Site-directed Mutagenesis Of Gene Encoding Thermostable Acidic α-Amylase And Its Expression In Bacillus Licheniformis

Thermostable α-amylase, widely used in starch saccharification, is one of themost important enzymes. Extracellular ? α-amylase (PFA) from Pyrococcusfuriosus is extremely thermophilic and of low optimal pH and the calcium ionindependent, which make it of potentially commercial application. The aim of thisstudy is to investigate the obstacle factor that affects on the heterologousexpression level of PFA encoding gene.Using Escherichia coli as host cells, we studied the effect of the composite oftRNAs on expression of PFA. The recombinant plasmid pET-PFA was applied totransform E. coli DE3 CodonPlus-RIL cells. Modification of intracellular tRNApool improved the expression level of PFA with about nine folds highercomparison to E. coli DE3 as a host cell.Consequently, we comprehensively reassembled the codons appeared in thegene encoding PFA according to codon usage in Bacillus licheniformis. Codonsused seldom in B. licheniformis were exchanged with its synonymy. With the aid ofthe computer analysis, total 78 oligonucleotides were designed and chemicallysynthesized. Assembly PCR followed by specific PCR obtained the complete genefragment. The amplified DNA fragment was subcloned into pBlueScript SK(-) andapplied to sequence. The nucleic acid sequence was 9.62% difference comparisonto the native sequence encoding PFA and completely identity to the sequence wepreviously designed.We studied the expression of amyF in B. licheniformis. pSK-amyF wasdigested by EcoR I and Sma I and the amyF fragment was recovered by gelextraction. The pBL-amyF was constructed by inserting the amyF fragment at thesites of EcoR I and Sma I of pBL-WZX. The PamyL-SamyL-amyF-Tart-?Km-3'amyL'cassette was obtained by PCR using pBL-amyF as template and subsequentlysubcloned into the plasmid pHY300PLK resulted in pHY-amyF. The purifiedpBL-amyF or pHY-amyF was transformed into B. licheniformis by electroporationand the positive transformants were selected on LB plates supplemented with the 5μg/mL kanamycin. The recombinant enzyme encoded by amyF produced by therecombinants was studied. It had a similar activity optimum at pH 5.0, and theoptimum temperature at 95-98oC to the wild PFA. Fore the more, the expressionlevel of the recombinant enzyme was much more improved.