Improvement Of The Stability Of Cold-adaption ?-amylase Through Rational Design

Compared with mesophilic amylases and thermostable amylases,cold-adapted amylases have high activity at low temperature.This property can be extremely useful,as it could avoid spoilage and remain the nutritional value and flavour of the original heat-sensitive substrates and products.In addition,the catalyzed reaction in the low temperature implies energy conservation and environmental protection.However,the cold-adapted amylases often has low thermosability.Compared with other industrial enzymes,there are relatively few studies on improving the stability of cold-adapted ?-amylase at home and abroad.In this study,two rational design strategies were used to improve the thermostability of a cold-adapted ?-amylase(AHA)from Alteromonas haloplanktis.In the first strategy,the online method of Po PMusic was used to predict the unfolding free energy change of AHA.The conserved residues and residues within the 5 angstroms to the active center were not selected in this study.As a result,the 8 mutations with significant changes in unfolding free energy were selected and the corresponding mutants were constructed by the two-step PCR mutagenesis method.After preliminary screening by the enzyme activity and Tm,two positive mutants N124 P and A413 G were identified.Compared to wild-type,they had the same optimal temperature and p H,but the Tm values of N124 P and A413 G increased by 1.02°C and 1.85°C,respectively.Moreover,after incubation at 40 for 80 min,the residual activit? ies of N124 P and A413 G increased by 28% and 113%,respectively.The second strategy is based on the molecular dynamics simulations and energy calculation.The structure of AHA was simulated and the positive mutations were seleced with the method of virtual screening.As a results,the 10 mutations that might increase the thermostability of AHA were identified and a site-directed mutagenesis technique based on the two-step PCR was used to construct the corresponding 10 mutants.After preliminary screening with the enzyme activity and Tm values,two positive mutants(S255K and S340P)with enhanced the thermostability were identified.In addition,the optimal temperature and p H of the two mutants were same to that of the wild-type.However,compared to wild-type,the Tm values of S255 K and S340 P increased by 1.52°C and 3.38°C,respectively.After incubation at 40 for 80 minutes?,the residual activities of S255 K and S340 P increased by 60% and 140%,respectively.Finally,based on the four single-point mutants obtained by the above two methods,four combined mutants(S255K/S340 P,S340P/A413 G,S255K/S340P/A413 G and N124P/ S255K/S340P/A413G)were constructed and the thermostability of the four mutants were determined.The best mutant among the four combined mutants was S255K/S340P/A413 G,which the Tm values increased by 6.38?.After incubation at 40 for 80min?,the residual activities of it improved by 184%,compared with wild-type AHA.In addition,the optimal temperature and optimal p H of the four combined mutants were also same to that of the wild type.In this study,the thermostability of cold-adaption ?-amylase AHA was improved through two rational design methods.However,the optimal temperature of these mutants did not change compared to the wild type.The success of this site-directed design methodology for AHA suggests that it was an efficient strategy for enhancing protein thermostability and suitable for protein engineering.Moreover,our study will be the foundation for cold-adaption ?-amylase AHA industrial application.