Heterologous Expression Of α-glucosidase For Efficient Synthesis Of Isomalto-oligosaccharides

Isomalto-oligosaccharides（IMOs）are functional oligosaccharides composed ofα-D-glucose groups linked byα-1,6 glycosidic bonds,typically having a degree of polymerization between 2-10.Isomaltose,pannose,and isomaltotriose are considered the main functional components of IMOs.IMOs find widespread use in the food and feed industry as prebiotics.The key enzyme for the synthesis of IMOs isα-glucosidase（EC3.2.1.20）,which converted to IMOs using maltose as a substrate However,the enzyme activity of the Aspergillus niger-derivedα-glucosidase used in industrial production decreases rapidly at temperatures above 50°C.Improving the thermal stability and working temperature ofα-glucosidase can effectively reduce the viscosity of the sugar solution,improve conversion efficiency,and save production costs.In this study,α-glucosidases from different microbial sources were screened and heterologously expressed in Bacillus subtilis 168 to obtain recombinantα-glucosidases with significantly improved thermal stability through molecular modification.Efficient synthesis of IMOs was achieved by optimizing the whole-cell catalytic conditions of the recombinant strain,after amplification of enzyme production and conversion experiments in a 5-L bioreactor.The main findings were as follows:（1）Theα-glucosidase genes gsj and agl from Geobacillus sp.HTA-462 and Thermoanaerobacter ethanolicus JW200 were cloned and heterologously expressed in B.subtilis 168 to obtain recombinantα-glucosidases GSJ and AGL.The transglycosylation activity of GSJ and AGL was assessed using maltose as the substrate.It was found that GSJ and AGL could convert 17.2%and 54%of maltose to IMOs,respectively.Given its higher transglycosylation activity,AGL was chosen for purification to investigate its enzymatic properties further.AGL exhibited the highest catalytic activity at 60°C and p H 6.0,while maintaining good stability in the p H range of 5.0-9.0.With regards to thermal stability,AGL was observed to be more stable at 60°C.However,at a temperature of 65°C,the enzyme activity of AGL decreased rapidly,with only 42.14%remaining after 42 h of incubation.（2）Enhancing the thermal stability of recombinant proteins based on molecular interactions of isopeptide bonds.To construct cyclizedα-glucosidases,three different Catcher/Tagsystems（Spy Tag/Spy Catcher,Snoopp Tag/Snoop Catcher,and Sdy Tag/Sdy Catcher）were used in addition to a novel short peptide pair（RIAD/RIDD）.The optimal temperatures for Spy Tag-AGL-Spy Catcher,Snoop Tag-AGL-Snoop Catcher,and Sdy Tag-AGL-Sdy Catcher were all increased by 5°C,and their residual enzyme activities after42 h incubation at 65°C were 53.7%,73.2%,and 54.2%,respectively.The thermal stability of all three cyclizedα-glucosidases was improved,with Snoop Tag-AGL-Snoop Catcher showing a 1.74-fold increase in thermal stability compared to the wild type.Optimal p H and p H stability did not change significantly.The formation and action of the isopeptide bond was further validated by 3D structural analysis and molecular dynamics simulations of wild-type and three cyclisedα-glucosidases.（3）The recombinant strain B.subtilis 168/p MA5-Snoop Tag-AGL-Snoop Catcher synthesized 182.5 g·L^-1IMOs after 12 h of whole-cell catalysis at 65°C and p H 6.0 by adding an appropriate amount of bacteriophage to a 300 g·L^-1maltose solution to give a system bacteriophage OD₆₀₀of 15.In a study of enzyme production using a 5-L bioreactor,the recombinant strain B.subtilis 168/p MA5-Snoop Tag-AGL-Snoop Catcher,which produces cyclizedα-glucosidase,reached a maximum cell density of OD₆₀₀93.1 and a maximum whole-cell enzyme activity of 101.2 U·m L^-1.After optimising the substrate concentration and the amount of bacteriophage added,the highest conversion rate of 63.29%was achieved.