two commercial bacterial culture media, tryptic soy broth (TSB) and Luria-Bertani (LB), were applied to the cultivation of Bacillus sp. CP-1. The strain secreted only one proteolytic enzyme (subtilisin CP-1) in the culture broth. The molecular weight of subtilisin CP-1 was estimated to be 28 kDa. Subtilisin CP-1 was optimally active at pH 9.0 and 45 o C, and exhibited high specificity for Meo-Suc-Arg-ProTyr-pNA (S-2586), a synthetic chromogenic substrate for chymotrypsin. The first eight amino acid residues of the N-terminal sequence of the enzyme are AQSVPYGI; this sequence is identical to that of subtilisin NAT and E.

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Purification and characterization of a halotolerant serine proteinase from thermotolerant Bacillus licheniformis RKK-04 isolated from Thai fish sauce

Cited by 49 publications

References 30 publications

Biosynthesis optimization and purification of a solvent stable alkaline serine protease from Halobacterium sp.

Biosynthesis optimization and purification of a solvent stable alkaline serine protease from Halobacterium sp.

Statistical Approach for the Production and Partial Characterization of Alkaline Stable Protease from a Newly Isolated Bacillus sp. IND6 for Silver Recovery

Effect of Tryptic Soy Broth (TSB) and Luria-Bertani (LB) Medium on Production of Subtilisin CP-1 from Bacillus sp. CP-1 and Characterization of Subtilisin CP-1

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