Cloning And Expression Of Fugal-derived GH13 Family Alpha Amylase Tcamy And Improvement Of Thermal Stability Of Amy13A21

Alpha-amylase is an important industrial enzyme and is widely used in various industries such as food,sugar,detergents,textiles,papermaking,and medicine.Among them,?-amylases capable of exhibiting high catalytic efficiency at low temperatures and alpha-amylases capable of being thermally stable at high temperatures are also required.Therefore,studies on the psychrophilic mechanism of psychrophilic amylases and the improvement of thermostability of amylase have become particularly important.The work of this dissertation is divided into two parts.The first is the cloning of a low-temperature ?-amylase encoding gene Tcamy from Thermoascus crustaceus JCM12803 and successful heterologous expression in Pichia pastoris.The optimum temperature of the protein Tcamy was 35°C.It retained 27% activity at 0°C and the specific activity at 262.56 U/mg at 35°C.The better pH stability determined that Tcamy had a good application prospect.Through homology modeling,the molecular mechanism of cold adaptability was analyzed from three aspects of N-glycosylation,amino acid composition and internal interaction forces,which deepened the understanding of the relationship between the structure and function of low-temperature ?-amylase.Secondly,the mutant engineered gene Amy13A21 constructed by replacing a Linker based on the alpha-amylase gene Amy13 A from Talaromyces leycettanus JCM12802 was used as the material.Based on the rational design of protein surface charge distribution optimization,the thermal stability of Amy13A21 was improved.Two sites Glu87 and Tyr280,which may be related to thermal stability,were determined by calculation of enzyme thermal stability system(ETSS).Single point mutations E87 K,Y280K and combinatorial mutations E87K/Y280 K were constructed for heterologous expression and characterization,and compared with wild-type Amy13A21.The thermal stability of the three mutants was higher than that of the wild type.The increase range was E87K/Y280K>Y280K>E87K.The T50 and Tm values of the combined mutant E87K/Y280 K compared to the wild type Amy13A21 were increased by 8°C and 6.75°C,respectively.Its catalytic activity increased from wild-type 80.97 U/mg to 161.175 U/mg.This paper provides a potential low-temperature ?-amylase Tcamy for the industry,and uses it as a research model to explore its cold adaptation mechanism.On the one hand,it can deepen our understanding of low-temperature enzymes.On the one hand,it can provide scientific basis for the improvement of enzyme molecules research based on the application requirements.The second is the rational design of the engineered gene Amy13A21 which has been successfully constructed in our laboratory.Based on the rational design of protein surface charge distribution optimization,the thermal stability of Amy13A21 is effectively improved without loss of enzyme activity,and the thermal stability of the enzyme is improved.Provides an effective strategy.