The Study On The Screening,catalytic Mechanism,and Application Of Cyclodextrinase

Cyclodextrinase（CDase）is one of the members of the 20th subfamily（GH13＿20,also termed as the neopullulanase subfamily）from the glycoside hydrolases family 13（GH13）,and can usually act on cyclodextrin（CD）,starch,pullulan and other substrates.Among them,the hydrolysis rate for CD is higher than that for other substrates.Malto-oligosaccharides are linear oligosaccharides composed of 2～10 glucose units linked byα-1,4 glycosidic bonds,which have good processing adaptability and nutritional properties.CDase is considered to have great application potential for its ability to open the CD ring backbone to generate malto-oligosaccharides with specific degree of polymerization.Currently,there are few reports on CDase with strong specificity for CD hydrolysis,and the produced malto-oligosaccharides are with low purity.In addition,the catalytic mechanism of CDase on the substrates is still unclear,and the studies on product compositions and properties of the modified substrates by CDase are limited,which further limits the application of this enzyme in the food field.This subject mainly screened CDases with high specificity for CD hydrolysis,analyzed its catalytic mechanism on substrates,and explored the application of these enzymes in the preparation of malto-oligosaccharides.Furthermore,based on the CD hydrolysis specificity of CDase,the application of CDase in starch modification was explored by combining CDase with cyclodextrin glycosyltransferase（CGTase）which uses starch as substrates to produce CD.The main research contents and results are as follow:1.The amino acid sequences of the reported CDases were used as template.Based on the bioinformatics analysis,two amino acid sequences were uncovered from the Genbank database,which were derived from the thermophilic archaea Palaeococcus ferrophilus and Palaeococcus pacificus.And they were termed AMPf and PpCD respectively.After the multiple sequence alignment,it was found that both AMPf and PpCD possess 7 conserved regions,which were identified from the neopullulanase subfamily,including the typical sequence of this subfamily“³⁷⁵MPRLN⁴⁰³”（calculated from AMPf）.The expression,purification,and enzymatic property characterizations were conducted on AMPf and PpCD.The molecular weight of AMPf is 70k Da,the optimum temperature is 50°C,and the optimum pH is 7.0.AMPf still maintained more than 60%of the original activity after the incubation at 45°C for 8 h.The molecular weight of PpCD is 80 k Da,the optimum temperature is 95℃,and the optimum pH is 6.0.PpCD maintained more than 90%of the original activity after 8 h incubation at 75℃and 85℃.Besides,both the enzymatic activities of AMPf and PpCD were influenced by some metal ions and organic solvents.2.The substrate specificity and product specificity of AMPf and PpCD were identified using substrates such as CD,starch,pullulan,and malto-oligosaccharides.It was found that AMPf has a high enzymatic hydrolysis activity on starch,while PpCD has a high hydrolysis specificity for CD.AMPf has obvious transglycosylation activity and can use maltose and maltotriose as substrates to produce maltotetraose and malto-oligosaccharides with higher degree of polymerization,while PpCD has no obvious transglycosylation activity.In addition,the High-performance liquid chromatography（HPLC）determination results indicated that the main components of starch hydrolysates catalyzed by AMPf were glucose,maltose,maltotriose and maltotetraose.PpCD can hydrolyzeα-CD,β-CD andγ-CD to their corresponding linear malto-oligosaccharides,including maltohexaose,maltoheptaose and maltooctaose.3.The PpCD,which has higher thermostability and hydrolysis specificity for CD,was selected for the following researches.HPLC was used to determine the hydrolysis products from CD by PpCD for various time to explore the hydrolysis mode of this enzyme.The results indicated that PpCD has similar hydrolysis patterns toα-CD,β-CD andγ-CD.Firstly,PpCD rapidly hydrolyzes CD into the corresponding linear malto-oligosaccharides.Secondly,malto-oligosaccharides are slowly degraded into oligosaccharides with a lower degree of polymerization.Finally,PpCD degrades all products into glucose and