PURIFICATION AND CHARACTERIZATION OF THE MAJOR BETA-1,4-ENDOGLUCANASE FROM THERMOMONOSPORA-CURVATA
PURIFICATION AND CHARACTERIZATION OF THE MAJOR BETA-1,4-ENDOGLUCANASE FROM THERMOMONOSPORA-CURVATA
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DOI：
10.1111/j.1365-2672.1995.tb03160.x
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发表时间：
1995-10-01
期刊：
影响因子：
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通讯作者：
STUTZENBERGER, FJ
中科院分区：
文献类型：
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作者：
LIN, SB;STUTZENBERGER, FJ
弯曲高温单孢菌（Thermomonospora curvata）的主要β-1，4-内切葡聚糖酶（EG）在纤维素上生长后的无细胞培养液中占总EG活性的80%以上。该酶经硫酸铵沉淀、离子交换层析和尺寸排阻HPLC纯化至电泳纯。当在70 ℃，pH 6.0下用2.5%（w/v）羧甲基纤维素（CMC）测定时，该单体酶的比活性为750 IU mg（-1）。在聚合度为3200的CMC上观察到最高活性。EG在60 ℃、pH 6.0下稳定48 h，在80 ℃下半衰期为30 min;最适温度和pH分别为70-73 ℃和6.0-6.5。摩尔wt为100000，pI为4.0。K-m和V-max值分别为7.33 mg ml（-1）和833 μ mol min（-1）。Fe ~（2+）、Hg ~（2+）、Ag ~+和Pb ~（2+）对EG活性有抑制作用，而Zn ~（2+）和二硫苏糖醇对EG活性有促进作用。N端前12个氨基酸残基为：Asp-Glu-Val-Asp-Glu-Ile-Arg-Asn-Gly-Asp-Phe-Ser，其中谷氨酸和天冬氨酸占24%，未发现氨基糖。
The major beta-1,4-endoglucanase (EG) of the thermophilic actinomycete, Thermomonospora curvata, contributed over 80% of the total EG activity recovered from cell-free culture fluid after growth on cellulose, The enzyme was purified to electrophoretic homogeneity by ammonium sulphate precipitation, ion-exchange chromatography and size exclusion HPLC. This monomeric enzyme had a specific activity of 750 IU mg(-1) when assayed with 2.5% (w/v) carboxymethyl cellulose (CMC) at 70 degrees C, pH 6.0. Highest activity was observed on CMC with a degree of polymerization of 3200. The EG was stable for 48 h at 60 degrees C, pH 6.0 and had a half-life of 30 min at 80 degrees C; temperature and pH optima were 70-73 degrees C and 6.0-6.5, respectively. The mol. wt was 100000 and the pI was 4.0. The K-m and V-max values were 7.33 mg ml(-1) and 833 mu mol min(-1), respectively. EG activity was inhibited by Fe2+, Hg2+, Ag+ and Pb2+, and enhanced by dithiothreitol and Zn2+. The first 12 amino acid residues at the N-terminus were: Asp-Glu-Val-Asp-Glu-Ile-Arg-Asn-Gly-Asp-Phe-Ser. Glutamic and aspartic acid constituted 24% of the total amino acid composition; no amino sugar was found.