Department of Chemistry, M. V. Lomonosov Moscow State University, Vorobyovy Gory, Moscow, Russia.
Biotechnol J. 2010 Aug;5(8):871-80. doi: 10.1002/biot.201000050.
Nine major cellulolytic enzymes were isolated from a culture broth of a mutant strain of the fungus Penicillium verruculosum: five endo-1, 4-beta-glucanases (EGs) having molecular masses 25, 33, 39, 52, and 70 kDa, and four cellobiohydrolases (CBHs: 50, 55, 60, and 66 kDa). Based on amino acid similarities of short sequenced fragments and peptide mass fingerprinting, the isolated enzymes were preliminary classified into different families of glycoside hydrolases: Cel5A (EG IIa, 39 kDa), Cel5B (EG IIb, 33 kDa), Cel6A (CBH II, two forms: 50 and 60 kDa), Cel7A (CBH I: 55 and 66 kDa), Cel7B (EG I: 52 and 70 kDa). The 25 kDa enzyme was identical to the previously isolated Cel12A (EG III). The family assignment was further confirmed by the studies of the substrate specificity of the purified enzymes. High-molecular-weight forms of the Cel6A, Cel7A, and Cel7B were found to possess a cellulose-binding module (CBM), while the catalytically active low-molecular-weight forms of the enzymes, as well as other cellulases, lacked the CBM. Properties of the isolated enzymes, such as substrate specificity toward different polysaccharides and synthetic glycosides, effect of pH and temperature on the enzyme activity and stability, adsorption on Avicel cellulose and kinetics of its hydrolysis, were investigated.
从一株产黄青霉突变株的发酵液中分离得到 9 种主要的纤维素酶：5 种内切-1,4-β-葡聚糖酶（EGs），分子量分别为 25、33、39、52 和 70 kDa，以及 4 种纤维二糖水解酶（CBHs：50、55、60 和 66 kDa）。根据短序列片段的氨基酸相似性和肽质量指纹图谱，分离得到的酶初步被分类为不同糖苷水解酶家族：Cel5A（EG IIa，39 kDa）、Cel5B（EG IIb，33 kDa）、Cel6A（CBH II，两种形式：50 和 60 kDa）、Cel7A（CBH I：55 和 66 kDa）、Cel7B（EG I：52 和 70 kDa）。25 kDa 的酶与先前分离的 Cel12A（EG III）相同。通过对纯化酶的底物特异性研究进一步证实了家族归属。发现高相对分子质量形式的 Cel6A、Cel7A 和 Cel7B 具有纤维素结合模块（CBM），而具有催化活性的低相对分子质量形式的酶以及其他纤维素酶缺乏 CBM。研究了分离得到的酶的性质，如对不同多糖和合成糖苷的底物特异性、pH 和温度对酶活性和稳定性的影响、在 Avicel 纤维素上的吸附以及水解动力学。
