Cloning And Expression Of AppA Phytase Gene And Nattokinase Gene

Phytases are hydrolytic enzymes that initiate the release of phosphate from phytate, the major phosphorus (P) form in animal feeds of plant origin. Phytases are found naturally in microorganisms and plants. Monogastric animals, such as pigs and poultry, are not able to utilize phytate phosphorus efficiently, since they have only low levels of phytase activity in their digestive tracts, phytate also acts as an antinutritional agent in monogastric animals by chelating various microelements needed by the animal.Escherichia coli phytase appA is an alternative enzyme with performance advantages over the conventional A. niger enzyme. The appA phytase displays several favorable characteristics: an acidic pH optimum close to the physiological pH range of the stomach of pigs and chickens, higher affinity to sodium phytate, greater resistance to pepsin than the commercially available A. niger PhyA, and higher catalytic efficiency for phytate than that of all other known phytases.In this study, an Escherichia coli strain with high phytase activity was screened from pig excreta. Using primers designed according to the sequence originally described by Dassa, appA gene was amplified by PCR technique. DNA sequencing of the appA gene showed an open reading frame of 1299 bp. The deduced appA phytase composed of 432 amino acids (predicted molecular mass, 47.06 kD) also contained the reserved active-site motif RHGXRXP, which is shared by other phytases and acid phosphatases. To obtain large amounts of appA phytase, the appA gene was subcloned into the prokaryotic expression vector pET-28a(+) and baculovirus transfer vector pVL-1393 under the control of the lac and polyhedrin promoter, respectively. The appA phytase was overexpressed in E. coli strain BL21 induced by lactose. The recombinant baculovirus harboring the appA gene was obtained after co-transfection and several screening rounds. The newly molted 5th instar larva of silkworm Bombyx mori was infected with the recombinant virus. Using a silkworm baculovirus expression vector system (BEVS), a large amount of appA phytase was obtained (up to 7710 per mL hemolymph). SDS-PAGE analysis revealed the molecular mass ofprokaryotic and eukaryotic derived appA phytase was approximately 50 kD and 53 kD, respectively. Both the two phytase had a optimal temperature and pH at 60 癈 and pH 4.5 except the baculovirus derived phytase seemed to be more thermostable. The enzymatic activity of the both prokaryotic and eukaryotic derived appA phytase were increased in the presence of Ca2+ and Mn2+ at a concentration of 1 mM. Due to the high expression efficiency and the enzymatic characteristics, BEVS derived appA phytase seemed to be a valuable candidate for the application of feed supplement.Nattokinase (NK) is a new fibrinolytic enzyme which cleaves directly cross-linked fibrin in vitro/in vivo. In the present study, the nucleotide sequence of the nattokinase gene was amplified from the genomic DNA of B.subtilis (natto) by PCR. DNA sequence analysis showed that the cloned nk gene was highly homologous to the sequence reported by Nakamura. The deduced amino acid sequence also had the conserved sequences (serine 221, histidine 64, and aspartic acid 32) which was essential for the catalytic center of serine proteases.The nk gene was then cloned into the transfer vector pVL1393. Then Bm cell line was co-transfected with parental Bm-NPV DNA and the transfer plasmid pVL-nk, the recombinant virus was then obtained subsequently with routine procedure. Nattokinase was expressed in silkworm larva by injection with the recombinant virus. As a serine proteases, the expression product was detected with a relatively high protease activity. A similar result was also obtained from fibrin plate assay, it revealed that the expressed NK was provided with fibrinolytic activity.