Heterologous Expression Of Lipase Genes From Penicillium Cyclopium’ Engineering Thermostability And Substrate Specificity Analysis Of The Enzymes

Lipases (EC3.1.1.3) catalyze the hydrolysis of long-chain acylglycerols at the oil–waterinterface, as well as the esterification, transesterification and interesterification in organicsolvents. According to the substrate specificity, they consist of triacylglycerol hydrolases,mono-and diacylglycerol hydrolases, and so on. Triacylglycerol hydrolases catalyze thehydrolysis of triacylglycerol, while mono-and diacylglycerol hydrolases catalyze thehydrolysis of mono-and diacylglycerol but not triacylglycerol. Penicillium cyclopium PG37strain can produce a triacylglycerol lipase (PcLipI) together with a mono-and diacylglycerollipase (PcMdl). In order to widen the application fields of PcLipI and PcMdl, the expressionsof PclipI and Pcmdl in Pichia pastoris GS115were firstly carried out. The recombinantPcLipI (rePcLipI) showed low thermostability. It is urgent to enhance the thermostability ofPcLipI by genetic engineering. The PcLipI and PcMdl showed different substrate specificities,it is necessary to understand these specificities especially in PcMdl substrate specificity. Inthis work, the synegistic reaction of rePcLipC(recombinant engineered thermostable PcLipI)and re (recombinant PcMdl) toward lipid was also investigated. The PG37strain is poor inproductive level, meanwhile the PcLipI has low thermostability.A777-bp cDNA fragment encoding a258-aa mature PcLipI from P. cyclopium PG37was amplified by RT–PCR, and expressed in P. pastoris GS115. One transformant resistant to4.0mg mL-1of G418, numbered as P. pastoris GSL4-7, expressing the highest rePcLipIactivity. When the P. pastoris GSL4-7was cultured under the optimized conditions, initial pH9.0, adding methanol of1.0%, inductive temperature26°C, the expressed rePcLipI activitywas up to407.3U mL-1. The rePcLipI was verified as a glycosylated protein with an apparentmolecular weight of about31.5kDa. The rePcLipI activity was significantly inhibited by Hg2+,Fe3+and Cu2+. The rePcLipI showed the highest activity at pH10.5and25°C, and was stableat a broad pH range of7.0–10.5and at a temperature of30°C or below. The rePcLipI belongsto an alkaline cold-active lipase and shows low thermostability. The specific activity ofrePcLipI was5,300U mg-1.By the methods of homologously modeling, disulfide bridge prediction, and moleculardynamics simulation, cysteine mutants of PcLipI predicted to have better thermostabilitiesthan the wild-type. The mutants were first expressed in Escherichia coli BL21(DE3) and then,for further investigation, in P. pastoris GS115. Based on the analysis of recombinant mutantPcLipIs, reE-PcLipV248C-T251C(expressed in E. coli) and reP-PcLipV248C-T251C(expressed in P.pastoris) both had enhanced thermostabilities with half-lives at35°C about4.5-and12.8-foldlonger than that of the parent PcLipI expressed in E. coli and P. pastoris, respectively. Thetemperature optima of reE-PcLipV248C-T251Cand reP-PcLipV248C-T251Cwere each5°C higherthan those of the parent PcLipI expressed in E. coli and P. pastoris.A full cDNA gene which encodes the mono-and diacylglycerol lipase from P. cyclopiumPG37was cloned, then the sequence was submitted to GenBank under the accession numberof HM135194. Based on the principle of the nest PCR, a cDNA gene, Pcmdl, encoding themature peptide of the mono-and diacylglycerol lipase from P. cyclopium PG37was cloned, then expressed in P. pastoris GS115. One transformant resistant to4.0mg mL-1of G418,numbered as P. pastoris GSM4-2, expressed the highest recombinant PcMdl (rePcMdl)activity. When the P. pastoris GSM4-2was cultured under the optimized conditions, initial pH6.5, adding methanol of1.5%, inductive temperature28°C, the expressed rePcLipI activitywas up to215.2U mL-1. The rePcMdl was also verified as a glycosylated protein with anapparent molecular weight of39.0kDa. The rePcMdl showed the highest activity at pH7.5and35°C, and was stable at pH range of6.5–9.5and the temperature below35°C. TherePcMdl activity was significantly inhibited by Hg2+and Fe3+. The rePcMdl was confirmed tobe strictly specific for1-monobutyrin (189.3U mg-1) and1,2-dibutyrin (344.9U mg-1), butnot tributyrin (0U mg-1).Using the methods of homology modeling, molecular docking, and molecular dynamicssimulation, the substrate specificity of PcMdl were investigated. The α-helix form lid in openconformation uncovers the catalytic center and leads to the catalytic triad Ser145-Asp199-His259exposing to solvent. The residue Ser83, together with the Ser145, constitutes the oxyanion holewhich stabilizes the tetrahedral intermediates. The catalytic pocket in open conformation,organized by residues Tyr21, Phe112, Leu146, Pro174, Val201, Phe256, Val266, and Asp267, exposesto solvent. Stereographic view of PcMdl docked with substrate (tri-or diacylglycerol)analogue indicated that the residue Phe256played an important role in conferring the substrateselectivity. The Phe256projected its side chain towards the substrate binding groove and madethe sn-1moiety difficult to insert in. On the contrary, sn-1moiety hampered the phosphorusatom (taking the place of carboxyl carbon) from getting to the Oγof Ser145and caused thefailure of triacylglycerol hydrolysis.The factors affecting lipid hydrolysis by rePcLipC(reP-PcLipV248C-T251C) and rePcMdlwere determined. The hydrolysis of olive oil and tributyrin was investigated, meanwhile partof the hydrolysis products were analyzed by HPLC. The rePcLipCand rePcMdl have optimalsynergistic initial pH10.5, temperature30°C, and dosage ratio3:1by the active unit.Comparing with the hydrolysis by CALB or rePcLipCalone, rePcMdl could increase thehydrolysis rate by synergy with CALB or rePcLipC. With the addition of rePcMdl, there weremore monobutyrin and dibutyrin released from tributyrin than that by rePcLipCalone. Theresult demonstrates there is synergistic interaction between rePcMdl and rePcLipCwhichaccelerates the hydrolyzing of tributyrin.