| Fungal ?-amylases are widely used in the production of maltose syrup,while additional production costs may be required in the syrup production process due to the loss of enzyme activity,and the syrup was frequently contaminated by common mesophilic microorganisms during long time treatment,because of the poor thermostability and acid-tolerant exhibited in this type of enzyme.Thus,enhancing the stability of fungal ?-amylase under acid or heat conditions may give a great help to improve the sugar processing to reduce the chance of syrup contaminated by mesophilic microorganisms and reduce the enzyme cost.In this paper,?-amylase(ROAmy)from Rhizopus oryzae was performed by protein structure modeling,multiple sequence alignment and bioinformatics analysis to improve its thermostability and acid tolerance.The Kluyveromyces lactis was used for the expression of the mutants,and then the physicochemical properties and protein structure and function analysis were applied on the mutants.Finally the residues related to p H and temperature tolerance of ROAmy were presented.The main research contents are as follows:(1)Seven mutants were constructed based on the protein structure analysis and the results of multiple sequence alignment between ROAmy,the thermostability and acid stability fungal ?-amylase and neutral fungal ?-amylase.The results indicated that the mutants A144 Y,V174R,T253 E and I276 P showed enhanced thermostability and acid resisting.For the mutant V174 R,its half-life at 55? was almost increased by 2.52-fold than that of the wild-type enzyme.Meanwhile,it displayed 1.55 times increase in half-life at p H 4.5.In addition,the catalytic efficiency(kcat/Km)of the mutants A144 Y,T253E and I276 P were about rised by 61.18%,65.50% and 24.25%,respectively.Through the analysis of the protein structure and function between the mutants and the wild-type enzyme,the enhanced hydrophilicity of protein surface,the increase of the number of hydrogen bonds,the formation of salt bond and the rigidity enhancement of the flexible regions may be responsible for the improved thermostability and acid-tolerant that found in these mutants.The results also meant the A144,V174,T253 and I276 may be the key residues that related to the thermostability and acid tolerance of ROAmy.(2)Single-point mutations and combined mutations that based on analysis of B-factor values and molecular dynamics simulations were carried out for amino acid residues G128,K269 and G393 of ROAmy by overlapping PCR.The results showed that all mutants obtained presented better thermostability than the wild-type enzyme,and the best mutant was G128L/K269L/G393 P which showed a 5.63-fold increase in half-life at 55? compared with the wild-type enzyme.Meanwhile,its optimum temperature increased from 50? to 65?,the maximum reaction rate(Vmax)and catalytic efficiency(kcat/Km)increased by 65.38% and 99.86%.By comparing and analyzing the protein structure and function between the mutants and the wild-type enzyme,it was found that the increase of the number of hydrogen bonds or the introduction of proline in special position may be the main reasons for the improved thermostability that found in the mutants.The results also meant the amino acid G128,K269 and G393 may be the key residues that related to the thermostability of ROAmy.(3)The results of aligning and superimposing the 3-D model structure of ROAmy with the heat resisting and acid resisting ?-amylase from Asperigillus niger(PDB code: 2aaa)showed that the existence of disulfide bonds in the A.niger ?-amylase but none in ROAmy.It has been shown the disulfide bonds plays an important role in the structural stability of protein.Therefore,three mutations were obtained by introducing disulfide bonds into the corresponding position of ROAmy based on the protein structure modeling and the analysis of the sequences.The results demonstrated that the thermostability of all mutants was improved,and the mutant ROAmyC30-C36 showed 2.31-fold in half-life at 55? greater than that of the wild-type enzyme.Meanwhile,all mutants exihibited greater catalytic activity and stability when the p H value is 3.5~4.5.Besides,the catalytic efficiency(kcat/Km)of mutants ROAmyC30-C36 and ROAmyC230-C273 increased about 20.17% and 37.19%,respectively,than that of the wild-type enzyme.The above results showed that the mutants have better thermostability and acid resisting than that of wild-type enzyme may be due to the enhanced stability of protein local structure by the introduction of the disulfide bond. |
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