Cloning, overexpression, and characterization of a novel organic solvent-tolerant lipase from Paenibacillus pasadenensis CS0611

We found a novel lipase gene in the Paenibacillus pasadenensis CS0611 strain.The lipase gene sequence was cloned into the pET-28a expression vector to construct a recombinant lipase protein containing 6×His tags at the C-and N-termini,respectively.High-level expression of the lipase in E.coli BL21(DE3)was obtained upon induction with IPTG at 20°C.The recombinant lipase activity was approximately 1631-fold higher than the wild type.His-tagged recombinant lipase was purified rapidly and efficiently by using Ni-charged affinity chromatography with 63.5%recovery and a purification factor of 10.78.The purified lipase was stable in a broad range of temperatures and pH values,with the optimal temperature and pH being 50°C and 7.0,respectively.Its activity was stimulated to different degrees in the presence of metal ions such as Ca2+,Mg2+,and some non-ionic surfactants.In addition,the purified lipase was activated by a series of water-miscible organic solvents such as some short carbon chain alcohols and was highly tolerant to some water-immiscible organic solvents.

Purification and characterization of alkaline chitinase from Paenibacillus pasadenensis CS0611

An extracellular chitinase produced by Paenibacillus pasadenensis CS0611was purified by ammoniumsulfate precipitation,HiTrap DEAE FF and HiLoad26/600Superdex200pg column chromatography.The apparent molecular mass determined by sodium dodecyl sulfate polyacrylamide gelelectrophoresis was69kDa.The optimum pH and optimum temperature of the chitinase were5.0and50°C,respectively.The enzyme showed high stability at alkaline pH values and temperaturesbelow40°C.Additionally,the metal ions Mn2+,Mg2+,and Co2+inhibited activity of the chitinase.Thechitinase was active on colloidal chitin with an apparent Km of4.41mg/mL and Vmax of1.08mg/min.Substrate spectrum analysis indicated that the chitinase reacted preferentially with the glucosidicbond between GlcNAc‐GlcNAc.The enzymatic hydrolysate was analyzed by high‐performance liquidchromatography and thin layer chromatography,and clearly showed that a subunit of(GlcNAc)2was the main hydrolysis product.

Using multiple site-directed modification of epoxide hydrolase to significantly improve its enantioselectivity in hydrolysis of rac-glycidyl phenyl ether

The epoxide hydrolase gene(SpEH) from Sphingomonas sp. HXN-200 was synthesized and expressed in robust Escherichia coli cells that had a dual protection system. The enantioselectivity(E-value) of the recombinant SpEH was 7.7 and the yield of the remaining(R)-PGE was 24.3% for the hydrolysis of racemic phenyl glycidyl ether(rac-PGE). To improve the catalytic properties of SpEH, the site-directed mutagenesis was carried out based on homology modeling, sequence alignment and molecular docking. Six residues(V195, V196, F218,N226, Q312, and M332) near the active site were mutated to hydrophobic amino acids and the positive mutations were selected for combinatorial mutation. The optimal mutant SpEH^(V196A/N226A/M332A) had an enhanced E-value of 21.2 and a specific activity of 4.57 U·mg^-1-wet cells, which were 2.8-, and 2.3-fold higher than those of wild-type SpEH. The optimal temperature and p H for purified Sp EHV196 A/N226 A/M332 Ato catalyze the hydrolysis of rac-PGE were 25 ℃ and 7.0 with 200 U·mg^-1. The enantioselectivity and yield of the remaining(R)-PGE of E. coliSpEH^(V196A/N226A/M332A)increased from 7.7 to 21.2 and 24.3% to 40.9%, respectively. The molecular docking and kinetic parameter analyses showed that SpEH^(V196A/N226A/M332A) has a greater affinity toward(S)-PGE than(R)-PGE, and that it was more difficult for the O-atom of ASP170 to achieve the nucleophilic attack on the Cα of(R)-PGE, resulting in its improved enantioselectivity.