| Lipase(EC 3.1.1.3)is a kind of serine hydrolases,which catalyzes the hydrolysis of triglycerides and converts them into glycerol and fatty acids.Compared with traditional catalysts,lipase has many significant advantages,such as interfacial catalysis and enantioselectivity.Lipases have been used in food.textile and pharmaceutical industries.Among them,the cold-adapted lipases from extremely cold environment have been paid increasingly attention for high catalytic activity and selective specificity at low temperatures.Firstly,the psychrophilic strain(Psychrobacter sp.ZY124)was isolated and identified,and Lipase ZC12 was produced from the supernatant of Psychrobacter sp.ZY124.It was found that Lip ZC12 had good catalytic activity,temperature stability and organic solvent tolerance.indicating that Lip ZC12 possesses promising application potential.In this paper,based on the structure of Lip ZC12.the influence of the lid domain on the substrate binding ability was studied,and the effect of the steric hindrance of the substrate channel on the catalytic efficiency was analyzed,thereby realizing the rational modification of Lip ZC12.By immobilizing Lip ZC12 in the form of organic-inorganic hybrid nano flower,the stability and reusability of lipase were enhanced.Finally,in order to explore the application potential of modified Lip ZC 12,the esterification ability of modified Lip ZC12 was evaluated by enzymatic synthesis of fructose laurate.The main contents of this paper were divided into four parts:(1)In order to overcome the shortage of long culture period and low yield,the cold-adapted Lip ZC12 was recombined and expressed based on the whole gene sequence.According to the sequence analysis,it was found that Lip ZC12 belonged to lipase family I.1.The residues of the catalytic triplets were Ser144.His324 and Asp302.The oxyanion hole was composed of Leu77 and Glnl45.Then,the recombinant plasmid pET28a(+)-lip was successfully constructed to obtain the soluble lipase in E.coli BL21(DE3).The study of enzymatic properties showed that the optimal reaction temperature of Lip ZC12 was 40?,and the optimal pH value of Lip ZC12 was 8.0.In addition.Lip ZC12.like Pseudomonas lipase in the lipase family I.1.also had Ca2+ activation.(2)Based on the enzymatic and sequence characteristics of Lip ZC12,in order to improve the substrate affinity and catalytic efficiency of Lip ZC12,the structural characteristics were analyzed.Compared with the homologous lipase.Lip ZC12 specifically had extra 8 amino acids(NTSGQPSN)in the non-conserved region of the lid domain.In order to explore the effects of the lid domain on the enzyme,the truncated lid region was obtained to construct the recombinant plasmid pET28a(+)-?lid by overlapping PCR method.Based on the enzymatic properties constants of modified lipases,the results showed that the Km value of lip-?lid(9.02×10-3 mol·L-1)was significantly lower than that of free lipase(13.12×10-3 mol·L-1),indicating that truncating the lid domain could significantly increase the binding energy between the enzyme and substrate.Furthermore,substrate channel also seriously affected the catalytic efficiency of enzyme.Therefore,it was necessary to modify the amino acids with the the larger steric hindrance which were located in this substrate channel.The key amino acid(Phel44)was found by bio informatics methods,and it was subjected to site-directed mutagenesis to obtain the recombinant plasmid pET28a(+)-F144A/V.The Km values of the mutant lip-F144A/V lipase were basically unchanged,and the kcat values of lip-F144A/V were determained to be 1436 s-1 and 1821 s-1,respectively.The kcat/Km value of lip-F144V was 1.5 times higher than that of free lipase,reducing the steric hindrance of the substrate channel could obviously enhance the enzymatic catalytic efficiency.(3)To further improve the stability and achieve reusability of cold-adapted lipase,a new type of Lip ZC12/Ca3(PO4)2 hybrid nanoflower(LHNs(Ca))was prepared for the immobilization of Lip ZC12 based on the Ca2+ activation of Lip ZC12.The immobilized lipase nanoflowers were confirmed to be stable nanoparticles(2-5 ?m)with uniformly distributed flower-like structure stacked in layers by observing the morphology of LHNs(Ca).By characterizing its structure,it was found that LHNs(Ca)had the only metal crystal Ca3(PO4)2 and organic components(lipase),and there was no covalent bond,which indicated that the self-assembled LHNs(Ca)nanoflowers were successfully prepared.Compared with free lipase ZC12,the LHNs(Ca)exerted enhanced enzymatic activity of 206%and 2.31-fold in kcat/Km value,especially high specific activity at low temperature,indicating that LHNs(Ca)showed better substrate affinity and higher catalytic efficiency.After 7 successive cycles,the LHNs(Ca)could still maintain its initial activity,demonstrating superior durability than the free lipase ZC12.Lip-Alid,lip-F144A and lip-F144V were immobilized by the same method,the kcat/Km value of LHNs(Ca)-lip-?lid was increased by 3.11 times higher than that of free lipase.(4)Both rational modification and immobilization have improved the catalytic efficiency of Lip ZC12.In order to further study its application,fructose laurate was synthesized with acetone as the reaction solvent and fructose and lauric acid as the substrate.The results showed that LHNs(Ca),LHNs(Ca)-lip-?lid,LHNs(Ca)-lip-F144A and LHNs(Ca)-lip-F144V were all able to catalyze the esterification reaction.The products were identified as fructose laurate monoester and fructose laurate diester by qualitative analysis,characteristic peak and functional group identification after separation and purification.By optimizing the conditions of esterification reaction,the maximum conversion rate was obtained at 40? for 72 h and the molar ratio of fructose to lauric acid was meastured to be 2:3.Under the same conditions,the results showed that the conversion rate of LHNs(Ca)-lip-?lid at 48 h(58.36%)was significantly higher than that of LHNs(Ca)(41.73%),which further indicated that the stability and enzyme activity of modified Lip ZC12 were significantly improved by immobilization and lid truncation.In summary,a cold-adapted lipase recombinant strain was constructed to obtain soluble lipase.The substrate affinity and catalytic efficiency of rationally modified Lip ZC12 were significantly improved by truncating the lid domain and reducing the steric hindrance of the substrate channel,respectively.Meanwhile,the Lip ZC12 was immobilized by a novel hybrid nanoflowers,LHNs(Ca)showed higher enzyme activity and better stability,and fructose laurate was synthesized,which effectively realized the application and development value of cold-adapted lipase. |
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