| Entomppathogenic fungi (EPF) contribute to the natural regulation of insect, tick and mite populations. Over750different species have been identified to date. Among them, the genus Metarhizium anisopliae is one of the most important and best-studied entomopathogenic fungus which is common and widely distributed in soil throughout the world, for controlling of a wide range of host insects, because of its effective pathogenesis and the minimal negative toxicological or environmental impact. For this reason, M. anisopliae has been used commercially in many countries as a biological control agent. Some reports suggested that its extracellular phosphatase might play a pivotal role in invading and killing the hosts. Especially the protein tyrosine phosphatase (PTPase) plays a role in insect immune system. So it will contribute to the research of M. anisopliae’’invadation and nosogenesis that studying the extracellular phosphatase further.1. Metarhizium anisopliae CQMa102biomass growth and extracellular phosphatase biosynthesis with different phosphorus sourcesThis study explored the effect of different phosphorus sources on biomass growth and extracellular phosphatase biosynthesis of M. anisopliae by using shaking flask methods. Those evaluated phosphorus sources are including inorganic phosphorus (KH2PO4), simple organic phosphorus (phytate sodium and C6H5Na2PO4·2H2O) and protein phosphorus (casein). The results indicated that:1) The fungus had the highest productivity when casein was used as the sole phosphorus source, and the biomass was up to4.94g/L in the culture; The highest extracellular protein biosynthesis was395.0mg/L, these values were2-7folds higher than the results obtained with other phosphorus sources; The highest ACP activity was1208.5U/μg, these values were2-10folds higher than the results obtained with other phosphorus sources. However, the specific activity of ACP and the activity of protein phosphatase were significant lower than the values in the medium of KH2PO4and C6H5Na2PO4·2H2O.2) There was no significant difference between the medium with KH2PO4(3.24g/L) and disodium phenyl phosphate (3.32g/L) on the biomass growth of M.anisopliae. But the extracellular protein biosynthesis and the activity of ACP were higher, the specific activity of ACP and rotein phosphatase were lower when KH2PO4was used than the other one.3) in terms of biomass growth (0.59g/L), production of acidic phosphatase and PTPase, phytate sodium yielded the worst result, but the protein biosynthesis was higher than C6H5Na2PO4·2H2O and the activity of protein serine/threonine phosphatase were higher than casein.4) The highest activity of protein phosphatase was observed in the medium with disodium phenyl phosphate (C6H5Na2PO4·2H2O) as phosphorus source. But it has the worst protein biosynthesis (53.95mg/1), which makes the specific activity of ACP ranked the first (5.99U/μg).The results indicated that, in terms of biomass growth, production of acidic phosphatase and extracellular protein, casein yielded the best result, followed by KH2PO4and C6H5Na2PO4·2H2O, wherein phytate sodium came at last. For purification of the extracellular protein phosphatase of M. anisopliae, the optimal phosphorus source is C6H5Na2PO4·2H2O, where there was the highest specific activity of protein phosphatase.2. Metarhizium anisopliae CQMa102phosphatase expression in Pichia pastoris, purification and its enzymatic characteristics analysisLi et al have purified a kind of acid phosphatase that the enzyme could specifically dephosphorylate trans-Golgi p230in vitro, and proved that the enzyme is PTPase by characterization, In order to study this PTPase further, We have identified and cloned the PTPase gene from a locust specific Metarhizium anisopliae Strain CQMa102(GenBank accession number EFY93239.1). Xin have cloned the PTPase gene by the method of RT-PCR and make the CQMa102PTPase expressed in Pichia pastoris to verify its protease activity.So this study screen the postive clones by the methods of geneticin resistance and PCR, then incubate the postive with methanol, in order to screen the high expression strain, purified the PTPase from the liquid media after the incubation with methanol. To characterize the recombinant proteins produced by the transformants, SDS-PAGE、IEF-PAGE and Western blot as well as purification with Ni-NTA and enzymatic characteristics analysis were performed.1) The molecular weingt (Mr) and the isoelecterc point (p) of the PTPase were about85kDa and6.15respectivity. The enzyme had optimum activity at about pH6.5, The optimum temperature for activity was determined to be about70℃.2) There was no obvious effect on PTPase activity was observed when Ca2+(50mM) and Ba2+(1mM) was used, phosphatase inhibitors Dithiothreitol,β-mercaptoethanol and N-ethylmaleimide were really inhibite the activity of enzyme. But PTPase activity was significantly inhibited by EDTA (32.04%inhibition at20mM), F"(34.44%inhibition at1mM), Ag+(37.21%inhibition at1mM), Cu2+(45.63%inhibition at5mM), Fe2+(24.89%inhibition at10mM), Zn2+(30.52%inhibition at1mM). In addition, PTPase activity was increased by Sodium molybdate (54.92%increase at10mM), Sodium tungstate (58.75%increase at20mM), EDTA (48.08%increase at10mM), Ca2+(68.62%increase at20mM), Fe2+(37.87%increase at1mM), Mg2+(65.16%increase at20mM), Mn2+(90.94%increase at20mM), Ba2+(45.02%increase at20mM), and Co2+(110.41%increase at10mM)3) At its optimal pH of6.5and optimal temperature of708, the protein showed the highest activity when using O-phospho-L-tyrosine and pNPP as substrate. The purified enzyme showed activity on O-phospho-L-serine%O-phospho-L-threonine. |
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