Cloning And Expression Of D-lactonohydrolase From F.moniliforme And Directed Enzyme Evolution

D-lactonohydrolase from Fusarium moniliforme CGMCC 0536 which could catalyze thestereospecific hydrolysis of chemically-made DL-pantolactone to generate D-pantoic acid,has attracted increasing attention as biocatalyst for stereospecific production of D-pantoic acid.Though the D-lactonohydrolase showed excellent stereoselective activity to D-pantolactoneand was called "supper catalyst", the activity decreased and half-life of the enzyme shortenedgreatly under low pH situation especially between pH 5.0 and pH 6.0 with the reactionresulting from the decrease of pH. It is necessary to improve the qualities ofD-lactonohydrolase, such as activity and tolerance to low pH, in order to make it suitable forthe industrial application. We studied the D-lactonohydrolase from Fusarium moniliformeCGMCC 0536 further, the contents included cloning and expression of D-lactonohydrolase indifferent hosts and directed enzyme evolution.First strand cDNA was synthesized by RT-PCR with Oligo(dT)15 using mRNA isolatedfrom F. moniliforme CGMCC 0536. The two strands cDNA of F. moniliforme CGMCC 0536encoding D-lactonohydrolase was amplified by the 3' race (rapid amplification of cDNA ends)with the primers which were designed based on the sequences of NH2-terminus and thecDNA encoding D-lactonohydrolase of F. oxysporum reported on the National Center forBiotechnology Information (NCBI) and the structure of mRNA respectively. The cDNAencoding D-lactonohydrolase was analyzed and primers were designed according to analyzingresult. The structural cDNAs encoding D-lactonohydrolase were amplified and ligated withpMD18-T vector by using the T/A cloning procedure, and then the structural cDNA weresequenced. The sequenced result of this structural cDNA has been submitted to Genebankdatabase, the accession number was AY728018.The structural cDNA encoding D-lactonohydrolase was inserted into the vectors pTrc99aand pET20b respectively, the recombinants were named pTrc99a-LAC and pET20b-LAC.The recombinant pTrc99a-LAC was introduced into host Escherichia coli JM109. Afterinduced by IPTG, D-lactonohydrolase gene was successfully expressed endocellular of host.The recombinant protein was checked by SDS-PAGE method, the result showed that about 40kD protein has been expressed in the host. The activity of D-lactonohydrolase was determined,and the average value of activity was 39 U/g (wet cells). The recombinant pET20b-LAC wastransformed into host E. coli BLR(DE3)pLysS to study the secret expression ofD-lactonohydrolase in the periplasmic space of E. coli. After induced by IPTG, the productsexpressed in host were transported into the periplasmic space of E. coli by N-terminal pelBsignal sequence, and the signal sequence was cut down by the signal identification system ofhost after the products expressed entering into periplasmic space, then the protein refolded tothe active form. The activity of D-lactonohydrolase expressed in the periplasmic space of E.coli was determined, and the result was 42 U/g (wet cells). Compared with the activity ofD-lactonohydrolase expressed in endocellular of host, there was no apparently improvementof activity of D-lactonohydrolase expressed in the periplasmic space of E. coli.In order to study the level of expression in eukaryotic host, the structural cDNAencoding D-lactonohydrolase was ligated with eukaryotic expression vector pYX212, and theligated plasmid pYX212-LAC was transformed into S. cerevisiae W303-1A byelectroporation, and transformants were selected on uracil-deficient plates. S. cerevisiaetransformants were grown in liquid selective medium for about 3 days, then centrifuged. Thepositive transformant containing recombinant pYX212-LAC was cultivated and collected bycentrifugation. The D-lactonohydrolase activity in recombinant S. cerevisiaeW303-1A/pYX212-LAC was measured, and the average value of activity was 62 U/g (wetcells). the activity of D-lactonohydrolase expressed in S. cerevisiae was higher than thatexpressed in E. coli.D-lactonohydrolase is useful in the procedure of resolution of racemic pantolactone toproduce D-pantolactone, but the activity and stability under low pH of the wild type enzymeare not satisfactory enough to be applied to industrial production. One goal of ourexperiments was to enhance the expected properties of wild type enzyme by directedevolution—evolving the enzyme by error prone PCR, DNA shuffling, followed by screeningand make this enzyme easy to use in the industry. Mutations were introduced into geneencoding D-lactonohydrolase by error prone PCR combined with DNA shuffling. The mostimportant step of any directed evolution experiment is the development of a simple and rapidmethod for screening the positive mutants from large numbers of variants for the desiredfeatures. The screening method is required to be sensitive enough to identify mutationsconferring even a small enhancement to achieve the desired result by subsequently combiningthese improved properties. To minimize the hard work and the length of time of thelarge-scale screen, a two-step screening method was designed as follows: a modified platemethod was developed for rapid crude screening and HPLC for re-screening. An aliquot ofthe culture was spread onto LB agar plates supplemented with chloramphenicol (34 μg/mL),tetracycline (12.5 μg/mL), ampicillin (100 μg/mL), and bromocresol purple (0.01% w/v). Inaddition, plates containing both 0.5% (w/v) of D-pantolactone and 0.5 mM IPTG wereincubated for 48 h at 30 °C. This modified procedure was based on the formation of yellowspots as a result of hydrolysis. The yellow spots produced by potential improved variantswere identified and positive clones were selected by the formation of yellow spots and theirsizes. We screened for the enzyme variants with improved activity and stability under low pHsituation using this selective plate based on the product formation and pH indicators, and thefield around positive colonies with improved properties could become yellow. However,negative mutants could not change the color of plate with purple background, and it was easyto inspect by eyes. After three sequential error prone PCR and one round DNA shufflingfollowed by screening, more than 40 positive mutants were produced. Among them, MutE-861, the best mutant with improved activity and stability under low pH situation wasobtained by re-screening method. Gene analysis of the Mut E-861 mutant indicated that themutant enzyme had A352C, G721A mutations and a silent mutation of position 1038.Moreover, the activity and stability of this best mutant were determined. The results showedthat the activity of this mutant was 5.5-fold higher than that of wild type, and the stabilityunder low pH was improved at no expense of D-lactonohydrolase activity. After incubated atpH 6.0 and pH 5.0 the activity of D-lactonohydrolase could be retained 75% to 50%, however,compared with 40% to 20% for wild type.We analyzed and compared the secondary structure of wild type and evolved enzyme.Mutations made the rate of α-helix of evolved D-lactonohydrolase decreased, but the rates ofβ-sheet and coils were raised respectively. These structure changes led to raise the flexibilityof evolved D-lactonohydrolase and adaptability to substrate. Simultaneously, because ofamino acid substitution, evolved enzyme could more readily adjust its tertiary and quaternarystructures by itself than wild type, and the stability of evolved D-lactonohydrolase wasimproved under the low pH situation. Perhaps, it is the main reason that make the optimal pHof Mut E-861 changed.