Characterization,Molecular Modification,and Application Of Key Enzymes In The DFA-Ⅲ Metabolic Pathway Of Inulin

Inulin is a natural fructan abundant in the Asteraceae family,which can be degraded into small molecules of sugar to provide energy for the organism and has excellent physicochemical properties and physiological functions.One of the metabolic pathways of inulin is the degradation of inulin by DFA-Ⅲ-forming inulin fructotransferase（IFTase-Ⅲ,EC 4.2.2.18）to produce difructose anhydrideⅢ（DFA-Ⅲ）.DFA-Ⅲis a functional sweetener and IFTase-Ⅲis the main enzyme used to produce DFA-Ⅲ.Although various sources of IFTase-Ⅲhave been identified,most of them suffer from low enzyme activity or thermal stability,which is not conducive to industrial production.DFA-Ⅲcan be further hydrolysed to inulobiose by difructose anhydride hydrolase（DFAase）,but there are few studies on inulin,probably due to the low yield of inulobiose.The low enzymatic activity of DFA-Ⅲase is the main reason for the low yield of inulobiose,which has prevented the research of inulobiose.Therefore,mining for IFTase-Ⅲwith excellent enzymatic properties and achieving DFA-Ⅲase with high activity is important for the production and research of DFA-Ⅲand inulobiose.In this work,two IFTase-Ⅲwere synthesized by genetic engineering techniques:Arthrobacter sp.ISL-85（As85-IFTase-Ⅲ）and Arthrobacter ramous（Ar IFTase-Ⅲ）,and their enzymatic properties were studied.Ac DFA-Ⅲase derived from Arthrobacter chlorophenolicus A6 was modified by site-directed mutagenesis and random mutagenesis to obtain positive mutants with increased enzyme activity and stability.Finally,these two enzymes were applied to an inulin-rich burdock system to generate DFA-Ⅲand inulobiose with research value,and finally the utilization value of burdock was improved.The main findings of this study are as follows:1.Both the constructed As85-IFTase-Ⅲand Ar IFTase-Ⅲrecombinant enzymes could convert inulin to DFA-Ⅲ.The enzymatic properties of the two enzymes differed significantly,with the specific enzymatic activity of As85-IFTase-Ⅲbeing 783.39 U mg^-1 and that of Ar IFTase-Ⅲbeing only 311.93 U mg^-1.The optimum temperature and p H of As85-IFTase-Ⅲwere 65℃and 6.5 respectively,while those of Ar IFTase-Ⅲwere 55℃and 5.5 respectively.The thermal stability of As85-IFTase-Ⅲwas higher than that of all the IFTase-Ⅲreported so far,maintaining 80%of the enzyme activity after being incubated at 80°C for 4 h,demonstrating the potential of the enzyme for industrial application,while Ar IFTase-Ⅲdisplayed a low thermal stability.Both recombinant enzymes are non-metal ion-conjugating enzymes and can generate DFA-Ⅲwith nistose（GF₃）as the minimum acting substrate.The K_m of As85-IFTase-Ⅲand Ar IFTase-Ⅲwere 5.96 and 2.95 mmol L^-1,respectively.When 100 g L^-1 of inulin was used as the substrate,the conversion rates of As85-IFTase-Ⅲand Ar IFTase-Ⅲwere 73.70%and 71.30%,respectively.2.The key amino acid residues in the active pocket of Ac DFA-Ⅲase and the loop above the active pocket were selected for site-directed mutagenesis,and 30 mutants were obtained.More than 2300 mutants were obtained by random mutagenesis.Using high-throughput screening techniques,E389A,E91K,D380A and D393A mutants were screened out,whose enzyme activities were increased to 3.63,2.57,1.63 and 1.23 times of Ac DFA-Ⅲase,respectively.Moreover,the mutants showed different degrees of improvement in thermal stability,with E389A showing the most significant improvement,whose half-life at 55°C increasing to four times that of the wild type.The mutants also showed different degrees of improvement in affinity,catalytic efficiency and conversion ratio.Homology modelling analysis suggested that the improved enzyme activity and thermal stability of the mutants was due to increased intramolecular interaction forces.3.Finally,the obtained As85-IFTase-Ⅲand E389A were applied to the burdock system to achieve an increase in the added value of burdock.Using burdock root as raw material,a simple hot water extraction was used to obtain 26.34 g of inulin from 100 g of burdock root（dry weight）,as well as a small amount of oligofructose.After the reaction with the addition of As85-IFTase-Ⅲ,15.71 g of DFA-Ⅲ,and small amounts of 1-kestonse（GF₂）,GF₃,and fructofuranosylnystose（GF₄）were produced,and these several sugars can act synergistically to enhance the physiological efficacy of burdock.After the addition of the E389A mutant,the inulobiose content of the system was increased to 4.93 g.This work provides a new reference for the industrial application of IFTase-Ⅲand DFA-Ⅲase and a new idea for the deep processing of burdock.