Studies On Enzymatic Properties And Site-Directed Mutagenesis Of Halophilic Alpha-Amylase From Klebsiella Sp.GXK-1

Halophiles are extremophilic microorganisms that live,grow,and multiply in highly saline environments,and can be a source of halophilic enzymes.Halophilic enzymes show optimal activity at high salt concentrations and are of interest in food production,environmental remediation and enzyme preparations.However,once in the absence of salt solutions,halophilic amylases can be drastically reduced or even inactivated,which greatly limits the production and application of enzymes.In present study,an approximately 1.5 kb halophilic alpha amylase gene named k3 was cloned from Klebsiella sp.GXK-1 which was screened from soil.The recombinant pSE380-k3 was introduced into E.coli JM109 and was induced to expression.The recombinant protein K3 was purified using Nickel column affinity chromatography according to the manufacturer's instructions.The size of K3 was about 55 kDa,which was consistent with the expected results and the enzymatic properties indicated K3 had maximum activity in 3 M NaCl at 50? and pH6.5,respectively.The enzyme activity measured in the presence of NaCl and without NaCl were 3512 U/mg and 97 U/mg,respectively.Under the optimal conditions,it showed that starch hydrolysis products were mainly maltose and maltotriose,respectively,as well small amounts of glucose,maltotetraose,maltopentaose and maltohexaose.Besides,In the presence of NaCl,the substrate were almost completely hydrolyzed into maltose and maltotriose accounted for more than 90%of the total product.However,it was corresponding to only 66.22%in the absence of NaCl.The study aimed to obtain a halophilic enzyme maintained high enzyme activity in the absence of salt so as to increase its industrial application value.As a result,halophilic amylase K3 was modified by site-directed mutagenesis.The homology modeling analysis was performed by the biological analysis software SWISS-MODEL,and K3 was site-directed and mutagenized according to the joint selection of three-dimensional structural analysis by Chmiera and PyMOL.Firstly,14 sites were mutated namely K3-161D,K3-183S,K3-185D,K3-204D,K3-204P,K3-204V,K3-204R,K3-204C,K3-205V,K3-208E,K3-327F,K3-329D,K3-204D-205V,K3-328A-329D.Then the enzymatic properties of mutants were tested.It was found that the enzymatic activity of K3-329 that changed from threonine(T)to aspartic acid(D)increased from 97.21 U/mg to 1517 U/mg,which was 15-fold as before in the absence of salt.Therefore,it was speculated that that 329 site had marked impact on the enzymatic activity of halophilic amylase under low salt conditions.Consequently,the study of saturation mutation in 329 site was conducted on the site.There total of 19 mutants were obtained,and then the activity of these mutants was determined.It was found that only acidic amino acids had a greater effect on the halophilicity of this site.To verify that K3-329 site had the common effect on other halophilic amylases,we selected three halophilic amylase in previous studies with more than 80%homology compared to the K3 sequence,namely Escherichia coli JM109 EAMY,E.cloacae amylase gene and Citrobacter freundii amylase gene f2,as experimental subjects.Then 329 site amino acid corresponding to K3 was mutated to acidic amino acid.The results were in line with expectations which indicate that the site does have a reference value for the molecular modification of halophilic amylase.Generally,the discovery broadens the application range of extreme halophilic enzyme,making it well applied in salt or salt-free conditions,and provides the reference for the molecular modification of other halophilic amylases.