Clone And Expression Of Hydantoin Oxoprolinase In Bacillus Lichenformis

With Bacillus lichenformis strain MR1229 as original strain, using the chromosome DNA of MR1229 as template, PCR was carried out. It was shown that specific amplified fragments of Hydantoin utilization protein A in Bacillus lichenformis. After separation and purification ,these fragments were ligated to the vector pGM-T, then transformed E.coli JM109. By usingα-complement test, extraction of plasmid, and restriction digestion analysis, sequencing. It was proved that clone pMH-2 has an insertion of the homologous fragment of Hydantoin utilization protein A in Bacillus lichenformis.The insertion fragment designated as hydantoin oxoprolinase(Hyu_O).Search Hyu_O in GenBank, using Blast, ClustalW software analysis and compare homology of its nucleotide sequence, amino acid sequence. The result show that this gene total length is 2106bp,coding 701 amino acid sequences, G+C content 49.85%, molecular weight 76.7KD,theoritic isoelectric point 5.24.Among the total hydrophobic amino acid content is 46.6%.Hyu_O has 99% nucleotide sequence similarity with Hydantoin utilization protein A in Bacilus lichenformis ATCC14580.There are 7 nucleotides have changed with 5 transitions and 2 transerversions. And it has 99% amino acid sequence similarity with hypothetical protein BLio1982 in Bacilus lichenformis ATCC14580 and 3 amino acids changed(Phe→Leu,Thr→Ile, Ser→Thr).Accroding to the conserved domain prediction of NCBI, Hyu_O has a complete hydantoin oxoprolinase and three domain. The first domain is from 9 to 188 amino acid (Hydan_A_N), Hydantoinase/oxoprolinase N-terminal region. The second domain is from 209 to 494 amino acid (Hydantoinese_A), Hydantoinase/oxoprolinase. This family includes the enzymes hydantoinase and oxoprolinase (EC 3.5.2.9). Both reactions involve the hydrolysis of 5-membered rings via hydrolysis of their internal imide bonds. The third domain is from 6 to 687 amino acid (HyuA), N-methylhydantoinase A/acetone carboxylase, beta subunit ,Amino acid transport and metabolism/Secondary metabolites biosynthesis, transport, and catabolism.Using bioinformatic analysis hydantoin oxoprolinase amino acid sequence structure and predict function. The result shows that from160 to 170, from 470 to 480 amino acid have two visible hydrophobicity region. In the 55th,510th,570th amino acid has fort tendency to formαhelix, and in the 170th,330th,380th amino acid has fort tendency to formβlaminar, and has random helix structure .From 510 to 530 amino acid has coiled coil. Hydantoin oxoprolinase has no N-terminal signal peptide,and have no transmembrane structure.