Heinzelman Pete, Snow Christopher D, Smith Matthew A, Yu Xinlin, Kannan Arvind, Boulware Kevin, Villalobos Alan, Govindarajan Sridhar, Minshull Jeremy, Arnold Frances H
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA.
J Biol Chem. 2009 Sep 25;284(39):26229-33. doi: 10.1074/jbc.C109.034058. Epub 2009 Jul 22.
A quantitative linear model accurately (R(2) = 0.88) describes the thermostabilities of 54 characterized members of a family of fungal cellobiohydrolase class II (CBH II) cellulase chimeras made by SCHEMA recombination of three fungal enzymes, demonstrating that the contributions of SCHEMA sequence blocks to stability are predominantly additive. Thirty-one of 31 predicted thermostable CBH II chimeras have thermal inactivation temperatures higher than the most thermostable parent CBH II, from Humicola insolens, and the model predicts that hundreds more CBH II chimeras share this superior thermostability. Eight of eight thermostable chimeras assayed hydrolyze the solid cellulosic substrate Avicel at temperatures at least 5 degrees C above the most stable parent, and seven of these showed superior activity in 16-h Avicel hydrolysis assays. The sequence-stability model identified a single block of sequence that adds 8.5 degrees C to chimera thermostability. Mutating individual residues in this block identified the C313S substitution as responsible for the entire thermostabilizing effect. Introducing this mutation into the two recombination parent CBH IIs not featuring it (Hypocrea jecorina and H. insolens) decreased inactivation, increased maximum Avicel hydrolysis temperature, and improved long time hydrolysis performance. This mutation also stabilized and improved Avicel hydrolysis by Phanerochaete chrysosporium CBH II, which is only 55-56% identical to recombination parent CBH IIs. Furthermore, the C313S mutation increased total H. jecorina CBH II activity secreted by the Saccharomyces cerevisiae expression host more than 10-fold. Our results show that SCHEMA structure-guided recombination enables quantitative prediction of cellulase chimera thermostability and efficient identification of stabilizing mutations.
一个定量线性模型准确地（R² = 0.88）描述了通过三种真菌酶的SCHEMA重组构建的54个已表征的真菌纤维二糖水解酶II类（CBH II）纤维素酶嵌合体家族成员的热稳定性，表明SCHEMA序列模块对稳定性的贡献主要是可加性的。31个预测的热稳定CBH II嵌合体中有31个的热失活温度高于来自特异腐质霉的最耐热亲本CBH II，并且该模型预测还有数百个CBH II嵌合体具有这种卓越的热稳定性。检测的8个热稳定嵌合体中有8个在比最稳定亲本至少高5℃的温度下能够水解固体纤维素底物微晶纤维素，其中7个在16小时的微晶纤维素水解试验中表现出卓越的活性。序列-稳定性模型鉴定出一个单一的序列模块，它能使嵌合体的热稳定性提高8.5℃。对该模块中的单个残基进行突变确定C313S取代是整个热稳定效应的原因。将此突变引入不具有该突变的两个重组亲本CBH II（里氏木霉和特异腐质霉）中，降低了失活，提高了微晶纤维素水解的最高温度，并改善了长时间水解性能。该突变还稳定并改善了黄孢原毛平革菌CBH II对微晶纤维素的水解，黄孢原毛平革菌CBH II与重组亲本CBH II的序列同一性仅为55 - 56%。此外，C313S突变使酿酒酵母表达宿主分泌的里氏木霉CBH II总活性提高了10倍以上。我们的结果表明，SCHEMA结构导向的重组能够对纤维素酶嵌合体的热稳定性进行定量预测，并有效鉴定稳定突变。
