Reverse Synthesis Characteristics And Application Of Pullulanase

Pullulanase (EC 3.2.1.41) is a member ofα-amylase family GH13. It specifically cleavesα-1, 6- glycosidic bonds in starch, amylopectin and pullulan. Reverse synthesis action of pullulanase was verifed at high substrate concentrations. The reversibility of hydrolysis for pullulanase has been employed to produce maltooligosaccharides and branched cyclodextrins. Maltooligosaccharides and branched cyclodextrins with wide application are new products of starch deep processing. Based on the related research of our group, pullulanase was purified from a commercial pullulanase and its reverse synthesis actions were studied in detail. Interaction between cyclodextrins and pullulanase was also investigated in the present study. 62-α-maltotriosyl-maltotriose and 6-α-maltotriosyl-β-cyclodextrin were synthesized by hydrolysis and reverse synthesis action of pullulanase using pullulan as substrate and their chemical composition and structure were elucidated by HPLC, ESIMS, FTIR, 1H NMR, 13C NMR and HMBC.Pullulanase was dialyzed overnight against acetate buffer to remove a great many saccharides and then concentrated against polyethylene glycol 20,000 at 4°C. The concentrating enzyme was further purified through HiTrap Desalting chromatography, HiPrep16/10DEAEFF ion exchange chromatography and HiPrep16/60Sephacryl S-200HR chromatography. All subsequent chromatographic steps were performed by the application of Akta Purifier 10 system. The purified pullulanase was analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and was certified a single band. The final purified pullulanase obtained a specific activity of 240.7 U/mg and there was a 5.77-fold.With glucose, maltose, maltotriose, maltoteraose and cyclodextrins as substrate, the specificity for reverse synthesis of pullulanase was studied. Reverse synthesis action of pullulanase was verifed at high substrate concentrations and the minimum substrate was maltose. With maltose as substrate, the optimum reverse synthesis pH of pullulanase was 4.5, and it was stable within the pH range of 4.0-6.0. The optimum reverse synthesis temperature of pullulanase was 65℃, and it was stable within the temperature range of 60-70℃. Metal ions such as Na⁺, K⁺, Li⁺ and Co²⁺ had no obvious effect on reverse synthesis of pullulanase, Hg²⁺, Sn²⁺, Cu²⁺, Ag²⁺ and Pb²⁺ inhibited the reverse synthesis action, Ca²⁺ and Mn²⁺ had obvious activation effects on reverse synthesis of pullulanase, Ca²⁺ also improved thermal stability for reverse synthesis of pullulanase.The hydrolysis activity and reverse synthesis action of pullulanase were strong inhibited byβ-CD. This inhibition was attributed to the interaction between hydrophobic cavities of cyclodextrins and pullulanase. The hydrophobic cavity was confirmed to encapsulate the groups of pullulanase molecules by the addition of competitive guests. Hydrophobic cavities ofβ-CD formed inclusion complexes with some groups of pullulanase molecules. The native conformation of pullulanase gradually unfolded, which led to obvious decrease of enzymatic catalysis. Inhibition ofβ-cyclodextrin on pullulanase depended on concentration and tended to be enhanced with the increase ofβ-cyclodextrin. The increase of intrinsic fluorescence induced byβ-cyclodextrin was observed, which was probably due to the formation of inclusion complexes between aromatic amino acid residues of pullulanase andβ-cyclodextrin. The circular dichroism spectroscopy was further employed to verify that changes of pullulanase secondary structure depended on theβ-CD concentration. The ratio ofα-Helix toβ-Sheet in pullulanase declined with the increase ofβ-CD, thus resulting in the loss of enzymatic activity.These results showed thatβ-cyclodextrin changed the secondary structure and microenvironment of pullulanase, accordingly leading to the loss of enzymatic activity. The geometric dimension of hydrophobic cavities was crucial for matching between cyclodextrins and pullulanase and steric hindrance caused by side chain led to the decrease of the interaction.β-CD showed more efficiently to interact with pullulanase molecules. The interaction betweenβ-CD and pullulanase molecules was also influenced by pH and temperature of reaction system, which was probably due to the change of binding site betweenβ-CD and pullulanase.Maltotriose syrup was obtained by hydrolyzing of pullulan with pullulanase. The optimum conditions were as follows: pullulan concentration, 5% (w/v); time, 8h; amount of pullulanase, 10U/g pullulan; pH, 5.0; temperature, 55°C. The content of maltotriose in the syrup was 90.35% by HPLC analysis. 62-α-maltotriosyl-maltotriose was prepared by reverse synthesis action of pullulanase using maltotriose syrup as substrate. The optimal reverse synthesis conditions were as follows: maltotriose syrup concentration, 70% (w/v); time, 44h; amount of pullulanase, 120U/g maltotriose syrup; pH, 4.0; temperature, 65°C. The products were further isolated by preparative HPLC on Lichrospher NH2 column. Structure of 62-α-maltotriosyl-maltotriose was elucidated by HPLC, ESIMS, FTIR, ¹H NMR, ¹³C NMR and HMBC.6-α-maltotriosyl-β-cyclodextrin was prepared by reverse synthesis action of pullulanase using maltotriose syrup andβ-cyclodextrin as substrates. The optimal reverse synthesis conditions were as follows: molar ratio of maltotriose andβ-CD, 12:1; time, 56h; amount of pullulanase, 200U/g maltotriose syrup; pH, 4.5; temperature, 65°C. The products were further isolated by preparative HPLC on Lichrospher NH2 column. Structure of 6-α-maltotriosyl-maltotriose was elucidated by HPLC, ESIMS, FTIR, ¹H NMR, ¹³C NMR and HMBC. These results strongly demonstrate that the synthesized product is a mono-6-O-α-D- maltotriosyl-β-cyclodextrin.