Cloning and expression of the L-aspartate β-decarboxylase gene from Alcaligenes faecalis CCRC 11585

• Main Authors: Chang-Chih Chen, 陳長志
• Other Authors: Chia-Yin Lee
• Format: Others
• Language: zh-TW
• Published: 1999
• Online Access: http://ndltd.ncl.edu.tw/handle/19955887141325137837

碩士 === 國立臺灣大學 === 農業化學研究所 === 87 === The DNA fragment containing L-aspartate β-decarboxylase gene was amplified by polymerase chain reaction (PCR). Six clones were isolated from Alcaligenes faecalis CCRC 11585 genomic library that was hybridized by the PCR-amplified fragment. One of the six clones harvesting plasmid pBK-asdAE1 could express L-aspartate β-decarboxylase activity and 3,003 bp of the cloned DNA fragment was sequenced. Sequence analysis revealed an open reading frame (ORF) consisting of 1,599 bp that encodes a 533 amino-acids polypeptide. The nucleotide sequence of the L-aspartate β-decarboxylase gene from Alcaligenes faecalis CCRC 11585 showed 84 % identity with that from Pseudomonas dacunhae CCRC 12623, and the amino acid sequence showed 93 % identity. The amino acid sequence of the L-aspartate β-decarboxylase from Alcaligenes faecalis CCRC 11585 showed 51-59 % homology with various aminotransferases. Comparison of the L-aspartate β-decarboxylase with 5 aspartate aminotransferases and 2 tyrosine aminotransferases revealed 17 conserved amino acid that include most of the conserved amino acid residues within PLP binding domain of aminotransferases. L-aspartate β-decarboxylase contains the aminotransferases class-I pyridoxal-phosphate attachment site motif including the lysine residue covalently bound to pyridoxal 5'-phosphate. L-aspartate β-decarboxylase also contains the (A,G)XXXXGK(S,T) motif, characteristic of ATP/GTP -binding site motif A (P-loop). The L-aspartate β-decarboxylase gene of Alcaligenes faecalis CCRC 11585 was cloned into expression vector pQE-30 to express the L-aspartate β-decarboxylase in Escherichia coli Nova-Blue. Intracellular soluble form protein and inclusion body of E. coli Nova-Blue / pQE-asdA1 exhibit L-aspartate β-decarboxylase activity. A protein band sized at 62 kDa is present on the SDS-PAGE gel of the inclusion body of E. coli Nova-Blue / pQE-asdA1 that is absent from the gel of E. coli Nova-Blue / pQE-30.