Screening Neutral Phytase Producing Strains And Construction Of Engineered Strains

Neutral phytases, which exhibit their desirable activity profile at neutral pH, good thermal stability and strict substrate specificity for the calcium-phytate complex and produce myo-inositol trisphosphate as the final product, have considerable potential in commercial and environmental applications. This investigation aims to screen out the neutral phytase-producing strains, optimize the fermentation parameters and clone the neutral phytase gene. On these bases, the further study aims to construct engineered strains producing neutral phytase for the industrialized production. The main results were listed as follows:1. Screening, identification and fermentation parameters of neutral phytase producing strainsA wild type strain of neutral phytate-degrading bacteria was isolated from soil samples of Zhejiang province, and identified as Bacillus licheniformis ZJ-6 according to the morphological, physico-chemical properties and 16S rRNA sequences analysis. The optimal fermentation parameters for producing neutral phytase by strain ZJ-6 were determined by single factor test and the results were as follows:1.0% dextrose used as carbon source,0.1%(NH4)SO4 as nitrogen source, initially pH7.5, incubation temperature 55℃. After incubation for 36h under these conditions, the activity of neutral phytase reached 0.267 U/ml with specific activity 0.701U/mg.2. Sequence analyses of the neutral phytase gene from B.licheniformis ZJ-6The phyC gene encoding the neutral phytase of B.licheniformis ZJ-6 was amplified by polymerase chain reaction (PCR) with designed primers according to the sequences of phytase genes from Bacillus. The amplified fragment was cloned into the vector pUCm-T. Sequencing results showed that the coding region was 1146 bp in size encoding a peptide of 381 amino acid residues, in which there were a signal peptide of 31 amino acids and a mature peptide of 350 amino acids. The deduced amino acid sequence of phyC showed significant similarity to the reported sequences of other neutral phytases from Bacillus. Moreover, it did not display the active site hepta-peptide motif RHGXRXP or the catalytically active dipeptide HD in acidic phytases. The mature protein of the PhyC had random coil (57.42%), extended strand (40.29%) and alpha helix (2.29%). The 3-D structure of the phyC was predicted by homologous modeling technology, the results indicated that the homology was 68.2% between the template protein (1h61 A) and the mature peptide of the phyC.3. The construction of neutral phytase-producing engineered strainsThe gene encoding B.licheniformis ZJ-6 neutral phytase (phyC) was cloned from vector pUCm-T phyCl by PCR. The phyC with original signal peptide was ligated into expression vector pET-30a(+) and transformed into Escherichia coli BL21 (DE3). The phyC was successfully expressed and the product, EPhyC5, was purified by Ni-NTA chromatography. The activity of purified EPhyC5 was 2.87U/mg. SDS-PAGE analysis showed that molecular mass of the purified EPhyC5 was 42.86kDa, which was similar to the the theory molecular mass,42.1 kDa.The phyCm fragment encoding the mature peptide sequence of B.licheniformis ZJ-6 neutral phytase was amplified and cloned into expression vector pPIC9K. The pPIC9K-phyCm plasmid was linearized and transformed into Pichia Pastoris GS115 strain by electroporation. Positive strain was screened and purified by culturing on MD/MM plate and G418. After 96h 0.5% methanol induction, the activity of recombinant phytase (PPhyCm6) in culture supernatant of pPhyCm6 transformant was 8.64 U/mg. SDS-PAGE analysis showed that the molecular mass of PPhyCm6 was 38.78 kDa, which was in good agreement with the theory molecular mass, 38.7kDa.4. Analyses of enzymatic properties of PhyC, EPhyC5 and PPhyCm6The optimum temperature and pH of the PhyC were 55℃and 7.0, respectively. After treated at 80℃, pH 7.0 for 10 min, the residual activity of PhyC was 57.36%. Over 80% of PhyC activity was retained after treatment of the enzyme by preincubation over a pH range of 6.5-9.0 for 1 h at 25℃.The optimum temperature and pH of the EPhyC5 were 60℃and 7.0, respectively. After treated at 80℃, pH 7.0 for 10 min, the residual activity of EPhyC5 was 68.26%. Over 80% of EPhyC5 activity was kept after treatment of the enzyme by preincubated over a pH range of 6.0-9.0 for 1 h at 25℃.The optimum temperature and pH of the PPhyCm6 were 60℃and 7.5, respectively. After treated at 80℃, pH 7.5 for 10 min, the residual activity of PPhyCm6 was 59.42%. Over 80% of PPhyCm6 activity was retained after the enzyme treated by preincubation over a pH range of 5.0-9.0 for 1 h at 25℃.The others enzymatic properties of the recombinant neutral phytase (EPhyC5 and PPhyCm6) were identical to those of the original enzyme. PhyC, EPhyC5 and PPhyCm6 were greatly inhibited by EDTA and metal ions such as Cd2+, Mn2+, Cu2+ and Ba2+. Their thermostabilities and pH stabilities could be improved in the presence of 1mmol/L Ca2+. All of them exhibit highly strict substrate specificity for sodium phytate and had no enzymatic activity on other phosphate esters tested. NaF and vanadate showed slight inhibition on their activities.