Wang H, Jones R W
Department of Botany & Plant Pathology, Purdue University, West Lafayette, IN 47907, USA.
Appl Microbiol Biotechnol. 1997 Aug;48(2):225-31. doi: 10.1007/s002530051042.
Conserved regions within a family-5 beta-1,4,-endoglucanase (Eg11), produced by the fungus Macrophomina phaseolina, have been modified through site-directed mutagenesis, resulting in production of an enzyme with novel substrate requirements. The engineered form was generated through mutagenesis of D232A, which lies within the substrate-binding cleft of Eg11. Wild-type Eg11 requires a minimum substrate size of five glucosyl units, while the engineered form requires a minimum of six glucosyl units. Screening was facilitated by the unique ability to obtain functional expression of the fungal endoglucanase in Escherichia coli. Wild-type and mutated Eg11 have equivalent activity on cellohexaose, and both release cellobiose from the reducing end of the cellodextrin. This is the first example of protein engineering of an endoglucanase that results in a novel minimum substrate requirement for cellohexaose. This substrate specificity has not been reported for any native endoglucanases, thus the modified Eg11 may prove useful in applications requiring specific hydrolysis of complex carbohydrates such as beta-glucans.
菜豆壳球孢真菌产生的家族5β-1,4-内切葡聚糖酶（Eg11）中的保守区域已通过定点诱变进行了修饰，从而产生了一种具有新底物需求的酶。通过对位于Eg11底物结合裂隙内的D232A进行诱变，生成了工程形式。野生型Eg11需要至少五个葡萄糖基单元的底物大小，而工程形式则需要至少六个葡萄糖基单元。在大肠杆菌中获得真菌内切葡聚糖酶的功能表达的独特能力促进了筛选。野生型和突变型Eg11对纤维六糖具有同等活性，并且都从纤维糊精的还原端释放纤维二糖。这是内切葡聚糖酶蛋白质工程的首个实例，其导致对纤维六糖产生新的最小底物需求。尚未有任何天然内切葡聚糖酶报道过这种底物特异性，因此修饰后的Eg11可能在需要特异性水解复杂碳水化合物（如β-葡聚糖）的应用中证明是有用的。
