Purification, characterization and secondary structure elucidation of a detergent stable, halotolerant, thermoalkaline protease from Bacillus cereus SIU1
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- 作者：Sanjay Kumar Singh ; Santosh Kumar Singh ; Vinayak Ram Tripathi ; Satyendra Kumar Garg
- 关键词：Thermoalkaline protease ; Purification ; Oxidants ; Detergents ; Secondary structure ; CD analysis
- 刊名：Process Biochemistry
- 出版年：2012
- 出版时间：October, 2012
- 年：2012
- 卷：47
- 期：10
- 页码：1479-1487
- 全文大小：1201 K
文摘
A thermoalkaline protease with a molecular weight of 22 kDa was purified from the Bacillus cereus SIU1 strain using a combination of Q-Sepharose and Sephadex G-75 chromatography. The kinetic analyses revealed the Km, Vmax and kcat to be 1.09 mg ml?1, 0.909 mg ml?1 min?1 and 3.11 s?1, respectively, towards a casein substrate. The protease was most active and stable at pH 9.0 and between a temperature range of 45-55 ¡ãC. It was fully stable at 0.0-2.0 % and moderately stable at 2.5-10.0 % (w/v) sodium chloride. Phenyl methyl sulfonyl fluoride, ethylene diamine tetra acetic acid and ascorbic acid were inhibitory with regard to enzyme activity, whereas cysteine, ¦Â-mercaptoethanol, calcium, magnesium, manganese and copper at concentration of 1.0 mM increased enzyme activity. Sodium dodecyl sulfate, Triton X-100, Tween 80, hydrogen peroxide and sodium perborate significantly enhanced protease activity at 0.1 and 1.0 % concentrations. In the presence of 0.1 and 1.0 % (w/v) detergents, the protease was fairly stable and retained 50-76 % activity. Therefore, it may have a possible application in laundry formulations. An initial analysis of the circular dichroism (CD) spectrum in the ultraviolet range revealed that the protease is predominantly a ¦Â-pleated structure and a detailed structural composition showed ¡«50 % ¦Â-sheets. The CD-based conformational evaluation of the protease after incubation with modulators, metal ions, detergents and at different pH values, revealed that the change in the ¦Â-content directly corresponded to the altered enzyme activity. The protease combined with detergent was able to destain blood stained cloth within 30 min.