Trudel J, Asselin A
Département de Phytologie, Faculté des Sciences de l'Agriculture et de l'Alimentation, Université Laval, Québec, Canada.
Anal Biochem. 1989 May 1;178(2):362-6. doi: 10.1016/0003-2697(89)90653-2.
Commercial Streptomyces griseus and Serratia marcescens chitinases and purified wheat germ W1A and hen egg white lysozymes were subjected to polyacrylamide gel electrophoresis under native conditions at pH 4.3. After electrophoresis, an overlay gel containing 0.01% (W/V) glycol chitin as substrate was incubated in contact with the separation gel. Lytic zones were revealed by uv illumination with a transilluminator after staining for 5 min with 0.01% (W/V) Calcofluor white M2R. As low as 500 ng of purified hen egg lysozyme could be detected after 1 h incubation at 37 degrees C. One band was observed with W1A lysozyme and several bands with the commercial microbial chitinases. The same system was also used with native polyacrylamide gel electrophoresis at pH 8.9. Several bands were detected with the microbial chitinases. The same enzymes were also subjected to denaturing polyacrylamide gel electrophoresis in gradient gels containing 0.01% (W/V) glycol chitin. After electrophoresis, enzymes were renatured in buffered 1% (V/V) purified Triton X-100. Lytic zones were revealed by uv after staining with Calcofluor white M2R as for native gels. The molecular weights of chitinolytic enzymes could thus be directly estimated. In denaturing gels, as low as 10 ng of purified hen egg white lysozyme could be detected after 2 h incubation at 37 degrees C. Estimated molecular weights of St. griseus and Se. marcescens were between 24,000 and 72,000 and between 40,500 and 73,000, respectively. Some microbial chitinases were only resistant to denaturation with sodium dodecyl sulfate while others were resistant to sodium dodecyl sulfate and beta-mercaptoethanol.
将市售的灰色链霉菌和粘质沙雷氏菌几丁质酶以及纯化的小麦胚芽W1A和鸡蛋清溶菌酶在pH 4.3的天然条件下进行聚丙烯酰胺凝胶电泳。电泳后，将含有0.01%（W/V）乙二醇几丁质作为底物的覆盖凝胶与分离凝胶接触孵育。用0.01%（W/V）荧光增白剂M2R染色5分钟后，用透射仪紫外光照射显示溶菌区。在37℃孵育1小时后，可检测到低至500 ng的纯化鸡蛋清溶菌酶。W1A溶菌酶观察到一条带，市售微生物几丁质酶观察到几条带。该系统也用于pH 8.9的天然聚丙烯酰胺凝胶电泳。微生物几丁质酶检测到几条带。相同的酶也在含有0.01%（W/V）乙二醇几丁质的梯度凝胶中进行变性聚丙烯酰胺凝胶电泳。电泳后，酶在含1%（V/V）纯化 Triton X-100的缓冲液中复性。如天然凝胶一样，用荧光增白剂M2R染色后用紫外光显示溶菌区。由此可以直接估计几丁质分解酶的分子量。在变性凝胶中，在37℃孵育2小时后，可检测到低至10 ng的纯化鸡蛋清溶菌酶。灰色链霉菌和粘质沙雷氏菌的估计分子量分别在24,000至72,000之间和40,500至73,000之间。一些微生物几丁质酶仅对十二烷基硫酸钠变性有抗性，而其他一些则对十二烷基硫酸钠和β-巯基乙醇有抗性。
