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Simple, fast and high-efficiency transformation system for directed evolution of cellulase in Bacillus subtilis.

Zhang XZ, Zhang Y- - Microb Biotechnol (2011)

Bottom Line: Supercompetent B. subtilis SCK6 cells were prepared by overexpression of the competence master regulator ComK that was induced by adding xylose.Both protein expression level and specific activity of glycoside hydrolase family 5 endoglucanse on regenerated amorphous cellulose were improved through this new system.To our limited knowledge, this study is the first report for enhancing secretory cellulase performance on insoluble cellulose.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Systems Engineering, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA.

ABSTRACT
Bacillus subtilis can serve as a powerful platform for directed evolution, especially for secretory enzymes. However, cloning and transformation of a DNA mutant library in B. subtilis are not as easy as they are in Escherichia coli. For direct transformation of B. subtilis, here we developed a new protocol based on supercompetent cells prepared from the recombinant B. subtilis strain SCK6 and multimeric plasmids. This new protocol is simple (restriction enzyme-, phosphatase- and ligase-free), fast (i.e. 1 day) and of high efficiency (i.e. ~107 or ~104 transformants per mg of multimeric plasmid or ligated plasmid DNA respectively). Supercompetent B. subtilis SCK6 cells were prepared by overexpression of the competence master regulator ComK that was induced by adding xylose. The DNA mutant library was generated through a two-round PCR: (i) the mutagenized DNA fragments were generated by error-prone PCR and linearized plasmids were made using high-fidelity PCR, and (ii) the multimeric plasmids were generated based on these two DNA templates by using overlap PCR. Both protein expression level and specific activity of glycoside hydrolase family 5 endoglucanse on regenerated amorphous cellulose were improved through this new system. To our limited knowledge, this study is the first report for enhancing secretory cellulase performance on insoluble cellulose.

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Related in: MedlinePlus

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Mentions: The transformed cells containing vector pNWP43N‐Bscel5 were spread on LBR plates containing 0.4% regenerated amorphous cellulose (RAC) and then were incubated at 37°C for 20 h. Because secretory BsCel5 can hydrolyse insoluble cellulose on solid agar plates, it resulted in transparent halos (Fig. 4A and B). The bigger and clearer halo zones indicate that the corresponding cells can secrete a more active enzyme and/or secrete a larger quantity of cellulase. Under the tested mutation conditions, about 48.8% of the clones were estimated to harbour the inactivated enzyme mutants. In Fig. 4A, a positive mutant with a bigger and clearer halo zone compared with wild‐type or negative clones was identified from the plate. Three mutants (MT1, MT2 and MT3) with bigger and clearer halo zones were screened from ∼16 000 colonies on about 20 Petri plates.


Simple, fast and high-efficiency transformation system for directed evolution of cellulase in Bacillus subtilis.

Zhang XZ, Zhang Y- - Microb Biotechnol (2011)

© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3815800&req=5

Mentions: The transformed cells containing vector pNWP43N‐Bscel5 were spread on LBR plates containing 0.4% regenerated amorphous cellulose (RAC) and then were incubated at 37°C for 20 h. Because secretory BsCel5 can hydrolyse insoluble cellulose on solid agar plates, it resulted in transparent halos (Fig. 4A and B). The bigger and clearer halo zones indicate that the corresponding cells can secrete a more active enzyme and/or secrete a larger quantity of cellulase. Under the tested mutation conditions, about 48.8% of the clones were estimated to harbour the inactivated enzyme mutants. In Fig. 4A, a positive mutant with a bigger and clearer halo zone compared with wild‐type or negative clones was identified from the plate. Three mutants (MT1, MT2 and MT3) with bigger and clearer halo zones were screened from ∼16 000 colonies on about 20 Petri plates.

Bottom Line: Supercompetent B. subtilis SCK6 cells were prepared by overexpression of the competence master regulator ComK that was induced by adding xylose.Both protein expression level and specific activity of glycoside hydrolase family 5 endoglucanse on regenerated amorphous cellulose were improved through this new system.To our limited knowledge, this study is the first report for enhancing secretory cellulase performance on insoluble cellulose.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Systems Engineering, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA.

ABSTRACT
Bacillus subtilis can serve as a powerful platform for directed evolution, especially for secretory enzymes. However, cloning and transformation of a DNA mutant library in B. subtilis are not as easy as they are in Escherichia coli. For direct transformation of B. subtilis, here we developed a new protocol based on supercompetent cells prepared from the recombinant B. subtilis strain SCK6 and multimeric plasmids. This new protocol is simple (restriction enzyme-, phosphatase- and ligase-free), fast (i.e. 1 day) and of high efficiency (i.e. ~107 or ~104 transformants per mg of multimeric plasmid or ligated plasmid DNA respectively). Supercompetent B. subtilis SCK6 cells were prepared by overexpression of the competence master regulator ComK that was induced by adding xylose. The DNA mutant library was generated through a two-round PCR: (i) the mutagenized DNA fragments were generated by error-prone PCR and linearized plasmids were made using high-fidelity PCR, and (ii) the multimeric plasmids were generated based on these two DNA templates by using overlap PCR. Both protein expression level and specific activity of glycoside hydrolase family 5 endoglucanse on regenerated amorphous cellulose were improved through this new system. To our limited knowledge, this study is the first report for enhancing secretory cellulase performance on insoluble cellulose.

Show MeSH
Related in: MedlinePlus