Studies On Auto-Activation Mechanism And Thermostability Improvement Of Aspartic Proteases From Filamentous Fungi

Aspartic protease,known as acidic protease,has been widely used in food,fermentation,medicine,and leather industries.However,the poor thermostability of aspartic proteases limits their application.Therefore,exploring new high temperature aspartic proteases or improving their thermal stability using molecular modification is of great practical significance to study the stable mechanism and broaden their application sphere.In this study,six aspartic protease genes(Tlap,Tlapa1,Tcapa,Bsapa,Psapa and Psapb)were cloned from Talaromyces leycettanus JCM12802,Thermoascus crustaceus JCM 12803,Bispora sp.MEY-1 and Penicillium sp.JJ-1,and successfully expressed in Pichia pastoris.The optimum pH of the six recombinant aspartic proteases(TlAP,TlAPA1,TcAPA,Bs APA,PsAPA and Ps APB)was between3.0 and 3.5,and all of them had high catalytic activity under the condition of pH 2.0-4.0.They were stable under acidic conditions.Among them,BsAPA derived from acidophilic fungus Bispora sp.MEY-1 was more stable under acidic conditions compared with others.The effect of temperature on the proteolytic activity and stability was varied among them.The optimum temperature of Ps APA was50 ?,while that of BsAPA was 75 ?.And the thermostability of BsAPA was significantly higher than that of the other five aspartic proteases and commercial acidic proteases.More than 80% residual activities remained after incubated at 70 ? for 1 h,which was significantly higher than other homologs.Therefore,BsAPA was an ideal material to study the thermostability of aspartic proteases.The specific activity of BsAPA was up to 6537 ± 109 U/mg under optimal conditions,which was higher than that of other aspartic proteases.BsAPA also showed high hydrolysis activity for some natural substrates,such as gliadin(627±73 U/mg)and gluten(449±57 U/mg).These properties make Bs APA attractive for potential application.Aspartic proteases are expressed and secreted in the form of zymogen,and they auto-activated to mature molecules under acidic conditions.In our research,we found that the activation process of Tl APA1 from T.leycettanus JCM12802 was different from the reported one-step activation process.It can be divided into two stages: the formation of activation intermediate and further cleavage of intermediate into mature enzyme.The formation of intermediates was completely mediated by the pH value of the buffer,while the next stage was depended on the proteolytic activity.Studying the auto-activation process of Tl APA1 not only confirmed the optimal conditions of aspartic protease auto-activation,but also contributed to understanding the auto-activation mechanism of aspartic protease family.The crystal structure of BsAPA was obtained by X-ray diffraction.Analyzing its three-dimensional structure,we found that Y282 might play an important role in the thermostability and catalytic activity of BsAPA.And then,saturation mutation was conducted on Y282,and the thermal stability and catalytic activity of mutants were analyzed.The thermostability of Y282 L was improved obviously,and the residual activity was increased to 75.3% from 47.2% after incubation at 75 ? for 5 min.The specificactivity of mutant Y282 H was increased from 6537 ± 109 U/mg to 7296 ± 114 U/mg.At present,mutant Y282 L of BsAPA has been applied in feed industrial.It was found that the inactivation of aspartic protease was related to the autolysis at high temperatures.Therefore,the cleavage site(L205-F206)of BsAPA was identified by analyzing the autolysis fragments of BsAPA and N-terminal sequencing.In order to improve the stability of BsAPA,the residues neighboring the autolysis site were selected and mutanted.The residual activities of mutants F193 W,K203E,K204 P and A371 V were 72.4%,65.3%,67.4% and 68.2% respectively after 5 min treatment at 75 ?,which were higher than that of wild type(47.2%).Studying the relationship of autolysis and thermostability of Bs APA provided a new idea for improving thermostability of protease.In summary,six aspartic protease genes were cloned and expressed in Pichia pastoris.The auto-activation process of zymogens in vitro was studied.The properties of the six recombinant aspartic proteases were determined,and an aspartic protease BsAPA with high thermostability,high activity was obtained.The catalytic activity and thermostability of Bs APA were improved further by mutation.At the same time,the relationship between the thermostability of Bs APA and autolysis was identified,and then their autolysis resistance and thermostability were improved by mutation.These studies provide important references for the improvement of thermostability and broaden the applied range of aspartic proteases at high temperatures.