| Myrosinase is a β-glucosinolates glucoside hydrolase, it can decompose glucosinolates glycosidase to produce glucose, sulfate, isothiocyanates, acetonitrile, thiocyanate, under certain conditions. The isothiocyanates not only play an important role in plant resistance to infection of pathogen and pest, but also can significantly improve efficacy of biofumigation on suppression of soil borne disease. So far, there are many reports about extracting myrosinase and gene cloning of myrosinase from plants, however, the development of large-scale, commercial production of myrosinase was slow. Using microbial fermentation to produce myrosinase has a great advantage such as high yield and stable quality. In order to resolve the practical problem, the Trichederma Atroviride T155 was studied to understand the enzymology properties, effects of metal ions and protectants on myrosinase, and to separate and purify of protein of myrosinase.By set different conditions we studied the best growth conditions of Trichoderma atroviride T155. In triangle flask, oat culture medium was more suitable for Trichederma Atroviride T155 growth, and the best culture conditions was:180 rpm/min,7 piece of hyphae, cultivating 3 days. Under the pilot the best condition in the 30 L fermentor for Trichederma Atroviride T155 was:0.3 MPa for the input pressure,0.15 MPa for tank pressure.The best condition for breaking wall was different under different amount of hyphae of Trichederma Atroviride T155. By setting different ultrasonic wall breaking conditions, the results indicated that the best condition for cell wall breaking was:200W,10 minutes,20s of interval.In addition, we studied the enzymatic properties by setting different temperature, pH and metal ions. The best reaction temperature and pH was 30℃ and 6 respectively, which indicated that the myrosinase could play a better catalytic role under slightly acidic condition. The Km is 2.364mmol/L and Vmax is 909.1mmol/(L·min). Ag1+, Zn2+, Pb2+, Mg2+, Hg2+, Fe3+ inhibited enzyme activity of myrosinase, Ca2+ promoted enzyme activity at the concentration of 0.069-17 g/L. The stable effect of EDTA, DTT and glycerinum on myrosinase activity was studied in this paper, the result showed that 1 mmol/L EDTA associated with 3 mmol/L DTT was the best treatment for keeping myrosinase activity, and 85% enzyme activity was retained after 27 days of preservation at 4℃. The experiment successfully made enzyme preparation with lime as a carrier, and at 4℃, it can keep 80% of enzyme activity for 3 months.A mutagenic strain T155-UV36 was obtained through ultraviolet mutagenesis, and the myrosinase activity was increased by 46%. And the cellulose activity, P-glycosidase enzymes activity, chitinase activity were also increased, especially the chitinase activity was increased by 1 time. The growing conditions and mycelium character of mutagenic strain T155-UV36 were similar with that of the original strain.The pure myrosinase was obtained through cell disruption, ammonium sulfate precipitation, dialysis, Sephadex G-100 chromatography, DEAE-52 chromatography and Sephadex G-200 chromatography from Trichederma Atroviride T155 and T155-UV36. Molecular weight of the myrosinase produced by T155 and T155-UV36 was about 150 kD. Moreover, the elution profiles and electrophoresis of both kinds of myrosinase was similar. So, it was speculated that the two strains belong to one species, but theyield of myrosinase of T155-UV36 was higher. |
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