Heterologous Expression And Catalytic Properties Of Oligo-1,6-Glucosidase From Paenibacillus Sp. STB16

Debranching enzymes are a class of hydrolytic enzymes that can efficiently and specifically hydrolyze theα-1,6-glucoside linkages in starch and related polysaccharides.It is a key enzyme preparation in starch processing.Debranching enzymes can assist other starch hydrolases to hydrolyze starch substrate efficiently,which greatly improves the conversion rate of starch raw materials.In the actual production process,although the addition of debranching enzymes can significantly improve the production efficiency,there is still a problem of low conversion of starch,this is mainly because the branched oligosaccharides produced at the end of the reaction,such as isomaltooligosaccharides,are difficult to be further hydrolyzed by existing debranching enzymes and other starch hydrolases.Therefore,the development of a new type of debranching enzymes that can efficiently hydrolyze theα-1,6-glucoside bonds in oligosaccharides to achieve further transformation of substrate can significantly reduce the production cost of bulk products such as starch sugars,which has great economic value.This study was focused on the oligo-1,6-glucosidase（OGA）derived from Paenibacillus sp.STB16.Firstly,the Escherichia coli and Bacillus subtilis expression system of OGA were established and the expression level of OGA in different systems were analyzed.Results showed that OGA expressed by recombinant E.coli BL21（DE3）（p ET-20b（+）/oga）and E.coli BL21（DE3）（p ET-22b（+）/oga）were existed as inclusion body;and OGA expressed by E.coli BL21（DE3）（p ET-28a（+）/oga）remained as soluble proteins in the cell and the enzyme activity was 276.40 U/m L.Interestingly,the recombinant B.subtilis WB600（p P43NMK/oga）could achieve the secretory expression of OGA and the enzyme activity was 101.46 U/m L.Secondly,the shaking flask fermentation conditions of B.subtilis WB600（p P43NMK/oga）were optimized.Results showed that the optimal medium was TB medium,the nitrogen source was 24 g/L yeast extract,the carbon source was 6 g/L maltose,and the initial pH was 6.0.The enzyme activity reached 162.33 U/m L after cultivation at the optimal temperature 25℃for 96 h,which was 1.60 times that of the initial culture condition.Next,the crude enzyme was purified by nickel column affinity chromatography and the enzymatic properties of the purified enzyme were analyzed.Results showed that the optimum reaction temperature of OGA was 50℃,and the thermostability decreased sharply after 60℃.The optimum pH was 6.0,more than 80%enzyme activity could be maintained at pH 5.0-7.5,and the initial activity could be maintained at pH 5.0-11.0 when stored at 4℃for 3 h.Mg²⁺,Co²⁺,Mn²⁺activated enzyme activity,while Fe³⁺,Cu²⁺significantly inhibited its activity.Glucose showed product inhibition on OGA,and its semi-inhibitory concentration IC₅₀ was 7.26 mg/m L.With 4-nitrophenylα-D-glucopyranoside as substrate,the kinetic parameter K_m was 2.06 m M.The results of substrate specificity showed that OGA had the highest activity towards isomaltotriose,very low hydrolysis activity for maltodextrin and dextran,and no activity for starch.Finally,the catalytic properties of OGA were studied by using different substrates,and compared with those of isoamylase and pullulanase.Results showed that OGA could effectively hydrolyze theα-1,6-glucoside bonds in isomaltooligosaccharides.The substrate conversion of isomaltotriose and panose reached 100%,and the substrate conversion of isomaltose,isomaltotetraose and isomaltopentaose approached nearly 100%,and the conversion rate decreased slightly with the increase of chain length.OGA was revealed to be exo-type when hydrolyzing isomaltooligosaccharides,that is,from the outside of the molecule to hydrolyze theα-1,6-glycoside bond one by one to produce glucose and oligosaccharides with one less glucose unit than the substrate.OGA had no hydrolysis effect on pullulan,glycogen,waxy corn starch and cassava starch,while it had hydrolysis effect on maltodextrin.As the molecular weight of maltodextrin decreased,the hydrolysis efficiency increased.Ion chromatography was used to analyze the change of substrate polymerization degree.Results showed that OGA could increase the content of DP 1 in maltodextrin,while there was no significant change in the content of other chains,indicating that OGA specifically hydrolyzed branched chain of DP 1 in maltodextrin.Unlike OGA,neither pullulanase nor isoamylase could hydrolyze isomaltooligosaccharides,isoamylase was more suitable for substrate with large molecular weight and long branched chain structure,while pullulanase was suitable for pullulan and maltodextrin with smaller molecular weight.Furthermore,OGA was applied to hydrolyze the mother liquor of glucose,it was found that the glucose content was increased,and the glucose content in the chromatography-separated tail liquor changed most obviously,which increased by 14.95%.Results showed that OGA could further hydrolyze the isomaltooligosaccharides which were difficult to be used in the mother liquor of glucose into glucose.