Characterization,Molecular Modification,and Catalytic Mechanism Of Inulin Lyase

Inulin is a common plant polysaccharide,consisting of fructosyl linked byβ-（2,1）glycosidic bonds.To make full use of inulin,it can be converted into functional oligosaccharides,such as difructose anhydride（DFA）,and other high value-added products by bio-enzymatic methods.With the increasing demand for low-calorie sweeteners,DFA have received extensive attention as a novel functional sweetener with low-calorie.Specifically,difructose anhydride I（DFA I）and difructose anhydride Ⅲ（DFA Ⅲ）can be synthesized from inulin by DFA I-forming inulin lyase（ILase I,E.C.4.2.2.17）and DFA Ⅲ-forming inulin lyase（ILase Ⅲ,E.C.4.2.2.18）,respectively,which belong to glycoside hydrolase 91 family（GH91）.Presently,the crystal structure and catalytic mechanism of ILase Ⅲ have been resolved and the catalytic mechanism of ILase I has also been studied.However,the product specificity of ILase I and ILase Ⅲ with similar catalytic function has not been elucidated.Additionally,most researches on the catalytic mechanism of polysaccharide hydrolases focuses on the reactions occurring in the catalytic pocket,while the catalytic process of substrate binding and product release is rarely reported.Therefore,we selected ILase I from a novel microorganism as the research object,and then enhanced the thermostability of ILase I by rational design.Subsequently,the crystal structure of the ILase I was resolved.In addition,the catalytic activity of ILase I was improved by the surface-modification strategy,based on the obtained crystallographic information.Finally,the crystal structure of ILase I complexed with inulin-type oligosaccharide was resolved,and the product specificity of ILase I and ILase Ⅲ was preliminarily investigated.Combined molecular dynamics simulations with the solved structures,the substrate binding pathway and product release channel of ILase I were proposed.The details and conclusions of the study are as follows:（1）Identification of novel ILase Ⅰ（SpILase）.A segment of the gene annotated as ILase derived from Streptomyces peucetius subsp.caesius ATCC 27952 in the NCBI database was selected.The gene encoding Sp ILase was synthesized and introduced into the vector to construct the recombinant plasmid p ET-22b（+）-Sp ILase.Subsequently,the recombinant plasmid was transformed into E.coli BL21（DE3）for protein expression,and Sp ILase was purified by affinity chromatography.Then,DFA formed by Sp ILase from inulin was identified as DFA I.The optimum p H and temperature of Sp ILase were 6.5 and 45°C,respectively,and the T_m value was 75.49°C.Additionally,Ba²⁺could slightly increase the catalytic activity of Sp ILase.Using inulin as substrate,the K_m and k_cat/K_m values of Sp ILase were 3.08 m M and199.09 m M^-1 s^-1,respectively,indicating that Sp ILase had a strong affinity with inulin.Sp ILase synthesized DFA I with a yield of 82.97%at p H 6.5 and 45°C after 6 h,with 10 g/L of inulin as substrate.（2）Rational design was used to improve the thermostability of SpILase.Based on the amino acid sequence and simulated structure,15 mutation sites were selected.Then,four mutants Q69L,E201I,Q234L,and K310G,with significantly improved thermostability,were selected.Subsequently,by combining positive mutation sites,two triple mutants Q69L/Q234L/K310G and E201I/Q234L/K310G were obtained,with the T_m values increased by 4.99°C and 4.50°C,respectively,and 45 times improvement in the approximate half-lives at70°C,compared to the wild type.Additionally,the optimum temperatures were 50°C and 45°C,and the relative enzymatic activities were 100.03%and 53.32%,respectively.Two triple mutants were used to produce DFA I from inulin at their optimal conditions,with a yield of about 84%after 6 hours.Molecular dynamics simulations indicated that the two triple mutants increased the local hydrophobic interactions,changed the surficial local electrostatic potential,and stabilized partial secondary structure,which contributed to the improvement of the thermostability of Sp ILase.（3）The apo structure of SpILase was resolved and molecular modification based on catalytic efficiency was performed.The crystal of Sp ILase was obtained by the classical vapor diffusion method,and the apo crystal structure（PDB accession code:8HSN）was resolved through X-ray diffraction,with a resolution of 1.69（?）.Subsequent analysis showed that Sp ILase was a typical right-handed parallelβ-helix protein,adopting a trimeric assembly mode under the crystallization condition.The trimer and monomer of Sp ILase exhibited a trigonal prism geometry with“loose bottom and tight top”.Additionally,each monomer mainly consisted ofβ-strand and loop,with the characteristic structures of both the“asparagine ladder”and the stacking of aliphatic amino acid residue.Based on the crystal structure of Sp ILase,a surface-modification strategy was used to improve the catalytic activity of Sp ILase.Finally,the triple mutant A282S/A254S/Q69I was obtained with significantly improved catalytic efficiency and substrate affinity,and its enzymatic activity was 636.28 U/mg which was 1.46 times higher than that of Sp ILase.The results of molecular dynamics simulations suggested that the mutant A282S/A254S/Q69I-GF4 had a lower binding free energy compared with Sp ILase,which explained its enhanced enzymatic activity.（4）The complex structure,substrate binding pathway,and product release channel of Sp ILase were resolved.The complex crystal of Sp ILase was obtained by soaking,and its crystallographic information was obtained via X-ray diffraction.The resolution of the complex structure（PDB accession code:8HUI）was 1.44（?）,and we observed that four molecules of fructosylnystose,two molecules of DFA I,and one molecule of fructose were bound as ligands,with a large number of hydrogen-bond interactions and hydrophobic interactions formed between Sp ILase and ligands.A comprehensive analysis of enzymes of the GH91 family showed that the overall shapes of their structures were similar,and their amino acid sequences were highly conserved.Furthermore,structural comparison revealed that the catalytic centers of ILase I and ILase Ⅲ each had three characteristic loops which respectively constituted the unique topology of their catalytic pockets.By substitution mutagenesis,seven mutants were obtained and it was found that their enzymatic activity were significantly decreased or lost,which indicated that these three characteristic loops were important for the catalytic reaction of Sp ILase.Additionally,by molecular dynamics simulations,we speculated that ILase I and ILase Ⅲ might adopt different channels to release the products into the bulk solvent.Based on ligands binding sites and predicted tunnels,twenty key amino acid sites were selected and divided into four residues groups,subsequently constructing alanine mutants at these sites.Eighteen alanine mutants exhibited different degrees of decrease in catalytic activity,while mutants of T247,S267,R296,and D298,which were far from the catalytic pockets,had about 90%decrease in enzyme activity,verifying that the selected key sites also have an important role in Sp ILase catalysis.Ultimately,the potential inulin substrate binding pathway and the potential DFA product release channel of Sp ILase were proposed by analyzing the complex structure and results of alanine mutants.