Overexpression Of Phytase Gene From B.amyloliquefaciens In Escherichia Coli And Pichia Pastoris

Bacillus amyloliquefaciens is one of thermophilic bacteria. The phytase found in B. amyloliquefaciens has shown the extremely thermostat)lity and considerable potential in large-scale production. In this study the phytase gene BAP from Bacillus amyloliquefaciens was cloned and fused in the expression plasmid pET-30a(+). then transferred into the host E.coli BL21.Further, the BAP with origin signal peptide sequence was cloned into the expression plasmid pPIC9K and had been overexpressed in Pichia pastoris GS115.Then the biochemical properties of two expressed phytase were investigated. The main results obtained are as follows:Bacillus amyloliquefaciens BA could produce phytase. Its genome was used as template for gene cloning, the phytase gene of BA was amplified by the touchdown polymerase chain reaction (TD-PCR) with primers (5BAP01 and 3BAP01) designed according to the conserved regions of the phytase gene in Genbank.The DNA fragment obtained was cloned into pGEM?T Easy Vector, generating pGEM?T Easy BAP Vector. Sequencing result showed that its nucleotide sequence was 1167 bp, containing an Open Reading Frame(ORF) from 11 bp to 1159 bp,encoding a peptide of 383 amino acids . with a signal peptide of 26 amino acids. The deduced molecular weight of enzyme was 41.9 kDa.Comparison to this sequence with phytase genes of the other Bacillus sources. The nucleotide sequence and amino acid sequence identity were 100% and 100% for AF029053 (B.subtillus); 96% and 97% for AF453255(5. amyloliquefaciens); 92% and 94% for m5968(Bacillus sp.DSU); 92% and 94% for AY220075(5. subtillus); 92% and 94% for AYQ55220(B.amyloliquefaciens); 92% and 93% for AYS\82Q8(Bacillus sp.), respectively.Based on two primers 5BAP01 containing a BamHI site and 3BAP01 containing a Xhol site, the pGEM?T Easy BAP Vector was double digested with BamHI and Xhol, and then cloned into the expression vector pET-30a(+) previously digested with the same enzymes. The resulting plasmid BAPpET30a(+)Vector was introduced into E.coli BL21(DE3). The expressed enzyme has a molecular mass of 50.2 kDa dectected by SDS-PAGE, which was larger than the 41.9 kDa deduced from amino acid sequence. The fused protein was purified by immobilizing metal affinity chromatography. The expression phytase has normal bioactivity, which was up to 480.6 U/ml (1.27 times as that of B. amyloliquefaciens), over 33.5% the total soluble protein of E. coli.A pair of expression primers, the sense primer 5BAPK01 containing a SnaBI site and antisense primer 3BAPK01 containing a NotI site were designed according to the Multiple Cloning Sites(MCS) of expression vector pPIC9K and the Open Reading Frame of BA gene. The PCR fragment were cloned into pGEM?T Easy Vector, generating subcloned vector of pGEM?T Easy BAPK Vector. After the vector was double digested with SnaBI and NotI it was cloned to pPIC9K, an expression vector double digested with the same restriction enzymes, and the recombinat plasmid BAP-pPIC9K was obtained. The plasmid was linearized with Bgl II ,then, introduced into the host Pichia pastoris GS115 by electroporation. The Pichia pastoris recombinants for phytase overexpression were screened by enzyme activity analysis and SDS-PAGE. It had an apparent molecular weight of 45 kDa as determined by SDS-PAGE.The result revealed that the phytase was overexpressed and secreted into the medium supernatant, its activity was up to 4120 U/ml, which was 10.9 times as that of B. amyloliquefaciens.The biochemical properties of the phytases produced by E.coli BAPE and Pichia pastoris BAPK showed that both of them had an optimum temperature of 65-70 ?and pH of 7.0-7.5 respectively, and it has 70% activity at pH 6.0-7.5. Effects of pH on stability of phytase showed that phytase BAPE and BAPK retained more than 85% treated containing for 1 h at pH 5.0-9.0. The thermostability of different phytase activities varied, the phytase BAPK retained 80.54% and 68.42% treated with containing for 2 min at 70?and 80 癈, and retained 70.03% and 50.69% treating with containing for 10 min at 70 ?and...