Karlsson Johan, Siika-aho Matti, Tenkanen Maija, Tjerneld Folke
Department of Biochemistry, Lund University, PO Box 124, SE-221 00 Lund, Sweden.
J Biotechnol. 2002 Oct 9;99(1):63-78. doi: 10.1016/s0168-1656(02)00156-6.
Trichoderma reesei produces five known endoglucanases. The most studied are Cel7B (EG I) and Cel5A (EG II) which are the most abundant of the endoglucanases. We have performed a characterisation of the enzymatic properties of the less well-studied endoglucanases Cel12A (EG III), Cel45A (EG V) and the catalytic core of Cel45A. For comparison, Cel5A and Cel7B were included in the study. Adsorption studies on microcrystalline cellulose (Avicel) and phosphoric acid swollen cellulose (PASC) showed that Cel5A, Cel7B, Cel45A and Cel45Acore adsorbed to these substrates. In contrast, Cel12A adsorbed weakly to both Avicel and PASC. The products formed on Avicel, PASC and carboxymethylcellulose (CMC) were analysed. Cel7B produced glucose and cellobiose from all substrates. Cel5A and Cel12A also produced cellotriose, in addition to glucose and cellobiose, on the substrates. Cel45A showed a clearly different product pattern by having cellotetraose as the main product, with practically no glucose and cellobiose formation. The kinetic constants were determined on cellotriose, cellotetraose and cellopentaose for the enzymes. Cel12A did not hydrolyse cellotriose. The k(Cat) values for Cel12A on cellotetraose and cellopentaose were significantly lower compared with Cel5A and Cel7B. Cel7B was the only endoglucanase which rapidly hydrolysed cellotriose. Cel45Acore did not show activity on any of the three studied cello-oligosaccharides. The four endoglucanases' capacity to hydrolyse beta-glucan and glucomannan were studied. Cel12A hydrolysed beta-glucan and glucomannan slightly less compared with Cel5A and Cel7B. Cel45A was able to hydrolyse glucomannan significantly more compared with beta-glucan. The capability of Cel45A to hydrolyse glucomannan was higher than that observed for Cel12A, Cel5A and Cel7B. The results indicate that Cel45A is a glucomannanase rather than a strict endoglucanase.
里氏木霉可产生五种已知的内切葡聚糖酶。研究最多的是Cel7B（EG I）和Cel5A（EG II），它们是内切葡聚糖酶中含量最丰富的。我们对研究较少的内切葡聚糖酶Cel12A（EG III）、Cel45A（EG V）以及Cel45A的催化核心的酶学性质进行了表征。为作比较，研究中纳入了Cel5A和Cel7B。对微晶纤维素（微晶纤维素）和磷酸膨胀纤维素（PASC）的吸附研究表明，Cel5A、Cel7B、Cel45A和Cel45A核心吸附于这些底物。相比之下，Cel12A对微晶纤维素和PASC的吸附较弱。分析了在微晶纤维素、PASC和羧甲基纤维素（CMC）上形成的产物。Cel7B从所有底物中产生葡萄糖和纤维二糖。Cel5A和Cel12A在底物上除了产生葡萄糖和纤维二糖外，还产生纤维三糖。Cel45A表现出明显不同的产物模式，以纤维四糖为主要产物，几乎不形成葡萄糖和纤维二糖。测定了这些酶对纤维三糖、纤维四糖和纤维五糖的动力学常数。Cel12A不水解纤维三糖。与Cel5A和Cel7B相比，Cel12A对纤维四糖和纤维五糖的k（Cat）值显著较低。Cel7B是唯一能快速水解纤维三糖的内切葡聚糖酶。Cel45A核心对所研究的三种纤维寡糖均无活性。研究了这四种内切葡聚糖酶水解β-葡聚糖和葡甘露聚糖的能力。与Cel5A和Cel7B相比，Cel12A对β-葡聚糖和葡甘露聚糖的水解作用略弱。与β-葡聚糖相比，Cel45A能够显著更多地水解葡甘露聚糖。Cel45A水解葡甘露聚糖的能力高于Cel12A、Cel5A和Cel7B。结果表明，Cel45A是一种葡甘露聚糖酶，而非严格意义上的内切葡聚糖酶。
