Discovery And Engineering Of An Esterase From Pseudomonas Putida ECU1011and Its Application In The Kinetic Resolution Of α-Hydroxy Acids

Owing to the dual functionality, optically pure hydroxy acids and their derivatives are versatile chiral synthons in pharmaceutical and fine chemical industries.(R)-2-Hydroxy-2-(2’-chlorophenyl) acetic acid is the key chiral intermediate in the synthesis of (S)-clopidogrel, a platelet aggregation inhibitor used for heart attack and stroke treatment.(R)-2-Hydroxy-4-phenylbutyrate is an important building block for the production of various angiotensin-converting enzyme (ACE) inhibitors, such as lisinopril and ramipril. In previous work, an esterase producing strain, Pseudomonas putida ECU1011, was isolated from soil for the enantioselective deacylation of (S)-2-acetoxy-phenylacetate.In this study, the corresponding esterase was hunted from the genome of P. putida ECU1011and its application potential was further explored by the combinatorial engineering of enzyme, substrate and operation mode. Finally, an efficient and universal bioprocess was developed for the preparation of optical pure hydroxy acids.Firstly, rPPE01was hunted from P. putida ECU1011and its catalytic properties were characterized. On the basis of the analysis of the genomic information and corresponding functional prediction of P. putida in the NCBI database,16genes encoding lipases or esterases were selected as candidates. As a result, rPPE01was identified from nine successfully expressed enzymes with the highest hydrolytic activity and excellent enantioselectivity towards the target substrate a-acetoxyphenyl acetate. All the tested a-acetoxy carboxylates were converted with approximately50%yield and excellent enantioselectivity (E>200) at a substrate concentration of100mM. The enzyme showed an optimal pH of6.2and exhibited the highest activity at50℃. In addition, rPPE01displayed relatively high thermostability and organic-solvent tolerance, which are always the key factor for the biocatalyst industrialization. These attractive properties make rPPE01a competent biocatalyst for enzymatic resolution of a-hydroxy acids in spite of its relatively low enzymatic activity and poor operational stability.Secondly, protein engineering was used to improve the specific activity of rPPE01. Semi-rational design based on homologous modeling and molecular docking provided a single-point variant, W187H, whose kcat/KM for sodium2-acetoxy-2-(2’-chlorophenyl) acetate (AcO-CPA-Na) was increased100-fold, while retaining its excellent enantioselectivity and broad substrate spectrum. To verify the improvements offered by the mutant W187H in practical applications, biocatalytic resolution of AcO-CPA-Na (300mM) with either mutant or wild-type rPPE01was conducted with a catalyst loading of0.5g/L of lyophilized cells. The reaction catalyzed by the mutant W187H reached49.6%conversion after3h, whereas the conversion in the case of the wild type was only4.4%at the same time.In the third part, the substrate was engineered to improve the resolution potential. The kinetic parameters towards the AcO-CPA and its ester revealed that rPPE01showed higher affinity to ester of AcO-CPA (7-fold decrease in KM) while much lower catalytic velocity (as much as250-fold decrease in kcat). Thus, AcO-CPA is a better substrate for the resolution. The AcO-CPA existed in the ionic form in the reaction system and the optimum cation was reported to serve as an inherent additive to stabilize the enzyme against substrate/product deactivation. Potassium ion was found to be the best substrate counterion to relieve the enzyme of deactivation. Substrate with K+as counterion (AcO-CPA-K) of500mM could be easily deacetylated with excellent eep (>99%) and ees (98.7%) within15h. A lower conversion was observed when the Na+(39.0%) and NH4+(9.4%) were used as counterion even the reaction time was prolonged to20h. Further enzymatic resolution of other important hydroxy acids at the100mL scale demonstrated that the rPPE01w187H-based bioprocess is versatile and practical for the large-scale preparation of chiral a-hydroxy acidsIn the fourth part, the operational stability of the mutant rPPE01w187H was further improved by the immobilization. A commercial resin, ESR-1, was selected as the best immobilization carrier according to the specific activities and operational stabilities of enzymes immobilized by different immobilization resins. The activity recovery and specific activity of rPPE01W187H@ESR-1were60%and90U/g, respectively. The thermostability investigation revealed that the immobilization significantly enhanced thermostability of rPPE01W187H. There was no obvious inactivation observed for the immobilized enzyme after incubation at30℃for1440h while the half-life of the free enzyme was only50.2h at the same temperature. And the immobilized enzyme can be reused for22batches (253h) in the resolution of AcO-CPA-K.Finally, we constructed a packed bed reactor using the immobilized enzyme to avoid the enzyme activity processing loss from the agitation, separation between the batches, etc. After the optimization of the important parameters including initial pH, H:D ratio, and flow direction, the space-time-yield of the packed bed reactor for the resolution of AcO-CPA-K reached as much as140g L-1h-1(3.36kg L-1d-1). Further investigation on the operational stability of the packed bed reactor indicated that it could operate stably for42days without obvious deactivation.