Expression Research Of Candida Antarctica Lipase B (CALB) In Pichia Pastoris GS115

Lipases is a special kind of ester bonds hydrolytic enzymes which can efficientlycatalyze water insoluble triglyceride convert into glycerin and fatty acids in oil-waterinterface，the reaction intermediates diglyceride and monoglyceride can be furtherconverted into glycerin and fatty acids. Lipases are widely existed in many animals,plants and microorganisms. Lipases can catalyze a variety of reactions in differentconditions, such as hydrolysis, esterification, transesterification, alcoholysis, andammonolysis reaction,etc. The excellent catalytic properties and characteristicsmaking lipases a great commercial value of enzymes, and has been widely used inmany fields, for example, food processing, leather production, new biologicalmaterials, pharmaceuticals, fine chemicals, detergents and paper making,etc.Candida antarctica lipase B(CALB) is separated from the Antarctic Candida.Compare with other lipases, CALB has many excellent features, so brought to moreand more attention. Due to its special structure, CALB has a very strong catalyticactivity for both water-insoluble and water-soluble substrates, and with a highstereoselectivity in hydrolysis and organic synthesis reactions. So far，CALB isthought to be the best enzyme to catalyze ammonolysis reaction. In addition, CALBhas better thermal stability and broader activity pH range than other lipases, the waterdemand in catalytic reaction is also very small, and still maintain good activity evenin a completely anhydrous solvents. Thus, CALB has a very attractive applicationprospect in biodiesel production, organic synthesis, chiral compounds split, drugproduction, pharmaceutical intermediates and other fields. But the cost of productionand separation of CALB is very high at present, so its large-scale use in industrialproduction is still restricted strictly. However, expression CALB by gene cloning inefficient expression system is a effective way to solve this problem, and studies focused on this aspect has also been sustained.Pichia pastoris expression system is a new eukaryotic expression systemdeveloped in recent years, due to its incomparable advantages such as high expressionand easy purification of heterologous proteins, it’s used more and more in researchand production. A variety of proteins has been successfully expressed in Pichiapastoris. In this study, we explored the expression of complete sequence and the maturepeptide of CALB in Pichia pastoris GS115, respectively. Through gene cloning weconstructed the recombinant plasmid pPICZaA-CALB and pPICZaA-m-CALB whichcontains CALB complete gene and the mature peptide coding gene m-CALB,respectively. Then using electroporation method, the complete gene CALB andm-CALB gene was successfully integrated into the GS115genome, so the Pichiapastoris expression system of CALB complete sequence GS115-pPICZαA-CALB wasconstructed, as well as the mature peptide sequence, GS115-pPICZaA-m-CALB.After optimization, we determined the optimal expression conditions:1%of methanol,fermentation temperature28°C, BMMY pH6.2, BMMY amount of about half ofBMGY. Under these conditions, we successfully expressed CALB complete sequenceand its mature peptide m-CALB in GS115, and using ion affinity chromatographypurified both of them. Our results shows that in early fermentation, the expression rateof CALB mature peptide m-CALB in GS115was slightly faster than that of completeCALB, but after six days of fermentation the expression level of m-CALB isrelatively lower than the complete CALB. We failed to purify CALB mature peptidem-CALB effectively by Ni2+column, the specific reasons are not clear yet.In summary, this essay explored the expression and purification of completeCALB sequence in Pichia pastoris GS115under the condition of pPICZαA as thevector, as well as its mature peptide m-CALB. We hope our works could be useful forthe future research of CALB efficient expression and purification.