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Gene cloning, expression, purification and characterization of rice (Oryza sativa L.) class II chitinase CHT11

Paper ID Volume ID Publish Year Pages File Format Full-Text
17991 42709 2008 6 PDF Available
Title
Gene cloning, expression, purification and characterization of rice (Oryza sativa L.) class II chitinase CHT11
Abstract

Class II chitinase CHT11 is one of the 12 chitinases identified in rice (Oryza sativa L.). In order to elucidate its enzymatic properties, the Cht11 gene was cloned and overexpressed in Escherichia coli BL21 cells as a glutathione-S-transferase (GST) fusion protein. The optimal culture temperature and the optimal time after induction were found to be 16 °C and 12 h, respectively. In the process of purification, a non-specific 70-kDa protein bound to the fusion protein was successfully removed from recombinant CHT11 by adding 3 mM ATP-Mg in wash buffer. In the culture conditions and purification procedures presented in this study, the final yield of recombinant CHT11 from 800 ml of the culture supernatant was 4.1 mg with 11.5-fold purification. The optimal temperature and pH of recombinant CHT11 were determined to be 35–40 °C and 5.5–6.5, respectively. It inhibited the hyphal growth of both avirulent and virulent strains of Trichoderma viride at 100 and 300 μg in medium, but the growth inhibition zone was subtle even when 300 μg of protein was applied. This indicates that the antifungal activity of recombinant CHT11 is considerably lower than other chitinases.

Keywords
Adenosine 5′-triphosphate (ATP); Antifungal activity; Chitinase; Glutathione-S-transferase (GST) fusion protein
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Gene cloning, expression, purification and characterization of rice (Oryza sativa L.) class II chitinase CHT11
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 43, Issue 1, 7 July 2008, Pages 19–24
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