Dissertation
Dissertation > Agricultural Sciences > Plant Protection > Pesticide ( chemical control ) > Various pesticides > Pesticides > Other pesticides

Purification, Characterization and Gene Cloning of a Novel Protein Tyrosine Phosphatase from Metarhizium Anisopliae

Author LiZhenLun
Tutor XiaYuXian
School Chongqing University
Course Biomedical Engineering
Keywords Metarhizium anisopliae var. acridum entomopathogenic fungus protein tyrosine phosphatase gene cloning fermentation condition
CLC S482.39
Type PhD thesis
Year 2006
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Metarhizium anisopliae var. acridum strain CQMa102 was used as material in this study. The strain produced one phosphatase with pI of 9.45 when the casein was added to the medium as only phosphorus. The properties of the purified phosphatase indicated that the phosphatase is an extremely thermostable enzyme. Therefore, the study provides a new material for studying stable mechanism of extremely thermostable enzyme. Although the putative amino acid sequence of the phosphatase shares identiy with some fungal alkaline phosphatases, its substrate specificity, inhibitor sensitivity and containing HCX5RS peptide indicate that the enzyme is a novel PTPase. Moreover, one protein (toll-like receptor) involved with signal transduction could be dephosphorized by purified PTPase in vitro, which suggested that the PTPase could interfere the immune responses. Therefore, the PTPase has the important theory and application value in constructing a novel entomopathogenic fungus.The main results of the paper were as follows:Fermentation condition research The effects of carbon, nitrogen and phosphorus source, air, pH, inoculum size and time of fermentation on acid phosphatase production and growth of M. anisopliae were investigated in shake flask culture. The results indicated that the AcP production by M. anisopliae were far more effective using organic nitrogen than inorganic nitrogen, glucose than other carbon sources and protein organic phosphorus (such as casein) than other phosphorus. A specific phosphatase isoform with pI 9.45 was produced by the strain when grown on casein as sole phosphorus source, which probably dephosphorylated the casein from the medium ensuring orthophosphates for the fungal growth. Therefore, we selected it as the research aim. Optimal culture condition for purification as follows: glucose, 20 g/L; (NH4)2SO4, 2g/L; casein, 4g/L; KCl, 0.5g/L; MgSO4, 0.5g/L; microelement mixture solution, 10ml/L; inoculum size, 1×107 spores; aeration rate, 2/5 volume and 10g/LMES (initial pH 6.0). The culture was incubated on a rotary shaker (160 rev/min) at 27°C.Purification and characterization of the protein tyrosine phosphatasePurified and homogeneous PTPase from culture supernatant of M. anisopliae was obtained by using ConA-Sepharose 4B, HighQ and 25S column chromatography with a 41.0% yield. Molecular mass and isoelectric point of the purified enzyme were to be

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