Directed Modification To Substrate Specificity Of Rhizopus Chinensis CTCCM201021 Lipase

As a typical filamentous fungus Rhizopus chinensis CCTCC M201021 was an important functional strain in the brewing industry, which could produce many kinds of enzymes. In our previous studies, Rhizopus chinensis lipase gene (proRCL,GenBank Accession No. EF405962) had been cloned and high-level expressed in pichia pastoris GS115. In this study, the GAP promoter was inserted into the inducible expression vector pPIC9K-proRCL in order to introduce the double-crossover integration of the lipase gene into the yeast genome based on the homologous recombinant region of pAOX1 gene, which also resulted in the constitutive expression of the single-copy lipase gene proRCL. Moreover olive oil-rhodamine B plate was used as an effective, sensitive and fast screening approach. Lipase lid region was mutanted by rational and semi-rational designs. A series of mutants were obtained with changed activity and substrate specificity. The results were as follows:(1) Construction of constitutive expression of lipase in Pichia pastoris. The GAP gene promoter was amplified from the plasmid of pGAPZαA and inserted into the inducible expression vector pPIC9K-proRCL resulting in constitutive expression of the lipase gene-proRCL. Based on the homologous recombinant region of AOX1 gene, proRCL was recombined into chromsome of P. pastoris by double-crossover integration event resulting in constitutive expression of the single-copy lipase gene from R. chinensis. The activity of lipase reached its peak (130 U/mL) after fermentation of 144 h.(2) An efficient method was established for high-throughput screening constitutive expression lipase gene in P. pastoris using olive oil-rhodamine B plate. The method was very convenient with advantages of shortened the screening cycle from 12 d to 3 d without the interferences of multi-copy mutants. It also established the foundation of the screening of library by directed evolution.(3) According to the semi-rational design, we selected 10 consecutive amino acid sites of the lid region in proRCL to introduce random mutations by splicing overlap extension PCR (SOE PCR). A series of mutants were obtained with different activity and substrate specificity .(4) Through rational design, the lid of the thermostable Rhizopus chinensis lipase proRCLS4-3 was replaced by that from Rhizopus oryzae lipase, Rhizomucor miehei lipase and Aspergillus niger Feruloyl esterase by SOE PCR and the mutants were named as proRCLS4-3O, proRCLS4-3M, proRCLS4-3N, respectively.(5) The proRCLS4-3O showed the similar kcat value to that of wild type, but its km value increased two times. The triglyceride lipase activity and phospholipase activity were declined, and the thermostability was also decreased. According to the lipase structure investigated by bioinformatics, Met213 played the role of changing the size of hydrophobic channel, which affected the enzyme binding affinity for substrate, and Thr216 combined with His229 and Ala230 made the configuration of lid more stable. The mutant Asn216 decreased the whole protein structural stability as well as the thermal stability. (6) The proRCLS4-3M preferred C8 fatty acid esters and the lipase activity towards triglyceride increased 51%. Compared with the parent, its kcat value increased 5.6 times, while exhibited the similar km value. According to the lipase structure analysis, the increased GRAVY value indicated that the more hydrophobic lid resulted in higher affinity for hydrophobic substrate. Moreover the shorten distance between substrate and the catalytic center implied that they contacted with each other more tightly. The mutation Ala209Trp narrowed the channel of the substrate towards the enzyme active site, which made it prefer C8 fatty acid esters.(7) The proRCLS4-3N changed its specificity to C2 chain fatty acid esters. Its kcat / km declined from 2.80 mM-1s-1 to 0.05mM-1s-1and the activity of triglyceride lipase and phospholipase also decreased sharply. The GRAVY value which declined from 0.200 to -1.086 suggested the substituted hydrophilic lid destroyed the hydrophobic environment in the catalytic center and reduced its catalytic efficiency. The lid always kept open conformation due to the N-glycosylation site. On the other hand, the narrow open conformation reduced the affinity for hydrophobic substrates but took its specificity to shorten chain fatty acids substrates.