Expression and characterization of a recombinant γ-cyclodextringlycosyltransferase from Bacillus clarkii 7364
The γ-CGTase gene sequence from Bacillus clarkii 7364 was optimized according to the biased condons of E.coli and its prokaryotic expression strain was constructed.The condition of expression of γ-CGTase and the purification method of γ-CGTase was established,and the catalytic characterization of γ-CGTase was studied.The γ-CGTase achieved highly soluble expression at 28 ℃ and the soluble expression protein accounts for 63% of the total protein,the enzyme activity reached 3 830 U/mL in the periplasm.The recombinant γ-CGTase was purified by ammonia sulfate precipitation,followed by affinity chromatography on a α-CD-Sepharose 6B affinity column,and the purification fold and yield was 12.97 and 20.31%,separately.After incubation with cassava starch,the majority (90.9%) of product CDs was γ-CD that was about 15% higher than the wild γ-CGTase,and nearly no α-CD was detected.The engineering strain was cultivated in 20 L fermentor and γ-CGTase activity could reach 4 375 U/mL,which provided the possibility for further industrialization.The recombinant γ-CGTase showed very high specificity of transformation and would have better perspective in industrialization.
γ-cyclodextrin glycosyltransferaseBacillus clarkiisoluble expressionpurificationspecificity of products
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