| Different applications of enzymes require screening of the unique properties of those enzymes,including the specific requirements of the enzyme activity,acid and alkali tolerance,thermal stability,oxidation stability,organic solvent tolerance and stereoisomeric selectivity,that are often found in different ecological environments.Therefore,the exploration of enzymes with special characteristics will enrich and continue to expand our understanding of the various mechanisms of enzymes so that we can better develop or even create new enzymes that will meet specific requirements.Anoxybacillus sp.are thermophilic alkaline bacteria that are mainly distributed in the thermal environments of volcanic hot springs and in composting.Accordingly,the protein derived from Anoxybacillus sp.has the same characteristics of heat resistance and is basophilic,making Anoxybacillus sp.a valuable microbial resource.At the same time,Anoxybacillus sp.plays a significant role in biomass energy and in environmental pollution treatment.Through the analysis of the biochemical characteristics of the alpha amylase of Anoxybacillus sp.GXS-BL isolated from hot spring environments,previous research shows that Ca2+ can improve the thermal stability of the enzyme,and its activity is inhibited by Zn2+.Meanwhile,its thermal stability is reduced by the mutation of the B domain.The analysis of the mechanism involving the related thermodynamics and dynamics is helpful not only for the understanding and utilization of the enzyme but also for the theoretical and experimental knowledge that is obtained.It can also be used to guide the development and application of other industrial enzymes.The main results are as follows:The amylase-producing strain Anoxybacillus sp.GXS-BL was isolated from soil samples of the Tengchong hot springs using M9 medium plates,with soluble starch as the sole carbon source.According to the sequence alignment and gene amplification,the a-amylase gene AGXA was obtained,connected with the expression vector pQE30 and then transferred into E.coli M15.Next,the expression products were separated and purified,and the characterization of the enzyme was analyzed.The results showed that with soluble starch as the substrate,this recombinant AGXA displayed optimal activity at a pH of 8.0 and a temperature of 60 ? and had an apparent Km value of 3.43 g L-1,Vmar of 0.933×10-5 M L-1S-1,Kcat of 2.54×102 s-1 and Kcat/Km of 74.10 s-1 g-1 L.Additionally,the values of T50,Tm and t1/2 at 70 ? were 63.3 ?,67.3 ?and less than 5 minutes,respectively.AGXA had a strong tolerance to organic solvents and active regents.Na+ and K+ had activation effects on AGXA,while Mg2+ and Ca2+ had no obvious effects on the enzyme activity.Cu2+,Co2+ and Fe3+ had obvious inhibition effects on AGXA,and Zn2+ completely inhibited its activity.Ca2+ could improve the thermal stability of AGXA.In the presence of Ca2+,the optimum reaction temperature of AGXA was 70 ?,and the values of Vmax,Km,Kcat,and Kcat/Km were 1.17×10-5 M L-1 S-1,2.20 g L-1,3.195×102 s-1 and 145.23 s-1 g-1 L,respectively.Furthermore,the values of Tso,Tm and t1/2 at 70 ? were 73.8 ?,77.8 ? and more than 200 minutes,respectively.Circular dichroism experiments displayed that the addition of Ca2+ at room temperature did not change the secondary structures of AGXA,although it did make the structures more compact.In the process of heating,the conformation changes of AGXA without Ca2+ occurred in the range of 60-70 ? and finished after 70 ?.The absorbance values increased continuously along with the temperature rise,which was indicative of aggregation of the enzyme.With Ca2+,the conformation changes of AGXA initially occurred at 70 ? and finished after 80 ?.The rise in absorbance was indicative of the enzyme aggregation,and the fall in absorbance at higher temperatures indicated precipitation.The differential scanning calorimetry experiments showed that the Cp value changed from 70 ?? to 80 ?,reaching a maximum at 77.8 ?;AH was 164.7 kCal.mol-1;and ?Cp was 1.35 kcal/mole/? in the presence of Ca2+.Otherwise,the Cp value changed from 60 ? to 70 ? with its highest value at a temperature of 67.3 ?,?H was 133.0 kCal.mol-1,and ?Cp was 1.83 Kcal/mole/? in the absence of Ca2+.The addition of Ca2+ ions to AGXA improved its thermal stability,as evidenced by the increase in thermal unfolding enthalpy change and the reduction in heat capacity change.Zn2+ could inhibit the activity of AGXA.After incubation with Zn2+ at 40 ? for 30 min,AGXA was completely inactive.CD experiments showed that the conformation of AGXA showed obvious change,and its original second structures were lost.By adopting a quantum chemical method to calculate the energy values of the five kinds of bonds about histidine residue in protein interactions,we found that the coordinate interaction energy of His-Zn2+ was higher than all other interaction pairs,and the cation-? energy of Zn2+-His was also larger than the van der Waals interaction energy and hydrogen bond interaction energy.Molecular dynamics simulations showed that the Zn2+ ion not only could form the coordination interaction with the conserved H217 residue but also could form the cation-? interaction with the benzene ring of the F178 residue.Therefore,the dihedral of C-CA-CB-CG of F178 was kept in the planar construction,and the active pocket of AGXA was in the closed state.These results show that the addition of Zn2+ ions could lead to the inactivation of AGXA.Molecular dynamics simulations have been performed at various temperatures,and the results showed that the conformation motions at 400 K were higher than those at 350 K;in addition,the region of the greatest fluctuation from the equilibrium position was in the domain B.The mutation sites were selected according to the RMSF values between 350 and 300 K,and the replaced residues were chosen according to the virtual saturation mutation results using the mutation module of the DS software.Based on the experimental analysis of the targeted mutation,it was found that D358I improved the thermal stability of AGXA and that other mutation modifications in domain B decreased the activity and thermal stability of the enzyme. |
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