Rational Design And High Expression Of Candida Antarctica Lipase B

Lipase B from Candida antarctica(CALB)shows high catalytic activity in both aqueous and non-aqueous systems.It is widely used in the range of organic synthesis,food processing and biodiesel preparation,etc.In order to further improve the enzymatic characteristics of CALB and enhance its expression level,and to extend its application fields,the rational design on CALB was conducted in this study.A molecular modified lipase CALB with higher activity and higher thermo stability was obtained,and its high-level expression in Pichia pastoris strain was achieved.The main works were conducted as following:Firstly,the effect of signal peptides from the lipase CALB itself and α-factor on the secretory expression of CALB was compared.It was found that compared with the wild type signal peptide of CALB,the α-factor signal peptide could much more efficiently improve the secretory expression of CALB,and with a 27% higher lipase activity.In order to further improve the activity and thermo stability of CALB,the rational design of CALB was conducted basing on the 3-D structure of CALB.After bioinformational analysis,a series of amino acid sites of CALB were selected and mutated.The sites were listed as: T105 A,N110Y,M154 V,A173G,L188 F,V260A.The mutated lipase gene calb2 a was obtained.Compared with the wild-type CALB,the expression level of lipase CALB2 a was improved significantly,with the hydrolysis activity increased by 794.4%.Moreover,the optimum reaction temperature of mutant lipase CALB2 a was increased to 65℃.After being incubated at 50℃ for 3 hours,it still kept 75% enzyme activity.The designed lipase gene calb2 a was cloned into p AO815,and the plasmid carrying1-to 4-copy of expression cassettes of calb2 a were constructed.Plasmids were then transformed into P.pastoris,to obtain the yeast recombinants carrying 1-to 4-copy of calb2 a gene.Compared with 1-copy recombinants,the activity of 4-copy recombinant was increased by 139.3%.Under the high-density fermentation in a 50-L fermentor,after 168 h inducible cultivation,the enzyme activity in the supernatant reached 3200 U/ml.In this study,a molecular modified lipase CALB with higher activity and thermo stability was obtained after rational design.The successful high-level expression has also facilitated its further industrial produce and application.