LYTICASE - ENDOGLUCANASE AND PROTEASE ACTIVITIES THAT ACT TOGETHER IN YEAST-CELL LYSIS
LYTICASE - ENDOGLUCANASE AND PROTEASE ACTIVITIES THAT ACT TOGETHER IN YEAST-CELL LYSIS
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DOI：
10.1128/jb.142.2.414-423.1980
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发表时间：
1980-01-01
影响因子：
3.2
通讯作者：
SCHEKMAN, R
中科院分区：
文献类型：
--
作者：
SCOTT, JH;SCHEKMAN, R
从O.在以不溶性酵母葡聚糖作为C源的基本培养基上生长的解黄嘌呤菌。溶菌活性由两种协同酶活性组成，它们在羧甲基纤维素和Sephadex G-150上共纯化，但在Bio-Gel P-150上分离。第一个成分是β- MW为55，000的1，3-葡聚糖酶。酵母葡聚糖的Km为0.4mg/ml，海带多糖的Km为5.9mg/ml。水解β- 1，3-葡聚糖是内溶的，产生从葡萄糖到10或更多的低聚物的产物的混合物。产品的大小分布是pH值依赖性的，较小的寡聚物占主导地位，在较低的pH值。葡聚糖酶是无法裂解酵母细胞，没有2-巯基乙醇或第二裂解组分，一个ankaline蛋白酶。这两种试剂对多糖底物上的葡聚糖酶活性都没有任何影响。该蛋白酶的分子量为30，000，可水解Azocoll和多种变性蛋白。这种酶很不寻常，因为它对葡聚糖凝胶有亲和力。虽然活性是最不敏感的蛋白酶抑制剂，它是多糖的影响，酵母甘露聚糖是一种有效的抑制剂。该酶不具有任何甘露聚糖酶活性。链霉蛋白酶和胰蛋白酶都不能替代这种蛋白酶促进酵母细胞裂解。用单一纯化步骤容易获得的酶的部分纯化级分具有高的裂解比活性，并且在核酸酶、蛋白酶和几丁质酶污染方面优于商业制剂上级。溶胞酶应用于原生质球、膜和核酸的分离，并被证明在酵母转化过程中是有用的。
Yeast lytic activity was purified from the culture supernatant of O. xanthineolytica grown on minimal medium with insoluble yeast glucan as the C source. The lytic activity consisted of 2 synergistic enzyme activities which copurified on carboxymethyl cellulose and Sephadex G-150, but were resolved on Bio-Gel P-150. The 1st component was a .beta.-1,3-glucanase with a MW of 55,000. The Km for yeast glucan was 0.4 mg/ml; that for laminarin was 5.9 mg/ml. Hydrolysis of .beta.-1,3-glucans was endolytic, yielding a mixture of products ranging from glucose to oligomers of 10 or more. The size distribution of products was pH dependent, smaller oligomers predominating at the lower pH. The glucanase was unable to lyse yeast cells without 2-mercaptoethanol or the 2nd lytic component, an ankaline protease. Neither of these agents had any effect on the glucanase activity on polysaccharide substrates. The protease had a MW of 30,000 and hydrolyzed Azocoll and a variety of denatured proteins. The enzyme was unusual in that it had an affinity for Sephadex. Although the activity was insensitive to most protease inhibitors, it was affected by polysaccharides; yeast mannan was a potent inhibitor. The enzyme did not have any mannanase activity. Neither Pronase nor trypsin could substitute for this protease in promoting yeast cell lysis. A partially purified fraction of the enzymes, easily obtained with a single purification step, had a high lytic specific activity and was superior to commercial preparations in regard to nuclease, protease and chitinase contamination. Lyticase was applied in spheroplast, membrane and nucleic acid isolation, and proved useful in yeast transformation procedures.