| Pig liver carboxylesterase(PLE) is an enzyme family consisted of several isoenzymes and exists in pig liver. The PLE extracted directly from pig liver is a mixture of many kinds of isoenzymes, and extractions from different batches of PLE varied in component and activities. So far, there are 7 kinds of PLE isoenzymes reported in literatures, PLE1, PLE2, PLE3, PLE4, PLE5, PLE6 and APLE, respectively. PLE has a high selectivity to hydrolyze enantiomers, one of the enantiomers in the reaction can be selectively hydrolyzed to obtain a single enantiomers product. It plays an important role in the industrial production of functional single enantiomers and pharmaceutical intermediates, so it becomes the leading role in the field of organic chemistry synthesis.Our previous study showed that Tong cheng swine and large white swine had a variety of PLE isoenzymes in the liver of pig, their expression levels were significantly correlated with age and varieties. In this study, the PLE recombinant protein was obtained by prokaryotic functional expression and the hydrolysis activity of PLE recombinant protein towards the common substrate and specific substrate was determined to obtained hydrolysis characteristics of PLE isoenzymes which lay a foundation for the study that PLE hydrolyzed veterinary medicine. At the same time, selecting the PLE isoenzymes which has the smallest difference of amino acid and largest difference of activity to carry out gene site directed mutagenesis to find the key amino acids which affect the enzyme activity. So the determinants of the hydrolysis activity of PLE were discussed.In this study, we analyzed the expression abundance based on the cloning ORF of a variety of PLE isoenzymes to select the main subtypes of PLE isoenzymes(PLE1, PLE2, PLE3, PLE4, PLE5, PLE6, APLE, PLE12, PLE13, PLE18) and carried on the prokaryotic functional expression. Primers were designed according to the c DNA sequence of PLE1 in Gen Bank. The signal peptide nucleotide sequence was removed in the upstream primer, 7 protective bases GGAATTC and Nde I enzyme cutting site CATATG were added to 5’ sequence. 12 nucleotides encoding endoplasmic reticulum retention signal(CATGCTGA GCTG) was removed in downstream primers, 3 protective bases CCG were added to 5’ end sequence, as well as Xho I enzyme cutting site CTCGAG and termination codon TCA.Using the recombinant p MD18T-PLE as template constructed previously, the ORF of PLE was amplified by the primers. PLE gene was inserted into vector p ET15 b to construct prokaryotic expression vector p ET15b-PLE. The expression vector and molecular chaperone plasmid p Gro7 were transformed in Origami?2(DE3) to coexpress. Firstly, L-Arabinose was added to induce the expression of molecular chaperone p Gro7, and then the expression of PLE gene was induced by the addition of IPTG. The supernatant was collected after centrifugation and purified by nickel ion affinity chromatography column. The purified protein was analysis by SDS-PAGE gel electrophoresis and the activity of PLE was detected by hydrolyzing common substrate p-nitrophenyl acetate(p-NPA). The activities of PLE1, PLE2, PLE3, PLE4, PLE5, PLE6, APLE, PLE12, PLE13, PLE18 to hydrolysing p-NPA were: 3.5U/mg, 1.75 U/mg, 1.19 U/mg, 1.41 U/mg, 1.78 U/mg, 6.72 U/mg, 2.91 U/mg, 1.83 U/mg, 1.73 U/mg, 1.71 U/mg, respectively. The activities of PLE1, PLE2, PLE3, PLE4, PLE5, PLE6 and APLE toward the specific substrate methyl butyrate and hexyl butyrate were determined by automatic potentiometric titration. The results showed that PLE1, PLE2, PLE3, PLE4, APLE preferentially hydrolyzed methyl butyrate, while PLE5, PLE6 preferentially hydrolyzed hexyl butyrate.The author analyzes all PLE subtypes that have been reported in the literature and others newly discovered, it is found that all the subtypes have the same catalytic triplet as the activity center(Ser204-Glu336-His449). But the results of the previous studies showed that there was a difference in the hydrolysis activity of different isoenzymes, which indicated that the activity center of PLE could not completely determine the activity of the enzyme. So the author discussed the factors that may influence the hydrolysis activity of PLE. In this study, we have cloned 54 kinds of isoenzymes, which including those reported in the present study. According to the similarity of amino acid sequence of isoenzymes, they were divided into seven categories which are expressed by capital letter A~G, each major category was differentiated by Arabia digital. During the above 54 kinds of isoenzymes, the 5 groups of amino acids with the least difference between each other were screened out, PLE-A8/PLE-A9, PLE-C2/PLE-C3, PLE-D4/PLE-D5, PLE-F3/PLE-F4 and PLE-G6/PLE-G7, respectively, and then functional expression. Their activity toward p-NPA was determinated and results were as follows: 1.17U/mg/5.9U/mg, 0.99U/mg/0.63U/mg, 40.27U/mg/0.83U/mg, 7.84U/mg/ 13.29U/mg, 1.57U/mg/0.8U/mg, respectively. The results showed that the enzyme activity of PLE-D5 and PLE-D4 in this group was the largest. The PLE-D4 and PLE-D5 amino acid sequences were compared and found that there were four amino acids differences in different locations, which located in 43, 92, 150, 305. Using PLE-D4 and PLE-D5 as the template, the amino acids in four positions were site-directed mutagenesis, and obtained eight mutants. Functional expression of the mutants was performed, the enzyme activities of L43 P, T92 I, A150 D and M305 V in PLE-D4 toward p-NPA were 0.48 U/mg, 7.68 U/mg, 1.07U/mg, 2.85U/mg, respectively. Compared with PLE-D4, the enzyme activities decreased by 84.5 times, 5.2 times, 37.7 times and 14.1 times respectively. The enzyme activities of P43 L, I92 T, D150 A, V305 M toward p-NPA in four mutants of PLE-D5 were respectively 1.33U/mg, 2.0U/mg, 0.38U/mg and 0.57U/mg. Compared with PLE-D5, P43 L, I92 T, the enzyme activities respectively increased 1.6 times, 2.4 times, D150 A, V305 M, respectively, decreased by 2.2 times, 1.5 times. Through the analysis to the mutants, the activity of PLE-D4 43 position leucine L and 92 position threonine T can significantly increase the activity of PLE to the common substrate p-NPA. It can provide theoretical basis for the study of the PLE activity by site-directed mutagenesis. |
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