Secretory Expression Of Candida Antarctica Lipase B In Aspergillus Niger And Its Application In Diatomite Immobilization

Candida antarctica lipase B?CALB?is a tricylglycerol acyl hydrolyase of strong regionselectivity,stereoselectivity and broad substrate specificity.It is able to play excellent catalytic activity in both aqueous and organic phases.Therefore,CALB played an important role in the field of enantioseparation,pharmaceutical ingredients preparation and organic synthesis.However,commercial CALB is expensive,making the large-scale application with high cost.Recombinant expression of CALB could not yet decrease the cost.In this study,based on the improved protein modification system and strong extracellular protein secretion capacity of Aspergillus niger,the CALB gene was integrated into the genome of A.niger for recombinant expression,and a CALB engineering strain with high enzyme activity was screened out.The enzymatic properties of recombinant CALB and the growth performance of the strain were studied.The diatomite-CALB immobilized enzyme was prepared using diatomite as the carrier and used for the synthesis of ethyl hexanoate.The main results are as follows.?1?Recombinant expression of CALB in A.niger and growth performance of recombinant strain.The PglaA-CALB and PnaII/tpi-CALB expression vector were constructed using promoter PglaA and promoter PnaII/tpi,respectively.After the expression vector was transformed into A.niger,a positive CALB transformant LB3ang02E?CALB?-2with PnaII/tpi as promoter was screened out and its enzyme activity reached 117 U/mL after72 h fermentation.The growth performance of the strain was studied,and addition of 2%glucose to the traditional fermentation medium increased the enzyme activity by 46%?171U/mL?.?2?Purification and enzymatic properties of the recombinant CALB enzyme.The recombinant CALB was purified by nickel column affinity chromatography.After purification,the specific activity reached 32 U/mg with a purification factor of 6.4 fold.SDS-PAGE analysis showed that the molecular weight of the recombinant CALB was 33 kDa,which was consistent with the theoretical values reported in the literature.The optimum reaction temperature and pH of recombinant CALB were 50? and 8.0,respectively.The enzyme was very stable at pH 6.0⁹.0 and below 45?.The activity of CALB was strongly inhibited by10 mmol/L Cu²⁺,Zn²⁺and 0.1%?w/v?SDS,while 1 mmol/L Ca²⁺and 0.1%?w/v?sorbitol played a significant role on activating the enzyme.?3?Immobilization of CALB enzyme using diatomite as the carrier and its application in ester synthesis.The optimum immobilization conditions of 1 g diatomite in 50 mL CALB crude enzyme system were as follows:the amount of enzyme was 70 U/mL,the temperature was 40?,the pH was 7.5-8.0 and the immobilization time was 3 h.The activity of immobilized enzyme reached 187 U/g.The immobilized enzyme was used to catalyze the reaction of ethanol and hexanoic acid to synthesize ethyl hexanoate in solvent-free system.The optimum reaction conditions were as follows:the molar ratio of acid to alcohol was 1:1,the initial water content was 10%⁵0%of hexanoic acid,the amount of immobilized enzyme was 1.2 g,and the reaction temperature was 50?.The yield of ethyl hexanoate reached 91%in 4 h under the optimum condition.After three times of catalytic reaction,the activity of the immobilized enzyme still remains 80%.In conclusion,a CALB expressing strain with high enzyme activity was obtained by using A.niger expression system,and a diatomite-CALB immobilization system was constructed.The high-efficiency esterification of the immobilized enzyme in solvent-free system was completed,which provided data support for improving the recombinant expression level of CALB and its application.