Gene Cloning, Heterologous Expression Of A Yeast α-amylase And Its Application For Preparation Of Maltotriose

Maltooligosaccharides, which are a new nutritious source of sugar with low sweetness,good taste, and strong moisture, are used as base material for local and internationalfunctional food. They are widely used in the production of drinks, liqueurs, candies, babyfood, and supplements. With the suitble sweetness and highest moisture in themaltooligosaccharides, maltotriose is potential for production of sports drinks. Currently theproduction process of maltotriose and the enmymes used in which is monopolized by severalforeign componies,-amylase from Saccharomycopsis fibuligera is desired to preparemaltritose, which content up to50%or more in the maltooligosaccharides obtained byhydrolysis of starch. In this research, the maltotriose-forming-amylase gene fromSaccharomycopsis fibuligera was cloned and expressed in a heterologous system. Therecombinant enzyme was used to prepare the maltooligosaccharide maltotriose.(1) The-amylase gene from S. fibuligera was cloned and expressed in Pichia pastorisGS115and Kluyveromyces lactis GG799. Scale-up fermentation was performed using ashake flask. Based on the enzyme production curve, the activities of the recombinantamylases were found to be36.4and5.64U/mL, respectively. The recombinant amylase fromP. pastoris was purified using ammonium sulfate fractional precipitation, HiPrep16/10phenyl hydrophobic interaction chromatography, and Superdex75gel filtration. The finalprotein solution was verified using SDS-PAGE, with the result showing only one band. Themolecular mass of the purified recombinant amylase was approximately60KDa. Its specificactivity, purity, and yield were145.16U/mg protein,10.88fold, and21.6%that of the crudeextract, respectively.(2) The recombinant amylases from different host expression sources had the sameproperties. The optimal reaction temperature was45°C; high stability was achieved attemperatures below50°C. The optimal reaction pH was5.0; the recombinant amylasesmaintained excellent stability between pH4.5and7.0. The recombinant amylases wereactivated by most divalent cations, such as Cu2+, Mg2+, Mn2+, Ca2+, and Fe3+. They werestrongly inhibited by EDTA but strongly activated by5.0mmol/L Co2+and Fe2+. The KmandVmaxof the recombinant amylases were9.30mg/mL and2.41mg/(min·mL), respectively.(3) The main end-products formed by the recombinant amylases from starch weremaltotriose and maltose. Using dextrin as substrate, we determined the effect ofmaltooligosaccharide content generated at different enzyme dosages, reaction temperatures,and pH values. The maltooligosaccharide maltotriose was prepared under the followingconditions: substrate,15%dextrin; pH4.5; recombinant amylase,0.2U/g dextrin; reactiontemperature,40°C; and reaction time,32h. The dextrin conversion rate was60.8%, and themaltotriose content in the maltooligosaccharide was52.4%. Using001×7strong acid-typecation exchange resin to isolate maltotriose from the maltooligosaccharide, the purity andyield of the obtained maltotriose product were88.2%and81.3%, respectively.