Modification And Enzymatic Properties Analysis Of Lipase From Penicillium Cyclopium

Lipases (triacylglycerol hydrolases, EC 3.1.1.3) can catalyze the hydrolysis of triacylglycerols to yield free fatty acids, diacylglycerols, and monoacylglycerols at the oil-water interface and have a function of the synthesis of lipids by transesterification,esterification, and alcoholysis in organic solvents. So far, lipases have been widely used in industrial applications including food, detergent, papermaking, and textile. Microbial lipases have advantages of short production cycle and high-yield, however, most of wild-type lipases showed poor thermostability, which limited them to application in industrial processes with higher temperatures. Based on these reasons, Penicillium cyclopitum alkaline lipase I (PCL) was chcosen to construct the mutant PCLs with improved thermostability by molecular modification in the present study. Then, the enzymatic properties and structure-function relationship of the mutant PCLs were investigated. The main results were as follows:Total RNA was extracted from P. cyclopium followed by reverse transcriptase epolymerase chain reaction. PCL gene (pcl) was amplified by PCR using the cDNA from P.cyclopium. The PCR-amplified product was cloned into pET-22b(+), and the recombinant plasmid pET-pcl was transformed into E. coli BL21(DE3). Then, the recombinant protein PCL (WT) was expressed by BL21/pET-pcl.The pET-pcl was used as a template for overlap extension PCR. 26 mutants were obtained. 11 mutants with activity showed no chang in thermostability.The pET-pcl was used as a template for error-prone PCR. After screening, one mutant with improved thermostability was selected. The nucleotide sequence alignment of the selected mutant PCL gene with the WT displayed the substitution of Leu41Pro and Gly47Ile in amino acid sequence, named L41P/G47I. Also the single-site mutation with L41P and G47I was generated by overlap extension PCR, respectively.The purified WT, L41P, G47I, and L41P/G47I were obtained via affinity chromatography. Then the enzymatic property of WT and mutant PCLs were investigated.L41P,G47I and L41P/G47I showed the highest activity at 30 ℃,which was 5 ℃ higher than that of the WT. Moreover, the T1/2 at 45 ℃ of L41P, G47I, and L41P/G47I were 7-, 13-and 9-fold higher, respectively, than that of the WT. The optimal pH for WT and mutant PCLs was found to be 10.0, and the pH stability of the mutant PCLs was similar to that of WT at pH 9.0, 10.0, and 11.0. The Km, kcat, and kcat/K, of L41P, G47I, and L41P/G47I showed no obvious change compared with those of WT, respectively.In addition, the optimized formulation for the synthesis of ethyl propionate from ethanol and propionic acid by G47I was summarized. Under the ideal conditions for synthesis, the conversion of ethyl propionate catalyzed by G47I reached 45%. Meanwhile,conversion of 27%, 23%, and 8% were obtained under the same conditions for L41P/G47I,L41P, and WT,respectively.The relationship between the structure and function of mutants were analysed. The residue at position 41 is located on the surface of the protein. The Leu to Pro substitution replaced a hydrophilic side chain with a more hydrophilic group, moreover, Pro reduced the flexibility of loop with the conformation change caused by pyrrolidine, causing the enhancement of the stability in high temperature. The residue with position 47 is located in the interior of the protein. Gly was substituted by Ile with a more hydrophobic group. In addition, Ile has a higher propensity with forming the β-strand in comparision with Gly,which was beneficial to stabilize the β-strand, resulting in the increase of its structural rigidity for improving the overall thermostability.