Purification and characterization of two exo-cellobiohydrolases from the brown-rot fungus Coniophora puteana (Schum ex Fr) Karst.
Purification and characterization of two exo-cellobiohydrolases from the brown-rot fungus Coniophora puteana (Schum ex Fr) Karst.
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两种来自褐腐真菌 Coniophora puteana (Schum ex Fr) Karst 的外切纤维二糖水解酶的纯化和表征。
DOI：
10.1006/abbi.1993.1076
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发表时间：
1993
影响因子：
3.9
通讯作者：
G. Canevascini
中科院分区：
文献类型：
--
作者：
D. Schmidhalter;G. Canevascini
将两种胞外外切纤维二糖水解酶（EC 3.2.1.91）从褐腐真菌Coniophora puteana（Schum ex Fr）Karsten菌株EMPA 62的培养物滤液中纯化至均一。纯化方案包括三个连续的色谱步骤，即Q Sepharose fast flow、Superose 12和Fractogel TSK DEAE-650 S。这两种酶，命名为纤维二糖水解酶（CBH）I和CBH II，分别纯化了4.6和3.9倍，分别与总的9和19%的活性回收率，分别。在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳中，两种酶均以单一条带迁移，CBH I的M（r）为52，000，CBH II为50，000;通过FPLC凝胶过滤（TSK G3000 SW），M（r）更高（65，000和60，000）。这两种酶都是糖基化的，具有相似的等电点（pI 3.6和3.55）和几乎相同的活性最适pH值接近5。内切糖苷酶H消化得到两种不同的多肽，其中CBH I的分子量降低了6.5 kDa，CBH II的分子量降低了2.5 kDa。水解对硝基苯基乳糖苷（pNPL）的比活性几乎相同的两种酶（0.46与0.40 μ mol mg-1 min-1，在40 ℃）和Km值（6.8和4.3 mM，在30 ℃）也非常接近。这两种酶都被纤维二糖竞争性抑制：以pNPL为底物，确定CBH I和CBH II的Ki值分别为1.2 mM和2.4 mM。这两种酶以相同的方式作用于纤维素（无定形或结晶）和纤维糊精，主要释放纤维二糖，但对染色的羧甲基纤维素无活性。纤维二糖不水解，而纤维三醇水解成等摩尔量的纤维二糖和葡萄糖醇：这些结果支持这些酶是外切纤维二糖水解酶的解释。它们在褐腐真菌中的存在是一个新的事实。
Two extracellular exo-cellobiohydrolases (EC 3.2.1.91) were purified to homogeneity from the culture filtrate of the brown-rot fungus Coniophora puteana (Schum ex Fr) Karsten, strain EMPA 62. The purification scheme involved three successive chromatographic steps, namely Q Sepharose fast flow, Superose 12, and Fractogel TSK DEAE-650S. The two enzymes, named cellobiohydrolase (CBH) I and CBH II, were purified by a factor of 4.6 and 3.9, respectively, with an activity recovery of 9 and 19% of total, respectively. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis both enzymes migrated as single bands according to a M(r) of 52,000 for CBH I and 50,000 for CBH II; by FPLC gel filtration (TSK G3000 SW) the M(r)'s were higher (65,000 and 60,000). Both enzymes were glycosylated, had similar isoelectric points (pI 3.6 and 3.55) and nearly identical pH optima for activity close to 5. Endoglycosidase H digestion gave two distinct polypeptides where the molecular weight was lowered by 6.5 kDa for CBH I and by 2.5 kDa for CBH II. The specific activities for the hydrolysis of p-nitrophenyllactoside (pNPL) were nearly identical for both enzymes (0.46 versus 0.40 mumol mg-1 min-1 at 40 degrees C) and the Km values (6.8 and 4.3 mM at 30 degrees C) were also very close. Both enzymes were competitively inhibited by cellobiose: with pNPL as substrate, Ki values of 1.2 mM for CBH I and 2.4 mM for CBH II were determined. The two enzymes acted in an identical fashion on cellulose (either amorphous or crystalline) and on cellodextrins, liberating mainly cellobiose, but were inactive on dyed carboxymethylcellulose. Cellobiose was not hydrolyzed whereas cellotriitol was hydrolyzed to equimolar amounts of cellobiose and glucitol: these results support the interpretation that these enzymes are exo-cellobiohydrolases. Their presence in a brown-rot fungus is a new fact.