| Summary: | 碩士 === 國立中興大學 === 分子生物學研究所 === 88 === Original research direction is cloning the thermostable N- Carbamoyl-D-amino acid amidohydrolase(nca) gene. Therefor, designing the degenerate primer is based on highly consensus amino acid sequence region from N- Carbamoyl-D-amino acid amidohydrolase. To use of nested PCR with genomic DNA from thermophilic bacterium Bacillus kaustophilus CCRC11223 and Bacilius sp. TS23 as template DNA would find two fragments with 250 bp in length but different in DNA sequences. Using those DNA fragments was found in good alignment with conserved amino acid sequence region from the Agrobacterium radiobacter N- Carbamoyl-D-amino acid amidohydrolase gene. The similarities of two DNA sequences from B. kaustophilus were 58% and 60%, and two other DNA sequences from Bacillus sp. TS23 were 50% and 56% with A.rediobacter nca, respectivily. Using those DNA fragments as probe to screen two genomic libraries of B. kaustophilus and Bacillus sp. TS23 would not find nca gene.
So we use the B. kaustophilus DNA fragment obtained previously to design a pair of specific primers and screen B. kaustophilus genomic library with PCR amplification. But all were found only the leucine aminopeptidase(lap) (EC 3.4.11.1) DNA fragments. To discus the reason, we find the last 7 nucleic acid bases on 3'' end of designed primer was identical to the DNA sequence of leucine aminopeptidase gene. Because it is not found in any commercial application, and the thermostable leucine aminopeptidase has its industrial potential. So we switch the reseach direction to clone the thermostable leucine aminopeptidase gene.
Using this DNA fragment of leucine aminopeptidase as probe to screen the thermostable leucine aminopeptidase gene from B. kaustophilus genomic library. We got 2 pure clones that both contain the leucine aminopeptidase gene. After DNA sequencing , we found they are the same sequence of leucine aminopeptidase gene. Then we transformed this gene into E. coli and found to expression the enzymatic activity of leucine aminopeptidase. The ORF of leucine aminopeptidase gene is 1,494 bp and translated into a peptide of 497 residues with molecule weight 53.7 kDa and its enzyme was found to be intracellular. The similarity was found to be 62% in amino acid sequence comparison between B. subtilus and B. kaustophilus. Amino acid alignment with the know lap genes were found this B. kaustophilus leucine aminopeptidase has two zinc cneters.
The B. kaustophilus leucine aminopeptidase gene was cloned into pQE30 vector and then expressed in E. coli Nova Blue. With the recovery of this pure enzyme, we find the optimal temperature is 65℃ and optimal pH is 10. In the situation with no metal, this enzyme was not found any catalytic activity. But the addition of Co2+、Mn2+及Ni2+ will promote the enzymatic acyivity know as a metalloezyme. When useing L-leucine-p-nitroanilide as substrate, the km is 0.028 mM and kcat is 7.90min-1.
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