High-efficiency Expression Of Soybean-derived Antioxidant Pentapeptide SHCMN In Bacillus Subtilis And Its Hygroscopic Behavior Regulation

Food-borne antioxidant peptides are a class of safe,healthy,low molecular weight,low cost,high activity,easy to absorb compounds,which are mainly obtained from enzymatic hydrolysate of different protein.However,the industrialized production of enzymatic preparation is difficult,with low yield,high cost and complex separation and purification.In order to meet the technical requirements of industrial-scale production of antioxidant peptides,in this study,the soybean-derived antioxidant pentapeptide SHCMN was used as the research object,and DNA recombinant technology was used to express antioxidant peptide.The expression systems of bacillus subtilis and pichia pastoris were constructed.In addition,conditions for efficient expression of target protein by recombinant Bacillus subtilis 1A751-pMA5-20 bt was optimized and the enzyme digestion products were analyzed.The hygroscopic properties of the target peptide and regulation of hygroscopicity with Zn²⁺ were investigated.This study provides technical support and research basis for the subsequent development of biosynthesis technology of food-borne bioactive peptide.The target pentapeptide SHCMN was ligated with arginine to construct different copy tandem multimer as His6R(SHCMNR)_n.The target gene fragment was respectively recombined with the shuttle plasmid pMA5 and the plasmid p PIC9 K to construct the Bacillus subtilis expression system and the Pichia pastoris expression system.These two expression system were analyzed by Tricine-SDS-PAGE,Western-blot,UPLC-MS-MS and RP-HPLC.The results showed that the ten-copy tandem multimer,His6R(SHCMNR)10,was expressed in Bacillus subtilis system,but not in Pichia pastoris GS115 system.In addition,recombinant strain 1A751-pMA5-15 bt and 1A751-pMA5-20 bt can efficiently express the target protein,His6R(SHCMNR)15 and His6R(SHCMNR)₂₀,and these two target proteins existed in the intracellular supernatant.The target protein His6R(SHCMNR)₂₀ expressed by recombinant strain 1A751-pMA5-20 bt is not only present in a single form but also in the form of multimer.The protein His6R(SHCMNR)₂₀ was purified by Ni-NTA-Sepharose affinity chromatography and G-75 gel chromatography.After digestion by trypsin and carboxypeptidase B,the soybeanderived antioxidant pentapeptide monomer SHCMN could be released.Single factor experiment and response surface experiment were used to optimize the fermentation medium,inoculation amount,fermentation time and temperature at the shaking bottle level,and the cell density(OD600 value)as well as Western-blot gray value of the target protein was measured.Finally 2×SR medium was selected as the experimental fermentation medium,and the combination of fermentation parameters was inoculation ratio 2.43%,temperature 28.1°C,fermentation time 34.2 hours.Under the above conditions,the final expression yield of His6R(SHCMNR)₂₀ was 40.2 mg/L.Hydroxyl radical scavenging rate,ABTS radical scavenging capacity,DPPH radical scavenging capacity and oxygen radical absorption activity(ORAC)were used to evaluate the antioxidant activity of the enzymatic hydrolysate of His6R(SHCMNR)₂₀.The results showed that the hydroxyl radical clearance rate was 13.22±1.27% at 0.5mg/m L with DPPH radical scavenging rate 74.7±0.5%(Trolox equivalent 69.1 ?M).When the sample concentration was 0.125 mg/m L,ABTS·+ radical clearance rate was 67.3±1.6%,and its Trolox equivalent was 49.6 ?M.The Trolox equivalent calculated by net AUC at 0.1 mg/m L was 67.27 ?M.In conclusion,all the four evaluation methods can prove that enzymatic hydrolysate of His6R(SHCMNR)₂₀ have certain antioxidant activity,among which the scavenging activity of oxygen free radical is the highest.The soybean-derived antioxidant pentapeptide SHCMN was synthesized by F-moc solid phase and used as the research object.The dynamic vapor adsorption(DVS)apparatus and low field nuclear magnetic(LF-NMR)were used to investigate the water vapor sorption kinetic and distribution of adsorbed water.It was found that the antioxidant pentapeptide SHCMN had a relatively significant hygroscopic property and its mass increased by 17.67% when the relative humidity RH was 95%.The results of LF-NMR showed that the adsorbed water existed mainly in the form of bound water.During the research of Zn²⁺ targeted regulation of soybean-derived antioxidant pentapeptide moisture absorption site,pentapeptide SHCMN synthesized by F-moc solid phase and its zinc chelate SHCMN-Zn were studied.UV spectroscopy,elemental analysis,X-ray diffraction,mid-infrared spectroscopy and SEM were used.The water adsorption capacity of pentapeptide and SHCMN-Zn complex was studied by DVS and LF-NMR.The results showed that the zinc ion mainly chelated with carboxyl oxygen atom and amino nitrogen atom to form SHCMN-Zn complex,which exhibited spherical crystal structure.From the DVS results,when the relative humidity RH was 95%,the moisture adsorption capacity of SHCMN-Zn complex was lower than that of the SHCMN peptide powder.The results of low field nuclear magnetic experiments showed that the mass change of pentapeptide and SHCMN-Zn complex were 22.69% and 10.61% after being placed for 220 min at 93.4% relative humidity,which provides technical support for the subsequent research on controlling the hygroscopic behavior of peptide powder.