Purification, Properties And Antifungal Activity Of β-1, 3-Glucanase

Plant diseases by soil-borne were more important in all of diseases, whichcould be spread by ploughing, irrigating and other farming working. If thediseases came on, it would spread rapidly and do serious harm to plants. In1980s, various plant diseases by soil-borne were more and more seriousbecause the planting system and structure of industries had been adjusted andthe economy communion extended gradually, such as fusarium wilt byFusarium oxysporum f. sp. Vasinfectum, verticillium wilt by Verticilliumdahliae and root rot disease by Rhizoctonia solani. Biological control of plantdieases by soil-borne had attached importance to domestic and oversea experts,especially, strains of biological control were modified genes by means oftechnology and method of molecular biology in order to obtain high-poweredly transgenetic strains, which would bring into play potention ofbiological control perfectly, because the period of resistance breeding was toolong, resistance was single and genetic characters were not stable, which couldnot satisfy the needs of production. Using too much chemical pesticide hadresulted in pollution in the environment and did harm to human being anddomestic animals. Meanwhile, it induced resistance of pathogen to chemicalpesticide. β-1,3-glucanase was an important pathogen-related protein. It couldcatalyze the hydrolysis of β -1,3-glucan that was one of the majorcomponents of most pathogenic fungal cell walls, which made cell inclusion 3β-1,3-葡聚糖酶的纯化,特性及其抗菌活性overflow and dead at last. It showed that this enzyme had effect on pathogensby the test of inhibiting fungi in vitro. It not only inhibited the growth offungal mycelium but also released evocator of fungal cell walls whichindirectly promoted to accumulate phytoalexin in host and enhance resistance.Meanwhile, in the light of few β-1,3-glucan in plant cell walls, this enzymewould play important roles in resistance to fungal diseases. One strain of Streptomyces producing β-1,3-glucanase was studiedsystemically about purification,properties and antifungal activity of thisenzyme, which aimed to give a base on biological control of plant diseases bysoil-borne. 1. Soil samples were collected from the roots of Chinese cabbage in thesuburb of Tai'an. By the test of clearing zones on pachyman agar medium, 37strains producing β-1,3-glucanase are obtained, including 18 bacteria, 7fungi and 12 actinomycetes. Four strains of Streptomyces spp., R8, R15, R31 andAS, produced clearing zones were the biggest, the clearest and the mostpermanent. Streptomyces R15 produced the most β-1,3-glucanase. R15 wasprimarily identified to Streptomyces aureo chromogenes. 2. Enzyme extracts were obtained from the strain R15 in pachyman liquidmedium by centrifugation. The enzyme activity to degradeβ-1,3-glucanasewas measured. The enzyme activity was the highest in the eleventh day, so thatit was the optimum time producing enzyme.β-1,3-glucanase was purifided tohomogeneity, by using 10%-75% ammonium sulfate fraction, hydroxylapatitchromatography and DEAE-52 chromatography. Purification folds and yieldwere13.98 and 4.031%, respectively. The molecular mass of this enzyme wasestimated to be 41.3kDa by 12%SDS-PAGE. 3. The results showed that the optimum temperature for the enzymeactivity was 55℃ and the optimum pH was 5.5. Theβ-1,3-glucanase wasmore stable when temperature was at 45- 55℃ and pH was at 4.5- 7.5, theenzyme activity was above 70%. Thermal stability of the enzyme was under55℃. The enzyme almost lost activity at 65℃, and the lost ratio of enzyme 4山东农业大学硕士学位论文activity was 90.3%. pH stability of the enzyme was under 7.5, the enzymeactivity was above 85%. When pH was 8, the level of activity was 55.97%.Fe3+, K+, Cu2+, Hg2+ had inhibitory effect on the enzyme activity, while Ca2+,Fe2+, Ba2+, Mn2+, Al3+ and Zn2+ could stimulat...