Characterization Of A Novel Starch Branching Enzyme MfGBE From Myxococcus Sp.V11 And Its Application

Starch branching enzyme（EC 2.5.1.18）is a member of the glycoside hydrolase GH13family.Its mechanism of action is to hydrolyze theα-1,4 glycosidic bond through glycoside hydrolysis to generate a non-reducing end sugar chain,and then connect the C₁end of the free sugar chain to the C₆position adjacent to the glucose unit through glycosyl transfer to formα-1,6 branches.Using starch branching enzymes to process starch can improve the degree of branching of starch,generate slow-digesting starch,and prevent starch from aging.Therefore,starch branching enzyme is an important starch industrial enzyme.However,limited by the low activity of starch branching enzymes currently studied,commercial and industrial starch branching enzymes are still rare.Mining and identifying starch branching enzymes with high activity and high catalytic rate is of great significance to promote starch processing and resource utilization.In the previous work,a Myxococcus sp.V11 was isolated and screened in our laboratory.After genome sequencing and annotation,it was found that there was a gene orf1993 encoding branching enzyme in its genome,and its amino acid sequence was less than 60%identical to the reported starch branching enzymes.In this study,the recombinant pure enzyme was obtained through gene cloning,heterologous expression and protein purification,and its catalytic properties were characterized.Amino acid sequence analysis found that the starch branching enzyme encoded by orf1993 had a partial sequence missing from the N-terminus,so it was named MfGBEΔ（N1’）.By constructing the p ET-28a（+）-mfGBEΔ（N1’）expression vector and introducing the recombinant plasmid into E.coli BL21（DE3）,the efficient soluble heterologous expression of MfGBEΔ（N1’）was achieved after induction.The recombinant enzyme was purified by separation and ion exchange chromatography.Using the purified MfGBEΔ（N1’）as material,the optimum substrate and optimum reaction temperature were determined respectively.The results showed that the optimum substrate for MfGBEΔ（N1’）was tapioca starch,the optimum reaction temperature was 35℃,the optimum reaction p H was 7.5,and the measured specific enzyme activity was 5,895.98 U/mg.MfGBEΔ（N1’）is not stable to high temperature,but it can maintain good stability at the optimum temperature and p H of 7-10.Under the optimum reaction conditions,Ca²⁺and Mg²⁺can activate to a certain extent,but most organic solvents can inhibit the enzyme activity to a certain extent.Bioinformatics analysis found that the mfGBEΔ（N1’）gene contains 2022 base pairs,encoding 674 amino acids,and its protein has a molecular weight of 72 k Da.Site-directed mutation of starch branching enzyme gene was carried out,and the active center of MfGBEΔ（N1’）was found to be composed of triplet D353-E405-D474 by observing the enzymatic properties of the mutant enzyme.This result indicates that MfGBEΔ（N1’）is a glycoside hydrolase belonging to the GH13 family.We reanalyzed the MfGBEΔ（N1’）structure and sequence to find the missing domain N1.After the recombinant holoenzyme was purified by salting out,ion exchange chromatography and hydrophobic chromatography,the effect of the N-terminal domain on the enzymatic activity and stability of MfGBE was revealed by comparing the enzymatic properties.The enzyme activity assay found that the specific enzyme activity of the enzyme under optimum conditions was 18,338.98 U/mg,which was 3.11 times that of MfGBEΔ（N1’）.MfGBE can maintain good stability at p H 5-11 and improve the tolerance to high temperature.The results indicated that the starch branching enzyme domain N1played an important role in enhancing the enzyme activity and maintaining the stability of the enzyme.The analysis of the enzymatic hydrolysis products of MfGBE showed that MfGBE could effectively improve the branching degree of tapioca starch.The generation of starch amylopectin decreased the binding ability of starch and iodine solution,and the maximum absorption wavelength shifted from 620 nm to 550 nm.However,a shift of the maximum absorption wavelength from 580 nm to 600 nm occurred during the decrease in absorbance.At the same time,in the analysis of the length distribution of the side chain,it was found that after the reaction for 20 minutes the degree of polymerization（DP）of 20-40 is increased and gradually decreased with the reaction,causing the content of DP<14increased.This experiment shows that starch reacts with enzymes to cleave long chains and transfer them to short chains.Based on the study of starch branching patterns,this experiment also evaluated MfGBE in enhancing the anti-retrograde properties of tapioca starch and the content of slow-digesting starch and resistant starch.We explored the changes in molecular structure,macro-rheology and thermodynamics of starch induced by starch branching enzymes.The cold water solubility,Fourier transform infrared spectroscopy,rheological properties and differential scanning calorimetry analysis showed that after the MfGBE reaction,the reduction of intermolecular hydrogen bonds of starch reduced the recrystallization ability of starch,thereby delaying the aging of tapioca starch.The content of starch with different digestive properties was determined by Guyara method,and it was found that the content of slow-digestible starch increased from 27.70%to 32.33%;this result proved that enzymes have certain development potential in food health care.