Cloning, Expression And Characterization Of Twoα-amylases From Marine Bacterium Zunongwangia Sp

Starch is an important industrial raw material. Alpha-amylases （endo-1,4-α-D-glucan glucanohydrolase, EC3.2.1.1） are endoenzymes which can randomly hydrolyze a-1,4-glycosidic bonds in starch molecules to give diverse products, a-amylases are widely used in many fields of starch processing, food, paper, textile and pharmaceuticals et al, with significant economic value. In this study, two novel amylase genes was amyplified from marine bacterium Zunongwangia sp.. One of them is a typical a-amylase, and the other is a cyclodextrin-hydrolyzing a-amylase. The genes were cloned into the vectors pGEX-6P-1and expressed in Escherichia coli BL21（DE3）. Then, the obtained proteins were purified and characterzed for further study. The main conclusions are listed below.The gene of the cyclodextrin-hydrolyzing a-amylase （amyZl） is of1848bp （GC content37.55%） and encodes a polypeptide of615amino acids with a predicted isoelectric point （pI） of4.65and molecular mass of71.15kDa. The polypeptide was predicted to has a N-terminal signal peptide of21amino acids by SignalP4.1Server and the mature protein was composed of594amino acids with the predicted pI of4.60and a molecular mass of68.86kDa. Sequence alignment revealed that AmyZl showed clear homology to a-amylases belonging to glycoside hydrolase family13and the highest identity （72%） with the predicted a-amylase （WP₀10227850） from Gillisia sp. CBA3202. Besides the conseved domains of a-amylase, cyclomaltodextrinase domains were also found at the N-and C-terminus of AmyZl, showing45%identity with the cyclomaltodextrinase （WP₀22339125） from Bacteroides sp. CAG:714. The purified AmyZl showed the maximum activity at35℃, pH7.0, and retained approximately39%of its initial activity at0℃, indicating its cold activity. In addition, extreme salt-tolerance was observed in AmyZl with the retention of93%initial activity at4M NaCl. The salt optimum was found at1.5M NaCl, with an increase of26%activity. The Km and kcat values of AmyZ1were2.74mg ml-1and316.49s-1, respectively, using soluble starch as substrate. While when the reaction mixture contained1.5M NaCl, the two values were2.30mg ml-1and329.58s-1, respectively and the corresponding Km/kcat values increased from115.51mg ml-1s-1to143.30mg ml-1s-1. Besides, the enzyme thermostability was significantly improved by the pre-incubation of enzyme solution with1.5M NaC1or1mM CaCl2at35℃for120min, which led to a13.2-or11.4-fold increase in the residual activity. Substrate specificity experiments revealed that AmyZl was a cyclodextrin-hydrolyzing a-amylase, not only displaying the maximum activity towards soluble starch, but also showing activity towards cyclodextrin.The gene of the a-amylase AmyZ2, amyZ2, is of1446bp （GC content38.31%） and encodes a polypeptide of481amino acids with a predicted pI of4.24and a molecular mass of55.17kDa. AmyZ2was predicted to has a N-terminal signal peptide of24amino acids by SignalP4.1Server and the mature protein was composed of457amino acids with the predicted pI of4.14and molecular mass of52.13kDa. Sequence alignment revealed that AmyZ2showed clear homology to a-amylases belonging to glycoside hydrolase family13and the highest identity with the characterized a-amylase （PDB ID1MWO） from Pyrocoocus Woesei. The purified AmyZ2showed the maximum activity at50℃, pH7.0. Alkali stability was observed in AmyZ2, retaining approximately143and126%initial activity after5d of incubation （25℃） at pH10.0and11.0, respectively. In addition, AmyZ2exhibited extreme salt-tolerance with a salt optimum at2M NaCl （138%initial activity） and100%initial activity at3.5M NaCl. AmyZ2was relatively stable in0-4M NaCl and its thermostability was significantly improved by the pre-incubation of enzyme solution with2.0M NaCl at50℃for3h, which led to a53.6-fold increase in the residual activity. With soluble starch as substrate, the kinetic parameters of AmyZ2, Km, kcat and kcat/Km, were11.71mg ml-1,1449.43s-1and123.78ml mg-1s-1, respectively. What’s more, AmyZ2exhibited a specific activity of1662.4U/mg towards soluble starch.