棉花黄萎病拮抗细菌胞外酶检测及其酶活测定

Detection of Extracellular Enzymes of Antagonizing Cotton Verticillium wilt Bacteria and Determination of Enzyme Activity

【目的】研究Bacillus vanillea SMT-24、Bacillus velezensis BHZ-29、Bacillus subtilis SHT-15、Bacillus atrophaeus SHZ-24菌株产真菌细胞壁降解酶能力,为4株拮抗菌的机理研究及开发利用提供依据。【方法】通过平板透明圈法初步测定4株拮抗菌产胶体几丁质酶、蛋白酶、葡聚糖酶、纤维素酶种类;采用DNS法定量几丁质酶活、纤维素酶活、葡聚糖酶活,采用福林-酚法定量蛋白酶活。【结果】平板透明圈法显示:SMT-24菌株产蛋白酶、葡聚糖酶、纤维素酶,BHZ-29菌株产蛋白酶、纤维素酶、葡聚糖酶,SHT-15菌株产蛋白酶、葡聚糖酶、纤维素酶,SHZ-24菌株产几丁质酶、蛋白酶、葡聚糖酶; DNS法、福林酚法定量结果显示:在发酵期间酶活总体呈现先增加后减小的趋势,与细菌生长对数期增长趋势相同(酶活低于3. 00 IU除外); 4株拮抗菌酶活比较中,SHZ-24菌株产几丁质酶活最高,酶活最高为(12. 37±0. 15) IU; SHZ-24菌株产蛋白酶活最高,最高值达(24. 22±0. 45) IU,SMT-24、SHT-15菌株产葡聚糖酶活较高,最高酶活分别(12. 29±0. 23) IU、(12. 07±0. 59) IU,SMT-24菌株产纤维素酶活最高,酶活最高达(6. 43±0. 24) IU。【结论】4株拮抗菌均产几丁质酶、葡聚糖酶、纤维素酶、蛋白酶,酶活随发酵时间呈现先增大后减小的趋势,在细菌对数期,酶活与细菌数量呈正相关关系(酶活低于3. 00 IU除外)。

【Objective】To investigate species of fungal cell wall degrading enzymes of Bacillus vanillea SMT-24,Bacillus velezensis BHZ-29,Bacillus subtilis SHT-15 and Bacillus atrophaeus SHZ-24 strains in the hope of providing basis for the study of the mechanism of four antagonistic bacteria and their application in agriculture.【Method】The chitinase,protease,glucanase,and cellulase enzymes from four antagonistic bacteria were preliminarily determined by the plate transparent circle method;the chitinase activity,cellulase activity,and Glucanase activity were quantified by DNS method,and protease activity were quantified by Folin-phenol.【Result】The transparent circle method showed that:SMT-24 strain produced protease,glucanase and cellulase,BHZ-29 strain produced protease,cellulase and glucanase,SHT-15 strain produced protease,glucanase and cellulase and SHZ-24 strain produced chitinase,protease and glucanase;the results of DNS method and Folin-Phenol method showed that during the fermentation,the total enzyme activity first increased and then decreased,and the number of logarithmic growth phases of bacteria was the same as the growth trend of enzyme activity(except that the enzyme activity was lower than 3.00 IU);Among the four antagonistic enzyme activities,SHZ-24 strain produced the highest chitinase activity,with the highest activity of(12.37±0.15)IU,the protease activity of SHZ-24 was the highest,with the highest value of(24.22±0.45)IU,the highest glucanase activity was observed in SMT-24 and SHT-15 strains,the highest enzyme activities were(12.29±0.23)IU and(12.07±0.59)IU,respectively,SMT-24 produced the highest cellulase activity and the highest enzymatic activity was(6.43±0.24)IU.【Conclusion】The four antagonistic bacteria produced chitinase,glucanase,cellulase and protease,the enzyme activity first increased and then decreased during fermentation time.In the bacterial logarithmic phase,there was a positive correlation between enzyme activity and bacterial count.(except that enzyme activity was lower than 3.00 IU).