The Cloning And Expression Of Rhodococcus Sp. Partial Gene For Putative Cyclopentanone Monooxygenase

Biological-chemical conversion catalyzed by biocatalysis systems such as cells, recombinants is an emerging technology in the biochemistry development, namely biocatalysis. Attractive features of biocatalysis include high efficiency, diversity, substrate specificity, regioselectivity, chemoselectivity, enantioselectivity, as well as mild reaction conditions.Enzymes can catalyze a variety of reactions, which include a significant oxidizing reaction—Baeyer-Villiger reaction. In 1899, Baeyer and Villiger found that in the presence of peracetic acid, non-cyclo ketones can be converted into esters, while cyclo-ketones can be converted to lactones. This organic synthesis reaction contains a very high application potential. As a result of the recent pressing need of chiral chemistry compounds, catalysts of Baeyer-Villiger reaction for chiral synthesis have had some breakthroughs. These chiral catalysts are mainly transition metal complexes. However, they have many flaws and shortcomings, such as unstable, multi-products with plenty of side reactions; high toxicity, low yield, low optical purity and the intensive, harsh reaction conditions.Cyclopentanone and cyclohexanone are two important industrial organic compounds. Cyclopentanone is widely used as a intermedium of acridine, spices, bio-pharmaceuticals, and pesticides; cyclohexanone is widely used in the manufacture of caprolactam, it is also the intermedium of fiber, rubber, paraffin, paint, and pharmaceutical intermediates. Both are flammable, when encountering fire, heat or contacting with oxidants, there will be a risk of combustion and explosion. Cyclopentanone can irritate the eyes and skin, and it can harm the body by inhaling, ingesting and skin contact. In view of the wide industrial applications of cyclopentanone and cyclohexanone and their obvious danger to human, as well as the environment requirement of the production modernization, it is necessary to take certain measures on the industrial waste degradation.Cyclopentanone 1,2 - monooxygenase (CPMO) is considered as a potential enzyme with high application value for cyclopentanone degradation, CPMO (NCIMB 9872) can work with many substrates with high speed and little pollution. Lucubrating this enzyme or finding new CPMO can play an important role in the environment protection and biotransformation. In 2003, Beilen selected 25 strains from alicyclic compounds medium and amplified DNA fragments from 12 strains by the use of highly degenerate primers, some of which had been proved to be effective BVMO sequences, and some were classified as putative BVMO sequences. The study found that all of these segments had homology with published BVMO sequences. T3G6, AE12 was classified as putative cyclopentanone monooxygenase cpmA gene fragments, and no more report followed. In this study, the total length of 825 bp DNA sequence was obtained from Rhodococcus sp, including the gene sequence which had 97% homology with T3G6 and AE12, and was expressed in Escherichia coli.The contents of this paper are focused on the following parts:(1)Cloning of Rhodococcus sp. partial cpmA gene for putative cyclopentanone monooxygenaseRhodococcus rhodochrous genomic DNA was extracted as a template for PCR to obtain the DNA fragment of about 800bp length. The DNA fragment was connected to the pMD19-T vector, and was transformed into JM109 competent cells. Positive colonies were screened by PCR and digestion to verify whether the gene is inserted. The DNA sequencing results showed that the inserted fragment length was 825 bp. (2)Bioinformatics analysis of the cpmAThe sequence was analyzed by BLAST, it turned out that the sequence included a 425 bp sequence of Rhodococcus sp. partial cpmA gene for putative cyclopentanone Baeyer-Villiger monooxygenase (97% homology with the two partial cpmA gene for putative cyclopentanone monooxygenase sequence T3G6 and AE12). The entire sequence had a high degree of homology with the genomic sequences which had the features of the monooxygenase family. By the prediction of primary structure and secondary structure of the sequence, the sequence coded 274 amino acids. Analyzed by ANTHEPROT 5.0 software, its theoretical molecular weight was about 31 kD, and the theoretical isoelectric point (PI) was 5.245. In the secondary conformation, the helix was 42%, folded and random coil were 28% and 30% respectively, and there was no turn. Analyzed by BLAST, the amino acid sequence had 93.7% and 95.1% homology with putative cyclopentanone monooxygenase sequence AE12 (AJ515360), T3G6 (AJ515361) respectively. The proteins with higher amino acid sequence homology were all BV monooxygenase family members, of which the highest scores were cyclopentanone 1,2-monooxygenase, then were the other BV monooxygenases. This fragment contained two "GXGXXG" sequences, which could become non-covalent binding sites of FAD and BVMO; it also contained a characteristic fingerprint "FXGXXXHXXXW" of BVMO family. Therefore, the fragment could also be classified as a putative cyclopentanone monooxygenase fragment and it is more complete than AE12 (AJ515360) and T3G6 (AJ515361).(3)The expression of Rhodococcus sp. partial cpmA gene for putative cyclopentanone Baeyer-Villiger monooxygenaseThe gene was from the prokaryote. Analysis of its rare codons of E. coli amino acid, the rare codons's occurrence was relatively low. Therefore, E. coli expression system and the current powerful expression vector pET were selected for this study. The insert containing DNA was prepared via PCR according to the vector pMD19-T-cpmA. A Pair of primers were designed with restriction site NdeⅠand HindⅢrespectively. The PCR product and the pET-28a(+) vector were digested by the restriction enzymes (NdeⅠ/HindⅢ), and then integrated to construct recombinant expression vector pET-cpmA. The recombinant expression plasmid was transformed into E. coli BL21(DE3) cells, and positive strains were screened. The DNA sequencing results showed that the target gene sequence was correctly inserted into the pET-28a(+) vector, and were consistent with the previous results. The recombinant prokaryotic expression plasmid pET-cpmA were expressed in E. coli BL21 (DE3) and induced by IPTG. The results showed that the expression would be successful only in the IPTG-induced condition, which demonstrated that vector pET-28a(+) was strictly controllable. A molecular weight about 31 kD protein band was seen obviously by SDS-PAGE analysis, which was consistent with the theoretical prediction.In short, the search for new genes and their identifications are an important task in the post-genome era. Gene functions can be predicted through homology comparison. In this study, a Rhodococcus sp. partial cpmA gene for putative cyclopentanone Baeyer-Villiger monooxygenase was found in Rhodococcus rhodochrous genomic DNA, and the sequence structure was preliminary analyzed. The sequence was cloned and expressed in E.coli, and its biological function needed further study.