Study On The Mechanism Of Linkage Specificity Of The Product Catalyzed By Limosilactobacillus Reuteri 121 4,6-α-Glucosyltransferase GtfB

Limosilactobacillus reuteri 121 4,6-α-glucanotransferase enzymes（Lr121 4,6-α-GTase）GtfB is a novel amylase that uses starch/maltodextrin as a substrate to form soluble dietary fiber-Isomalto-malto polysaccharides（IMMPs）,which has high commercial value.The three-dimensional crystal structure of Lr121 4,6-α-GTase GtfB-ΔNΔV（N-terminally truncated and with deletion of structural domain V）has been reported and the amino acids near its acceptor subsite are essential for reaction and product（linkage）specificity,but the amino acid residues at the acceptor subsite have not been fully determined and their role in the linkage formation mechanism remains to be explored.Based on the above problems,through sequence alignment analysis and crystal structure analysis,the amino acid residues near the acceptor subsite in the homologous amino acid sequence of Glycoside hydrolase（GH）70 family were explored,mutants were constructed by site-directed mutagenesis,and the enzymatic properties such as enzyme activity,kinetic parameters,bond content changes and product specificity of the mutants were investigated to examine the effect of amino acids at the acceptor subsite on 4,6-α-GTase during linkage formation and product synthesis.The main research contents are as follows:（1）Selection of mutant sites and characterization of mutants:through sequence alignments between GtfB enzymes with different product linkage specificity（4,6-α-GTase and 4,3-α-GTase,with a high sequence identity）,amino acid residues in conserved amino acid sequences with unique sequence variation were screened.Amino acid residues located near the acceptor subsite were selected and mutants I1020M,S1057P,H1056G,Q1126I and I1020M-H1056G-S1057P-Q1126I were constructed.The changes in the product（α1→6）linkage content were explored using maltodextrin and maltoheptaose as substrates.The analysis of the results showed that H1056G and I1020M-H1056G-S1057P-Q1126I showed a decreased total activity and substrate affinity,which in turn affected the product（α1→6）content（9%,16%and 27%,18%decrease with maltodextrin and maltoheptaose as substrate,respectively）;mutant Q1126I increased substrate affinity,the content of the maltodextrin as substrate product（α1→6）by 11%.The other mutants（I1020M and S1057P）showed less variation in linkage specificity and product specificity.Therefore,site H1056 and Q1126 are the focus of subsequent studies.（2）Research on the influence of the key amino acid H1056,which has direct hydrogen bonding with the acceptor,on GtfB-?N reaction specificity and linkage specificity:Molecular docking model of GtfB-ΔN with maltoheptaose showed that there was a hydrogen bonding between the H1056 side chain and maltoheptaose in subsite+2.Mutants H1056R,H1056A,H1056V,H1056Y,H1056G,H1056F and H1056Q were constructed and heterologously expressed.The optimal temperature and optimal p H of mutants remained to be 40°C and 5.0,respectively.Enzyme activities of these mutants were determined to be 2.32,1.81,2.09,1.55,1.75,1.17 and 1.72 U/mg,respectively,which were all lower than that of GtfB-ΔN at 2.54 U/mg except for H1056R.The mutant H1056Y and H1056F had the highest enzyme loss.The substrate affinity of all mutants except mutants H1056R and H1056V was reduced;the catalytic efficiency(k_cat/K_m)of all mutants was reduced.The（α1→6）contents of the products with maltoheptaose as substrate were 44%,33%,29%,29%,26%,26%and 33%,which were lower than 53%of GtfB-ΔN except for H1056R.The results showed that the hydrogen bond interaction between H1056 and the substrate and the steric hindrance change of the side chain group would affect the enzyme activity and product specificity.（3）Research on the effect of changes in the the steric hindrance of Q1126,a key amino acid with an indirect effect on acceptor binding,on GtfB-?N reaction specificity and linkage specificity:Molecular docking model of GtfB-ΔN with maltoheptaose showed that Q1126 is located near the acceptor binding channel in the GtfB-ΔN substrate binding groove.Mutants Q1126F,Q1126V,Q1126R,Q1126Y,Q1126W,Q1126G and Q1126I were constructed and heterologously expressed.The optimal temperature and optimal p H of mutants remained to be40°C and 5.0,respectively.Enzyme activities of these mutants were determined to be 1.78,2.73,1.87,1.48,2.57,1.37 and 3.32 U/mg,respectively,which were lower than the 2.54 U/mg of GtfB-ΔN except for Q1126I,Q1126V and Q1126W.The results of kinetic parameters showed that the K_m values of Q1126I and Q1126V decreased and the substrate affinity increased,while the catalytic efficiency of other mutants decreased.The（α1→6）contents of the products with maltoheptaose as substrate were 29%,51%,20%,32%,38%,60%and 50%,respectively,and Q1126G was elevated compared to 53%of GtfB-ΔN.The results suggested that changes in the steric hindrance due to changes in the Q1126 side chain group at sites near the acceptor binding pathway affect the enzyme activity and its product linkage specificity.（4）Study on the effect of substrate structure on the bonding specificity and size of mutant products:Maltodextrin was chosen as the second substrate to investigate the effect of the H1056and Q1126 sites on the linkage specificity and size of the products of the structurally larger substrates.The NMR results showed that the（α1→6）linkages of the products of mutants H1056R,H1056A,H1056V,H1056Y,H1056G,H1056F and H1056Q were 33%,28%,26%,25%,26%,23%and 26%,respectively,which were lower than 35%of GtfB-ΔN except for H1056R.The degree and trend of decrease were the same as the trend of the linkage content of the product（α1→6）with G7 as the substrate.The molecular weight of the products of the mutants increased except for H1056R,which demonstrated that the H1056 site also had a significant effect on the ability to transglycosylateα-1,6 with a larger structure and a few branches of the substrate.The（α1→6）linkages of the products of mutants Q1126F,Q1126V,Q1126R,Q1126Y,Q1126W,Q1126G and Q1126I were 22%,32%,17%,24%,30%,30%and46%,respectively,and only Q1126I was increased compared with GtfB-ΔN of 35%.The results indicated that the side chain amino acids in the 1126 site would increase the（α1→6）linkages of the products substrates with a large structure and a few branched substrates and reduce the molecular weight;short side chain amino acids would increase the product（α1→6）content of shorter linear substrates.It indicated that the size of the side chain at the Q1126 site would have different effects on different structural substrates.