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Gene. 2015 Jul 10;565(2):221-7. doi: 10.1016/j.gene.2015.04.018. Epub 2015 Apr 10.

Screening, cloning and expression analysis of a cellulase derived from the causative agent of hypertrophy sorosis scleroteniosis, Ciboria shiraiana.

Author information

1
The State Key Laboratory of Silkworm Genome Biology, College of Biotechnology, Southwest University Chong Qing, People's Republic of China. Electronic address: lvruihua1987@163.com.
2
The State Key Laboratory of Silkworm Genome Biology, College of Biotechnology, Southwest University Chong Qing, People's Republic of China.
3
The State Key Laboratory of Silkworm Genome Biology, College of Biotechnology, Southwest University Chong Qing, People's Republic of China. Electronic address: yumd@163.com.

Abstract

A cellulase gene (KJ700939, CsCelA) from Ciboria shiraiana that is highly expressed during the infection of mulberry fruit was screened by quantitative real-time PCR (qRT-PCR). Using cDNA isolated from infected mulberry fruits as template, the full-length 1170-bp sequence of CsCelA was obtained, which encodes a 390-amino acid protein with a putative signal peptide of 24 amino acids. The 998-bp fragment encoding the mature peptide of CsCelA was cloned into the multiple cloning site of the pPIC9K vector and overexpressed as an active protein of 55.3kDa in the methylotrophic yeast Pichia pastoris. The specific activity of induced supernatants of the recombinant cellulase (CsCelA) was 17.44U/ml and 135U/g for freeze-dried powder. The Kmax and Vmax of CsCelA for sodium carboxymethylcellulose (CMC) were 4.6mg/ml and 107.2U/mg, respectively. The supernatant and freeze-dried powder of the recombinant cellulase exhibited stable activity from pH4.0 to 9.0, and at temperatures ranging from 30°C to 55°C. Finally, the activity of the recombinant cellulase was assessed by enzymatic hydrolysis of the cell walls of mulberry leaves. CsCelA showed an endo-cellulase mode of cleavage, as assessed by thin layer chromatography (TLC).

KEYWORDS:

Activity; Cellulase; Ciboria shiraiana; Expression; Pichia pastoris

PMID:
25865297
DOI:
10.1016/j.gene.2015.04.018
[Indexed for MEDLINE]

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