Study On Molecular Modification Of Phenylalanie Ammonia-lyase From Rhodotorula Glutinis And The Resolution Of Racemic DL-phenylalanine

Phenylalanine ammonia-lyase (PAL, EC4.3.1.5) catalyzes the non-oxidative deaminationof L-Phe to trans-cinnamic acid and ammonia at pH8-9, as well as the reverse reaction withthe trans-cinnamic acid and ammonia at pH10-11to produce L-Phe. PAL isolated from yeasts,such as Rhodotorula, has been applied in manufacture of L-Phe. Moreover, the PAL can beused in the chiral resolution of DL-Phe to produce D-Phe. The resulting D-Phe could be easilyseparated from the reaction solution, since the solubility of trans-cinnamic acid is low atacidic condition. In addition, the investigations of catalysis mechanism and substratespecificity are significant to provide theoretical basis for modification and improveapplication performance of PAL.In this study, the full-length RgPAL gene from R. glutinis was cloned and heterologouslyexpressed. The immobilization RgPAL with high stability was prepared to resolve DL-Phe toproduce the high value added D-Phe. According to the analyses of the catalysis mechanismand structure of RgPAL, the RgPAL was modified using site-directed mutagenesis, and amutant RgPAL with lower optimum pH was obtained, which improved the resolutionefficiency of DL-Phe. In addition, we found that the110Loop of RgPAL manipulated theregioselectivity, and obtained a mutant RgPAL with β-lyase activity. The main results weredescribed as follows:(1) The full-length gene of RgPAL was cloned, efficiently expressed in E. coli, and therecombinant RgPAL was characterized.The RgPAL from R. glutinis exhibited high activity, however, the intercellular PAL wasrapidly inactivated after logarithmic phase, and susceptible to protease and glycosylase,which is a disadvantage to industrial application of RgPAL. The disadvantage could beovercome by heterologous expression. Firstly, we cloned the full-length RgPAL gene from R.glutinis using RACE technology based on the3’ terminal sequence of RgPAL gene. TheRgPAL gene was found to be2,121bp, encoding a polypeptide with706amino acids and acalculated molecular mass of75.5kDa. Protein sequence alignment analysis showed that theprotein exhibited74%identity with RtPAL from R. toruloides. The recombinant expressionvector pET-28-pal was constructed and then transformed into E. coli BL21for RgPALexpression. The recombinant RgPAL was purified by ammonium sulfate precipitation, anionexchange, and gel filtration. The specific activity of the RgPAL was4.2U·mg-1at50℃andpH8-9, and exhibiting high stability at pH6-11and40℃.(2) An efficient enzymatic process was developed to produce optically pureD-Phenylalanine through resolution of the racemic DL-Phe using immobilized RgPAL.The DL-Phe was resolved to produce high optical D-Phe using immobilized RgPAL. Inorder to enhance the resolution efficiency and reuse the enzyme to lower the cost, we firstlyprepare the immobilized RgPAL with high stability. Mesoporous silica (MCM-41) was chosenas the immobilization support. To solve the leaching problem of the adsorbed enzyme,MCM-41was modified by3-aminopropyltrimethoxysilane and crosslinked by glutaraldehyde(GA), resulting in MCM-41-NH-GA-RgPAL. The optimum immobilized conditions were that the loaded amount of support was50mg·g-1and0.05%glutaraldehyde was used to crosslinkRgPAL. The specific activity of the MCM-41-NH-GA-RgPAL is90%of free enzym at55℃and pH9. The thermal and pH stabilities of MCM-41-NH-GA-RgPAL were significantlyhigher than those of the free enzyme and retained approximately80%activity after30reuses.The MCM-41-NH-GA-RgPAL was loaded into recirculating packed-bed reactor (RPBR) toresolve DL-Phe. Under optimal operational conditions, the volumetric conversion rate ofL-Phe and the productivity of D-Phe reached96.7mM·h-1and1.94mM·h-1, respectively. TheRPBR ran continuously for16batches, and the conversion ratio and volumetric conversionratedid not decrease. The reactor was scaled up to25-fold, and the productivity and yield ofD-Phe (eeD>99%) in the scaled-up reactor reached43.6mM·h-1and8.26g/L, respectively.These results suggest that the resolution process using MCM-41-NH-GA-RgPAL in an RPBRis an alternative method to produce optically pure D-Phe.(3) Based on analyses of structure and catalytic mechanism of RgPAL, a mutant withhigher activity at pH7.0was obtained using site-directed mutagenesis.In order to enhance the resolution efficiency of DL-Phe and relieve the inhibition ofproduct, the optimum pH of RgPAL was expected to be shifted to acidic side. The RgPAL is amember of the4-methylidene-imidazol-5-one (MIO)-dependent enzyme family. Themechanism underlying non-oxidative deamination catalyzed by PAL is currently notunderstood. The action of RgPAL was identified by through the following substrate:4-hydroxylphenylalanine (L-tyrosine) and3-hydroxylphenylalanine (m-tyrosine). This studyindicated that the RgPAL acts through the Friedel-Crafts-type mechanism in which the MIOattack the phenyl ring of the substrate. Based on structural analyses and molecular dockingsimulations, the136His and137Gln residues of RgPAL were found to form a hairpin motif toclamp the phenyl ring. When the137Gln was mutated to Glu, the optimum pH ofRgPAL-Q137E shfited to7.0and exhibited1.8-fold higher activity than that wild type at pH7.0. As revealed by mechanism of RgPAL, the improvement of the RgPAL-Q137E might bedue to the negative charge of Glu137which could stabilize the intermediate transition statesthrough electrostatic interaction. The RgPAL-Q137E mutant was used to resolve the racemicDL-Phe at pH7, and the conversion rate and the eeDvalue of D-Phe were increased by29%and48%, respectively.(4) The110Loop of RgPAL was mutated to decrease the flexibility, and β-lyase activitywas obtained.According to the structural analysis of RgPAL, a lid-like loop structure named110Loopcovers the active site. The110Tyrosine in the Loop is a key amino acid residue in thedeammino reaction and used as acceptor of proton. The molecular simulation results showedthat the Loop with high flexibility exhibited110Tyr-inner confguration and110Tyr-outerconfguration, respectively. The RgPAL exihited lyase activity only at110Tyr-innerconfguration. In order to decrease the flexibility of the Loop, two ionic bonds wereintroduced through mutating the107Ser and121Thr residues to Glu and Arg, respectively.The rereoselectivity of mutant RgPAL-S107E/T121R was transformed from α-lyase activityat40-45℃to β-lyase activity at55-60℃.