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Purification, characterization and secondary structure elucidation of a detergent stable, halotolerant, thermoalkaline protease from Bacillus cereus SIU1

Paper ID Volume ID Publish Year Pages File Format Full-Text
34809 45049 2012 9 PDF Available
Title
Purification, characterization and secondary structure elucidation of a detergent stable, halotolerant, thermoalkaline protease from Bacillus cereus SIU1
Abstract

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 °C. 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, β-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 β-pleated structure and a detailed structural composition showed ∼50% β-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 β-content directly corresponded to the altered enzyme activity. The protease combined with detergent was able to destain blood stained cloth within 30 min.

► It is a first report of a thermoalkaline protease of 22 kDa from B. cereus. ► Enzyme stability in broad pH range suggests it as a suitable detergent additive. ► NaCl stability of SIU1 protease paves the way for application in saline conditions. ► Oxidant/detergent stability strengthens the enzyme's claim as laundry additive. ► The secondary structure determination revealed the protease a β-protein. ► Interestingly, the ratio of β-content directly corresponded to protease activity.

Keywords
Thermoalkaline protease; Purification; Oxidants; Detergents; Secondary structure; CD analysis
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Purification, characterization and secondary structure elucidation of a detergent stable, halotolerant, thermoalkaline protease from Bacillus cereus SIU1
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Publisher
Database: Elsevier - ScienceDirect
Journal: Process Biochemistry - Volume 47, Issue 10, October 2012, Pages 1479–1487
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us