maltose.PpCD was utilized to prepare maltoheptaose withβ-CD as the substrate.When 8%β-CD is used as the substrate and the reaction time was 100 min,the ratio of maltoheptaose in the reaction product to all malto-oligosaccharides can reach 98.4%.When the reaction time was 180 min,the ratio of maltoheptaose in the reaction product to the total reaction products can reach 43.4%.In addition,the molecular simulation results indicated that the specific CD recognition of PpCD may be due to the aromatic amino acids around the active center.4.To explore the hydrolysis pattern of PpCD in the complex CD conditions,firstly,the kinetic parameters of PpCD were measured withα-CD,β-CD andγ-CD as substrates.The catalytic efficiency k_cat/K_m for the three CDs were 18.46,6.03,and 0.93 mg·m L^-1·min^-1respectively.This result showed that PpCD has significantly different catalytic efficiencies forα-CD,β-CD andγ-CD.Further,the substrate containing a mixture of CDs of various proportions was prepared and hydrolyzed by PpCD.It was found that PpCD had obviously selective hydrolysis effect for this substrate,and the selective hydrolysis effect was most significant on the mixed solution withα-CD andγ-CD.Whenα-CD andβ-CD were completely degraded in the mixed system,there was also a loss of about 20%to 50%ofγ-CD.In addition,temperature optimization experiments showed that PpCD has the best selective hydrolysis effect at 85℃.Finally,the product ofγ-CGTase acting on starch was used as the substrate for PpCD,and the results indicated that PpCD also has a favorable selective degradation effect in this system.5.PpCD and CGTase with CDs as products were used together for starch modification.The reaction times were 1,6,12,and 24 h.The results indicated that PpCD enhanced the release of reducing ends and glucose during the CGTase reaction process,and promotes the enzymatic hydrolysis efficiency of CGTase on starch.The HPLC was utilized to determine the composition of cyclic and linear malto-oligosaccharides in the reaction products.It was found that PpCD reduced the CD content in the CGTase reaction process and significantly increased the content of malto-oligosaccharides.The molecular structures of the reaction products during the modification process were determined.It was found that the average molecular weights（M_w）of the starch samples modified by single PpCD and CGTase for 1 h were 222.6×10⁵ g/mol and36.7×10⁵g/mol.However,the M_wof the starch samples modified by dual-enzyme treatment with PpCD and CGTase for 1 h was 15.0×10⁵g/mol,and PpCD had the effect of promoting degradation.As the reaction time increased,the degradation effect became more significant.Thus,there is a clear synergistic effect between PpCD and CGTase.In addition,the synergistic action of PpCD and CGTase significantly improved the anti-retrogradation property of starch.And,the retrogradation enthalpy of starch that stored at 4°C for 7 days can be reduced from5.65 J/g to 1.42 J/g.Based on the synergistic effect of PpCD and CGTase,simultaneous treatment and sequential treatment by PpCD and CGTase were selected to treat corn starch with various amylose content,including waxy corn starch（5%）,normal corn starch（25%）,and high amylose corn starch（45%）.The final compositions and the in vitro digestion properties of the products were determined.The highest conversion rate of cyclic and linear malto-oligosaccharides can be obtained when the normal corn starch was used as substrate,which can reach 45.7%.The fine structure characterization results of the modified starch indicated that CGTase and dual-enzyme treatments significantly reduced the chain length ratio of various corn starches with DP13～24,DP 25～36 and DP>37,while the chain length ratio of DP<13 increased significantly.The apparent improvement ratio was 50%～60%.CGTase and dual-enzyme treatments significantly improved the anti-digestible fractions of various corn starches.Among them,the resistant starch（RS）was improved most significantly.When the substrates were waxy,normal,and high amylose corn starch,the highest RS could reach 36.9%,40.0%,and 59.3%,respectively.When PpCD replaced the CD with malto-oligosaccharides for the CGTase products,the digestibility of the products was not significantly affected.