Acid Trehalase From Entomopathogenic Fungus Metarhizium Anisopliae: Purification, Characterization And Study On Its Gene

Metarhizium anisopliae, a kind of entomopathogenic fungus widely applied at home and abroad, plays an important role in biological control of insects. Compared with chemical insecticides, mycoinsecticides are not infective or toxic to human and can not pollute environment, but the slow insecticidal speed has been recognized as a potential drawback to prevent the utilization of these fungi against insects. Researches on mechanisms of fungal pathogenesis and host defense may suggest strategies for the development of more efficient anti-insect mycoinsecticides. Trehalose (α-D-glucopy- ranosyl (1-1) -α-D- glucopyranoside), a non-reducing disaccharide, exists in almost all kinds of insects as the predominant sugar (80-90% of total sugar) in the haemolymph of insects. Trehalose plays such an important role in insects'blood physiology that its exhaustion may be detrimental to the insects, which as a consequence must be viewed as a potential nutrient source for insect pathogenic fungi like Metarhizium anisopliae once the fungus infected into the haemolymph. Trehalose is impermeable substrate for some fungi, and corresponding glycosidase (acid trehalase and /orα-glucosidase) must be external to the cell membrane to hydrolyze it into glucose before use. Metarhizium anisopliae can produce acid trehalase once they get into the haemolymph of insects and the enzyme can effectively hydrolyze trehalose in the haemolymph of insects, which may suggested that the Metarhizium acid trehalase play an important role in the pathogenesis of insects.Much less is known about acid trehalase in fungi and at present only limited acid trehalase gene of four fungi has been cloned and identified. It does not show any homology between acid trehalases and neutral trehalases, and there also appears weak sequence identity among those reported acid trehalases, which enhance the challenge of the research on fungus acid trehalase. Also there has no relative report about acid trehalase and its gene in entomopathogenic fungus until now. In this research Metarhizium anisopliae var. acridum strain CQMa102 and the migratory locust, Locusta migratora manilensis was used, and the acid trehalase isozymes production condition was optimized. A Metarhizium acid trehalase was isolated through two step anion exchange chromatography (High Q Sepharoes and 25Q Sepharoes) followed by gel filtration and narrow Precast IEF. It is the first time that an acid trehalase was isolated from Metarhizium anisopliae and its properties were characterized in detail. Base on amino acid sequence analyses of the pure enzyme, An guess oligonucleotide...