Abstract
BackgroundGlucoamylase is an important industrial enzyme in the saccharification of starch into glucose. However, its poor thermostability and low catalytic efficiency limit its industrial saccharification applications. Therefore, improving these properties of glucoamylase is of great significance for saccharification in the starch industry.ResultsIn this study, a novel glucoamylase-encoding gene TlGa15B from the thermophilic fungus Talaromyces leycettanus JCM12802 was cloned and expressed in Pichia pastoris. The optimal temperature and pH of recombinant TlGa15B were 65 ℃ and 4.5, respectively. TlGa15B exhibited excellent thermostability at 60 ℃. To further improve thermostability without losing catalytic efficiency, TlGa15B-GA1 and TlGa15B-GA2 were designed by introducing disulfide bonds and optimizing residual charge–charge interactions in a region distant from the catalytic center. Compared with TlGa15B, mutants showed improved optimal temperature, melting temperature, specific activity, and catalytic efficiency. The mechanism underlying these improvements was elucidated through molecular dynamics simulation and dynamics cross-correlation matrices analysis. Besides, the performance of TlGa15B-GA2 was the same as that of the commercial glucoamylase during saccharification.ConclusionsWe provide an effective strategy to simultaneously improve both thermostability and catalytic efficiency of glucoamylase. The excellent thermostability and high catalytic efficiency of TlGa15B-GA2 make it a good candidate for industrial saccharification applications.
Highlights
Glucoamylase is an important industrial enzyme in the saccharification of starch into glucose
The poor thermostability of current glucoamylases results in great energy consumption and process inconvenience to undergo the saccharification process in starch industries
Several thermostable glucoamylases have been identified from A. wentii, A. oryzae, A. flavus, Fomitopsis palustris, Chaetomium thermophilum, Rhizomucor pusillus, Thermoanaerobacter tengcongensis, and Sulfolobus solfataricus, all of which exhibit optimal temperature or thermostability above 60 °C [3, 6,7,8,9,10,11,12]
Summary
Glucoamylase is an important industrial enzyme in the saccharification of starch into glucose. Its poor thermostability and low catalytic efficiency limit its industrial saccharification applications. Improving these properties of glucoamylase is of great significance for saccharification in the starch industry. Glucoamylase (1,4-α-d-glucan glucohydrolase, EC 3.2.1.3) is one of the most important and widely used industrial enzymes, exhibiting great application potential. The poor thermostability of current glucoamylases results in great energy consumption and process inconvenience to undergo the saccharification process in starch industries. Reconstructing enzymes by protein engineering is another efficient way to obtain glucoamylases with high thermostability. Due to the activity-stability trade-off, an increase in enzyme thermostability is accompanied by a decrease in catalytic efficiency [17, 18]
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