High-level Heterologous Expression And In Vitro Molecular Evolution Of Lipase Lip2from Yarrowia Lipolytic,and Its Application In Kinetic Resolution

The extracellular lipase Lip2from Yarrowia lipolytic, as an important industrial biocatalyst, has been widely used in esterification and transesterification reactions, chiral resolution of various chiral compounds and production of biodiesel because of its high reactive activity. For the characterization of lipase YlLip2and exploration of its practical applications, some researches have been done as follows:1.(±)a-phenylethylamine, broadly used as powerful intermediate in industrial asymmetric synthesis or chiral adjuvant, seems to have served as a test substrate as it has been subjected to various acylation conditions,In present study, we used the immobilized form of lipase YlLip2to catalyze the enzymatic kinetic resolution (KR) of (±) a-phenylethyl amine with acetic ester as the acyl donor in an organic medium containing cosolvent. Herein we discussed the effect of different cosolvents on the acylation of (±) a-phenylethyl amine in a non-aqueous medium. Seven solvents were chosen and tested for cosolvent in the hexane. We revealed that, with the presence of3%(v/v) dimethylsulfoxide (DMSO) in hexane, the immobilized lipase YlLip2mediated resolution of (±) a-phenylethyl amine was surprisingly effective and showed high enantioselectivity. This binary organic mixture markedly enhanced the enantiomeric excess of product (e.e.p) from0.35(in neat hexane) to0.96and, thus, improved the enantiomeric ratio (E) from2.5(in neat hexane) to190. Reaction parameters as cosolvent concentration, reaction temperature, amount of the immobilized lipase, and reaction time were optimized for further application. Finally, the recyclability of immobilized lipase in binary organic medium was also investigated. To our knowledge, YlLip2is reported on the resolution of amines for the first time. 2. Our research have introduced a simple and efficient method for the genetic manipulation in intact Bacillus subtilis. The gene replacement construct was generated based on multiple fusion PCR which involves fusing by PCR two flanking homology regions (upstream/downstream regions of amyE) with an antibiotic-resistance cassette. Rather than routinely using a double-stranded (ds) DNA cassette to transform naturally competent B. subtilis cells, the research investigated the feasibility of employing full-length single-stranded (ss) DNA constructs for chromosomal integration in B. subtilis via convenient electro-transformation. By taking advantage of B. subtilis endogenous homologous recombination machinery,5’end protected lagging-strand targeting ssDNA construct was demonstrated to be highly recombinogenic and recombined onto chromosome with an efficiency of two orders of magnitude higher than that of the normal substrate, i.e., dsDNA donor. By applying this strategy, a lipase gene from Yarrowia lipolytica was successfully integrated onto B. subtilis DL3p chromosome at the amyE locus and achieved functional expression. This method is rapid and straightforward and provides implications for genomic engineering in5. subtilis.3. YlLip2was successfully expressed in Pichia pastoris GS115under the control of the AOX1promoter. To further improve lipase production in P. pastoris, we screened multicopy lipase gene Lip2clones via high-Zeocin-resistance screening. Two clones which can resist>500g/ml Zeocin showed higher lipase production in shaking flasks. Southern blot analysis showed that three lipase genes were integrated into the genome of P. pastoris GS115. Low temperature (25℃) could also improve the extracellular lipase yield. A high lipase activity of11000U/mL (3.4g total protein and2.16g lipase per liter) was obtained using the three copy integration clone in fed-batch fermentation using basal salt medium, which was2.5-fold higher than that of using a single copy clone. The high yield of lipase YlLip2in P. pastoris could decrease the price of this biocatalyst and accelerate its industrial applications.4. The practical use is often hampered by the low thermal stability of lipase Lip2. Here three complementary protein engineering strategies were used to improve the thermostability of this enzyme. The first strategy was error-prone PCR based directed evolution, which resulted in a YlLip2variant with a2.5-fold longer half-life of thermal inactivation at50℃compared to the wild-type enzyme. The second strategy was semi-rational design using the so-called B-factor iterative test (B-FIT), which led to the discovery of two thermostable YlLip2variants that showed a half-life of thermal inactivation2-fold and5-fold longer than that of the wild-type enzyme, respectively, at50℃. The third strategy was to use site-directed mutagenesis to combinatorially combine all three thermostabilizing mutations identified in the first two strategies, which improved the half-life of thermal inactivation of YlLip2by7-fold compared to that of the wild-type enzyme. Such engineered lipases provide not only new insights on the protein structure and function relationship but also potentially useful catalysts for practical applications.