Cloning And Expression Of Hydantoin Oxoprolinase Gene B In Bacillus Lichenformis MV0658

Using the chromosome DNA of Bacillus lichenformis strain MV0658 which we preserved in our laboratory as template, through PCR the specific amplified fragments of Hydantoin utilization protein B in Bacillus lichenformis was obtained. After separation and purification, these fragments were ligated to the vector pUC19, then transformed E.coli JM109. By usingα-complement test, extraction of plasmid, restriction digestion analysis, and sequencing, we got six recombinant plasmids, which were named from pMV1000 to pMV1005. They all have the insertion of the homologous fragment of Hydantoin utilization protein B in Bacillus lichenformis. Thereinto, pMV1000 and pMV1001 are in the same direction with reading frame, the others are opposite. We sequenced pMV1000, and named the cloned gene fragment Hyu_Q. After searching in NCBI GenBank with Hyu_Q, using Blast and ClustalW software to analysis and compare the homology of its nucleotide and amino acids sequence, the result showed that the whole gene fragments were 1740bp long, coding 579 amino acids sequences, G+C content was 51.72%, theoretical molecular weight was 63KD, theoretical pI was 5.49. There were 35.8 percentage Hydrophobic amino acids in the sequence.Hyu_Q fragment separated from pMV1000 was ligated to an expressive vector pET28a, then transformed E.coli Rostta. After colony PCR test and a series of restriction digestion analysis, it was proved that recombinant plasmid pMV1006 contains the PCR amplification, and it was in the same direction with reading frame. After IPTG inducing expression, a corresponding Hyu_Q protein was obtained as predicted.Nucleotide sequence homology comparison of Hyu_Q with sequence of Hydantoin utilization protein B in Bacillus lichenformis ATCC14580 , the results showed that there were 8 nucleotides changing in Hyu_Q sequence, with 5 transitions and 3 transversions, the two sequences had 99% identity in a total of 1740 bp. Then homology comparison of Hyu_Q putative amino acids sequence with Hydantoin utilization protein B(YP₀79153.1)amino acids sequence in Bacilus lichenformis ATCC14580, it was shown that 6 amino acids changed in Hyu_Q putative amino acids sequence, the two sequences had 99% identity.Using bioinformatics software to analysis Hyu_Q putative amino acids sequence structure and predict its function, the results showed that amino acids had four hydrophobic region: from 64th to 72th, from 293th to 299th, from 339th to 343th and from 411th to 417th. In the 22th, 76th, 540th and 570th amino acids had fort tendency to formαhelix, and in the 55th, 265th, 380th and 468th amino acid had fort tendency to formβlaminar. There were many random helix structure in the sequence. Hyu_Q putative amino acids sequence had no N-terminal signal peptide and no trans-membrane structure. Then the tertiary structure prediction of Hyu_Q putative amino acids sequence was also obtained.