Structure Analysis And Molecular Engineering Of Oxidized Polyvinyl Alcohol Hydrolase

Polyvinyl alcohol (PVA), with its many desirable physical and chemical characteristics, iswidely used in textile sizing, adhesives, fiber coating, and et al. However, this extensive usage,particularly in the textile industry, has resulted in large amounts of PVA being discharged in thewastewater, causing environmental pollution due to the difficulty in degrading PVA. In contrast,PVA degradation using microbial enzymes is an environmentally friendly approach that cansave energy and reduce the problems caused by the treatment of sewage from the textiledesizing stage. However, the efficiency of PVA degradation by the isolated strains in thesestudies was relatively low. Microbial degradation of PVA entails a two-stepmetabolism. In thefirst step two neighboring alcohols are oxidized to form a diketone structure in the polymer byPVA oxidase or PVA dehydrogenase. In the second step oxidized PVA (OPA) is hydrolyzed byOPA hydrolase (OPH, also known as β-diketone hydrolase, BDH). The polymer isprogressively degraded by these enzymes into smaller fragments. In the dissertation, the ophgenes were expressed in engineering strains to enhance the production of the enzymes. TheOPH structures were determinated to improve the activities of OPHs. The main contents aresummarized as follows.(1) The high-level expression of OPH. The oph genes from Pseudomonas sp. VM15Cand Sphingopyxis sp.113P3(pOPH and sOPH) were modified with high-usage codons ofEscherichia coli and Pichia pastoris. Under optimized cultural conditions, sOPH activity insupernatant of E. coli BL21(DE3) reached47.5U·mL-1. The addition of glycine (200mmol·L-1) increased the productivity of sOPH to733U·L-1·h-1. Meanwhile, sOPH was produced in P.pastoris. The production of recombinant protein was performed by induction with14.4g·L-1methanol at22oC in a3-L bioreactor. sOPH activity and productivity have reached68.4U·mL-1and777U·L-1·h-1. pOPH fusing thioredoxin was expressed in E. coli BL21trxB (DE3).Cultural conditions were optimized to reduce formation of inclusion body. Finally, after48-hculture under20oC, production of pOPH was improved to15.7U·mL-1without induction ofIPTG.(2) Purification and crystallization of sOPH and phase calculation. The recombinantsOPH was purification with a hydrophobic interaction chromatography column and an ion-exchange chromatography column from the supernatant of E. coli and P. pastoris. The purifiedsOPH expressed in P. pastoris showed a higher activity, was concentrated to30mg·mL-1andcrystallized. After optimization of crystal conditions of sOPH, the crystals with regular shapewere obtained. The X-ray diffraction dataset of sOPH with resolution of1.90was collectedat National Synchrotron Radiation Research Center (NSRRC). Molecular replacement (MR),singlewavelength anomalous diffraction (SAD) and multiple isomorphous replacement (MIR) used to solve the phase problem did not work.(3) Purification and crystallization of pOPH and phase calculation. The mature pOPH waspurified with TEV protease digestion, Ni ion affinity chromatography column and Q anion-exchange chromatography column. The purified pOPH was concentrated to10mg·mL-1andcrystallized with Crystal Screen kits. During optimization of crystal conditions of pOPH,isopropanol was replaced by n-octyl-β-D-glucoside. Finally, the X-ray diffraction datasets ofpOPH (1.60and2.10) was collected at NSRRC. In the end the rhenium-containingderivative (K2ReCl6) was found effective in phasing by the SAD method. The structure ofpOPH S172C was determinated.(4) Analysis of structures of pOPH and sOPH. Based on the structure of pOPH S172C, thepOPH and sOPH structures were solved using method of MR. They shared65%amino-acidsequence identity. Their overall structures were similar. The lid was the most variable region inthe pOPH and sOPH structures. The complex structures of S172C/ACA and S172A/PNPC wereobtained by soaking pOPH crystals with inhibitors. Based on the structures, a13-carbon modelof OPA was constructed to elucidate enzyme-substrate interactions. The catalytic mechanismof hydrolyzing OPA was analyzed: the Ser172made a nucleophilic attack when deprotonatedby His298and Asp253. A double oxyanion hole was formed by hydrogen bonds among Ser66,Val67and Ser173to stabilize reaction intermediates, which unlike other α/β hydrolases with asingle oxyanion hole mechanism.(5) According to the complex structures of pOPH, the Trp255, Tyr270and Arg264residuesand a disulfide bond Cys257/Cys267locating the active site, were mutated to investigate thecatalytic ability. As a result, the mutants W255Y, Y270F and R264A improved activities of56%,41%and42%higher than those of wild-type. The results were explained with the crystalstructure of pOPH. Moreover, the combination mutants W255Y/R264A andW255Y/R264A/Y270A showed153%and166%higher activity respectively. Apparently theactivity improving effect of each mutant indeed acted synergistically by making double andtriple mutations. The gene of mutant W255Y/R264A/Y270A was inserted into the pET20b, andcultured under the refined conditions. The enzyme activity and productivity in supernatantreached20.9U·mL-1and249U·L-1·h-1respectively. The activity with PNP esters was measured.The Kmvalues of both OPHs for PNP esters increased with the chain length of the fatty acylsubstrates, whereas the kcat/Kmvalues tended to decrease with the chain length, except for PNPB(C4). The significantly lower activity for PNPB could be a result of product inhibition, assuggested by the complex crystal structures. The roles of tryptophan and cysteines in lid regionof OPH were analyzed.