| As one of the green additives, phytase has been used in feed industry more widely today,. Scientists found Bacillus phytase has the higher thermostablity than Aspergillus phytase. Moreover, its optimum reaction pH is between 6.5 and 7.5, which makes it be used carp with neutral intestinal tract. Up to now, there is no commercial neutral phytase on market yet.According to the Bacillus phytase gene sequences downloaded from GenBank, a pair of primers was designed to amplify the full sequence of phytase phyC gene from B. subtilis WHNB02 by PCR. The PCR product was cloned and sequenced. The sequence analysis results showed the full gene was 1152bp in length, and it coded a 383 amino acid multi-peptide. Compared the nucleotide and amino acid sequences with the other six phytases of Bacillus, their phylogenetic tree was drawn. The results indicated all the seven phytases from Bacillus had the high homology and belonged to two groups genetically. Furthermore, the specialized features of the encoded protein, including signal peptide cleavage site, N-glycosylation sites, codon bias, secondary structure, hydrophobicity and 3D structure, were predicted and analyzed. The gene and its protein sequences were accepted by GenBank with accession numbers AF220075 and AA043434.1.Based on the sequence of AF220075 and multi-clone site of E.coli expression vector pET-30b(+),the non-fusion and fusion expression fragments without signalpeptide sequence were amplified by PCR. The two fragments were cloned, sequenced and inserted into vector pET-30b(+) with lilac promoter. The recombined expression vectors pET30NFphyC and pET30FphyC were screened and transformed into E. coli BL21(DE3). The products with phytase activity were expressed successfully. Induction experiments showed the optimum induction temperature of non-fusion and fusion expression was 25℃ and 30℃ respectively, and the lower induction temperature was helpful to increase soluble protein expression. Their optimum induction concentration of IPTG and lactose was 0.4mmol/L and 1% respectively, but the peak value time of lactose was later about l-2h and lower than that of IPTG. The yield of non-fusion and fusion phytase came to 13% and 15% of total protein and their molecular weights were 40.13KD and 43.27KD, respectively.In order to perform the secretory expression of Bacillus phytase and get better enzymatic characters because of the post-translation modification of yeast, Bacillus phytase phyC gene was expressed in yeast primarily. According to the sequence of AF220075 and multi-clone site of P. pastorisi expression vector pPIC3.5K and pPIC3.5K, the intracellular and secretory expression fragments without signal peptide sequence were amplified by PCR. The products were cloned and sequenced. Then, two recombined expression vectors pPIC3.5KphyC and pPIC9KphyC were constructed. The plasmids were linearized by Bgl II and transformed into Pichia Pastoris GS115 strain by electroporation. Positive strains were screened and purified by culturing on MD plate, MM plate and PCR. Furthermore, they were cultured and induced by methanol. SDS-PAGE analysis showed that phytase was expressed intracellularly, and the yield came to 24% of the total soluble protein and its molecular weight was 42.01KD. However, the phytase expressed secretively was a kind of glycoprotein which had two molecular weight 53.5KD and 50.9KD because of the different glycosylation level. Additionally, in wort semi-synthetic medium, the phytase yield came to 2453U/ml after 48 hours induction, 10.5 times of original B. subtilis strain.All the four kinds of genetic engineering Busillus phytases, non-fusion and fusion phytases expressed in E. coli and intracellular and secretory phytases expressed in P. pastoris, were purified and performed to measure their optimum reaction temperature, thermostability and optimum reaction pH. The results indicated their optimum reaction temperature was 55 ℃, 75 ℃, 70℃ and 65 ℃ respectively. Comparison with the natural phytase, the temperature was lower 5℃ and higher 15℃ , 10℃ and 5...
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