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Molecular Cloning and Heterologous Expression of Laccase from Aeromonas hydrophila NIU01 in Escherichia coli with Parameters Optimization in Production.
MedLine Citation:
PMID:  23423657     Owner:  NLM     Status:  Publisher    
Abstract/OtherAbstract:
Prior studies disclosed that Aeromonas hydrophila NIU01 was a biodecolorization and bioelectricity bacterium which was isolated from a cross-strait of Taiwan. However, enzymatic function, laccase, involved in this strain had never been reported. This first attempt is to explore its laccase activity, the molecular cloning and heterologous recombinant expression in Escherichia coli. A full-length novel gene of 1,647 bp, LacA, encoding of 549 amino acids was successfully cloned by polymerase chain reaction. The recombinant pET-15b(+)-NIU-LacA expression was compared in different E. coli strains. By applying Taguchi's L9 in culture optimization, the soluble laccase increased to 22.7 %, in which the conditions were obtained at 22 °C with initial shaking speed at 200 rpm, addition of lactose of 0.2 mM and CuSO(4) of 0.5 mM to the medium, and shaking off while cell mass reached to OD(600nm) of 1.5. NIU-LacA was strongly inhibited by chloride ion. The optimal temperature was 60 °C and the optimum pH for ABTS (2,2'-azino-bis (3-ethylbenzthiazolinesulfonic acid) and 2,6-DMP (2,6-dimethoxyphenol) were pH 2.1 and pH 7.5 which enzymatic activity was 274.6 and 44.8 U/L, respectively. Further study in structural modeling of NIU-LacA showed the C terminal domain was the major variance in the three most closely A. hydrophila strains.
Authors:
I-Son Ng; Xia Zhang; Yu Zhang; Yinghua Lu
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Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2013-2-20
Journal Detail:
Title:  Applied biochemistry and biotechnology     Volume:  -     ISSN:  1559-0291     ISO Abbreviation:  Appl. Biochem. Biotechnol.     Publication Date:  2013 Feb 
Date Detail:
Created Date:  2013-2-20     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  8208561     Medline TA:  Appl Biochem Biotechnol     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Affiliation:
Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, China, yswu@xmu.edu.cn.
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