Directed Evolution Of Rhodococcus Sp. R04BphD And Characterization Of BphD Mutant Enzymes

BphD (2-hydroxyl-6-oxo-6-phenylhexa-2,4-dienoic acid hydrolase) is a key enzyme in the microbial degradation of polychlorinated biphenyls (PCBs) by the biphenyl catabolic pathway. The hydrolase catalyze the hydrolytic C-C cleavage of2-hydroxyl-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) to2-hydroxypenta-2,4-dienoic acid (HPD) and benzoate in the Rhodococcus sp. R04biphenyl degradation pathway. In recent years, the study of its structure-function and catalytic mechanism has become the hot research field.To obtain the better characteres of BphD, a method of the Molecule-directed evolution in vitro was used in this paper. DNA shuffling technology was used to successfully construct a mutant library of BphD. The mutant library was screened on a96-well microtiter plate by using enzyme activity. Six mutants with higher activity, E.coli DH5a/pBV220-bphD30、coli DH5a/pBV220-bphD41、E. coli DH5a/pBV220-bphD45、E. coli DH5a/pBV220-bphD48、E.coli DH5a/pBV220-bphD49and E.coli DH5a/pBV220-bphD53, were achieved. The comparison of sequences showed that Q20had been replaced in most of the sequences, and the mutation happened mostly in the range of1-20and40-120in the based sequence of BphD.The genes BphD and its mutant enzymes were cloned into expression vector pET21a, respectively. These recombinant plasmids were introduced into E. coli BL21(DE3), and expressed heterologously, then the expression products were purified by Q Sepharose and Phenyl FF. BphD and its mutant enzymes have the same thermal stability (T1/230min≈62.5℃). The km of these six mutant enzymes30#,41#,45#,48#,49#, and53#decreases by19.31-,2.8-,3.04-,2.14-,2.3-and1.34-fold, respectively.In addition, the site-directed mutagenesis of the hydrolase gene was also performed. The influence of replacements of Pro in BphD with others amino acids on the expression, thermostability, circular dichroism spectrum and fluorescence spectra of BphD was investigated. The soluble expression and specific activity of these mutant enzymes were lower than those of wild-type BphD. Specific activity of the mutants P43G and P43G/P163A decreased by4.23-and28.15-fold, respectively. The T1/220min of P43G/P163A, P241A, P145A, P253L, P43G, P161, P163A, P179Q and P214E were reduced13,9,6,6,4,3,3,3and1℃, respectively, compared with wild-type BphD. Circular dichroism of mutant enzymes showed that a-helix is dominant, and consistent with that of the wild-type enzyme. The T1/2CD of P43G/P163A, P241A, P253L, P161, P163A, P179Q, P214E, P145Aand P43G were reduced22,18.5,14,13,13,13,11,8.5and4℃, respectively, compared with wild-type BphD. The fluorescence spectrum analysis showed that replacements of Pro in BphD with other amino acids led to fewer change of molecular conformation of BphD. The results show that proline mutation affects soluble proteins expression level, enzyme activity, thermal stability (T1/220min, T1/2CD) of BphD and its molecular conformation, its enzymatic properties would also be recovery after proline reverse mutation.