The Catalytic Mechanism Of α-maltotriohydrolase And Its Application Research

The glucan 1,4-α-maltotriohydrolase（α-maltotriohydrolase,EC 3.2.1.116）is a member of the family 13 of glycoside hydrolases（GH13）,and this enzyme also belongs to the maltooligosaccharide-forming amylase.The maltooligosaccharide-forming amylase are defined as exo α-amylase and widely used in starch modification and bakery foods.At present,the α-maltotriohydrolase is widely believed as an endo α-amylase,and its hydrolytic mechanism is far from clarity.The confusing action mechanism of maltooligosaccharideforming amylases such as α-maltotriohydrolase limited their practical application in the food industry.In this work,α-maltotriohydrolase was purfied from commercial α-maltotriohydrolase to explore the hydrolytic mechanism.On the basis of hydrolytic mechanism,we tried to use this amylase to control retrogradation and digetibility of starch.In the end,we tried to use the newly discovered transglycosylation ability of this amylase to modify stevioside.We believed our research work was crucial to future practical application of the maltooligosaccharideforming amylase such as α-maltotriohydrolase in food industry.Firstly,the commercial enzyme（AMT 1.2L）was fractioned to purify α-maltotriohydrolase using dialysis,HiTrapTM desalting column,HiTrapTM Q fast flow cation-exchange chromatography and ultrafiltration concentration,yielding a single band in SDS-PAGE.The molecular weight of purified α-maltotriohydrolase was about 5.4×10⁴,and the enzymatic specific activity was 64.8 U/mg.The optimal temperature and pH were 50℃ and pH 6.5,respectively.The activity of this amylase depended on the Ca²⁺,and 5 mM Ca²⁺ could effectively improve its thermstability.We used TLC and HPLC to evaluate the hydrolysates from different substrates and found that this amylase could hydrolyze the cyclodextrins to produce maltotriose,which proved its endo-action.It could also be concluded that the specific structure of starch（amylose or amylopectin）determined the hydrolysis rate and the purity of maltotriose in hydrolysates.Then,the multiple attack actions on amylose of amylase were evaluated,Aspergillus oryzae α-amylase<pancreas α-amylase≈α-maltotriohydrolase<β-amylase.HPSEC and TLC were chosen to analyse the hydrolysates from amylose,p-nitrophenyl-α-D-maltopentaoside（pNPG5）and p-nitrophenyl-α-D-maltohexaoside（pNPG6）,and then demonstrated that this amylase firstly cleavaged the amylose by endo-action,and then produced maltotriose from the reducing ends through an exo-action.We also used TLC and HPAEC to evaluate the maltooligosaccharides hydrolysis catalyzed by the α-maltotriohydrolase,and discovered that it partially hydrolyzed maltooligosaccharides with DPn into products with DPn ± 3.Glucose,maltose,maltotriose and p-nitrophenyl-α-D-glucoside（pNPG1）were further valided as the donor in the transfer reactions.Secondly,the hydrolytic mechanism on amylopectin by α-maltotriohydrolase were evluated by HPSEC-MALLS-RI and HPAEC.In the initial hydrolysis stage,the exo-action of α-maltotriohydrolase was predominant with slow reduction in Mw and little changes in the internal chains of amylopection.With hydrolysis progressing,this enzyme cleaved the internal chains of starch via an endo-action,resulting in starch of low Mw,and then produced a nonreducing end could be further hydrolyzed into maltotriose.The β-amylase was selected to hydrolysis the amylopectin into different levels to prepare amylopectin with different external chain lengths（ECL）,and kinectic parameters analysis were performed.It was revealed that the Michaelis constant（K_m）was increased with the decreaed external chain lengths,whereas the catalytic efficiency(k_cat/K_m)was decreased.K_m of the β-dextrin-3（ECL 2.3）was eight folds than the amylopectin（ECL 17.8）,but its k_cat/K_m was only 6.5% of amylopectin,proved that α-maltotriohydrolase preferred the external chains to the internal chains for hydrolysis.Retrogradation behaviors of hydrolysates produced from amylopectin by α-maltotriohydrolase were evaluated using DSC.It could be concluded that α-maltotriohydrolase was effectively in retarding retrogradation of starch,and the retrogradation was completely inhibited after storage at 4℃ for 7 days when the DH higher than 15.9%.The α-maltotriohydrolase could inhibit the retrogration of amylopectin because of the increased shorter outer chains and maltooligosaccharids（maltotirose is in major）.Thirdly,the hydrolysis time of α-maltotriohydrolase was controlled to modulate the modification degree on normal maize starch with the purpose to improve SDS formation.The digestibility of α-maltotriohydrolase modified starches was evaluated,and demonstrated that the action of this amylase was beneficial for SDS formation.The highest amount of SDS of enzymatic modifies starch was 19.4%,which imporved 40.8% than the unmodified starch.The molecular fine struture was performed using HPSEC-MALLS-RI,HPAEC and ¹HNMR,and discovered that the SDS formation was improved due to that α-maltotriohydrolase prefered to hydrolysis the external chains of amylopectin,and then increase the high amounts of short chains（DP<13）and the ratio of α-1,6 linkages in enzymatic modified starches.After that,the α-maltotriohydrolase hydrolysis and pullulanase debranching were used in comibination to prepare resistant starch（RS3）in high amounts.The digestibility of retrogradaed starch demonstrated the action of α-maltotriohydrolase was effectively in promoting RS3 formation.Gelatinized maize starch was hydrolyzed by α-maltotriohydrolase at the concentration of 12 U/g for 2 h at 50℃ and pH 6.5,then debranched by pullulanase and retrograded for 48 h to produce 40.8% RS3 rich products.The crystal structure and chain length distribution were conducted with XRD and HPSEC,and Pearson correlations were used to explore the results.The action of α-maltotriohydrolase could significantly improve the amounts of DP 30-130 in debranched starches,determined that the transformation of crystal structure from B to B+V type,improved the relative crystallinity and anti-digestibility and promoted the RS3 formation.The molecular structure of α-maltotriohydrolase hydrolyzed starches were analyzed by HPSEC-MALLS-RI.These results demonstrated that the outner chains were firstly cleved through the exo action of α-maltotriohydrolase,and the long amylose and internal chains of amylopectin was hydrolyzed by endo action;and the amounts of DP 30-130 chains were significantly improved in the debranched starch,then promote the resistant starch formation.Finally,the transglycosylation was verified by HPLC and UHPLC-MS/MS,and the results revealed that the α-maltotriohydrolase could selectively modify the stevioside to produce the maltotriosyl-stevioside as the major product.The effects of reaction conditions on the stevioside conversion were evaluated,and the results showed that the highest conversion of stevioside was reached 46% with 40 U/m L α-maltotriohydrolase in 3 h at 55°C with 1.5 mg/m L stevioside and 15 mg/mL soluble starch in sodium phosphate buffer（pH 6.5）.The enzymatic modified stevioside was refined using the macroporous absorption resin D101,and sensory evaluation and storage stability analysis were conducted.The α-maltotriohydrolase modification induced a 1.2-fold higher sweetness and slight better quality of taste,compared to native stevioside.After 28 days of storage at 25℃,70.6% of enzymatic modified stevioside in aqueous solution（1%,w/v）was presented in a soluble form,compared to 49.8% for stevioside or 26.2% for rebaudioside A.These results suggest that AMTS may be efficient in the modification of sweetness of stevioside,weaken the bitter taste and significantly improve the storage stability in aqueous solution.