Extreme high maltose syrup,which is characterized by their low sweetness,low viscosity in solution,reduced browning capacity,and good heat stability,can be used as a new sweetener to replace the glucose and sucrose.It has been widely used in the food industries:brewing,baking,soft drink,canning,and confectionery etc.Extreme high maltose syrup was prepared from the enzymatic hydrolysis of starch,which involves its liquefaction with a-amylase,and saccharification with debranching enzymes (pullulanase etc.) and exo-amylase (β-amylase and maltogenic amylase).In recent years,our group studied pullulanase,maltogenic amylase and β-amylase.Pullulanase was heterologous expressed,and several mutants with improved thermostability were constructed.However,its limited ability in expressing soluble protein and extracellular secretion has impeded its application.Based on the hypothesis that pullulanase was an "aggregation-prone protein",several approaches were applied to enhance the extracellular production of this enzyme.As for the maltogenic amylase,it was found that Bacillus stearothermophilus maltogenic amylase not only has hydrolysis activity but also has transglycosylating activities,which hamper its application in the production of Extreme high maltose syrup.Site-directed mutagenesis at Trp 177,which was found in subsite +2 of the active deft,was performed.The mutant could dramatically reduce the maltotriose content.Furthermore,a β-amylase producing strain isolated from a vegetable field soil sample was characterized and identified as Bacillus flexus.The β-amylase encoding gene from B.flexus was cloned and expressed in B.choshinensis.To investigate the potential industrial utility of above enzymes,they were used in combination to hydrolysis starch to produce maltose.After about 36 hours of reaction,the maximal maltose content was 91.46%,and the glucose and maltotriose content was 3.0% and 0.3%,respectively.