Study On Expression, Modification And Application Of The Maltotetraose-Forming Amylase From Pseudomonas Saccharophila STB07

Maltotetraose is composed of four glucose units connected byα-1,4-glycosidic bonds,and as one of the maltooligosaccharides,it has excellent processing properties and physiological function,which can be obtained by the hydrolysis of starch or dextrin by the corresponding Maltooligosaccharide-forming amylase（MFA）,which has a wide application prospect in food,medicine and other fields.The enzyme activity and substrate conversion rate of MFA are important factors affecting the industrial production of maltooligosaccharides,so it is necessary to improve the enzyme activity and its ability to hydrolyze substrates.The enzyme activity of Maltotetraose-forming amylase from Pseudomonas saccharophila STB07(MFA_PS)constructed in our laboratory is only 100 U/m L and the substrate conversion rate is less than 70%,both of which largely limit its industrial application value.In this study,based on the MFA_PS:（1）Recombinant expression vector suitable for secretion and expression of MFA_PSenzyme is constructed by screening vectors and signal peptides;（2）Mutant with significantly higher substrate conversion and enzyme activity than MFA_PS is constructed by cutting off the Carbohydrate Binding Module（CBM）of MFA_PS;（3）Based on the mutant,the fermentation is optimized by shaking flask and fermenter to improve the level of secretory expression and shorten the fermentation time;（4）Optimize the enzymatic reaction conditions of this mutant and expand the optimized reaction system,then the Simulated Moving Bed（SMB）is used to purify the enzymatic reaction products to obtain high purity maltotetraose.Firstly,the recombinant expression vector p HT01-SP_STB01-MFA_PS、p HT43-SP_STB01-MFA_PS、p P43NMK-SP_STB01-MFA_PS were constructed by optimizing several expression vectors stored in our laboratory.After fermentation,the recombinant p P43NMK-SP_STB01-MFA_PS showed the highest enzyme activity of 185 U/m L,which was 2.28 times higher than that of the p ST-SP_STB01-MFA_PS recombinant vector previously constructed in the laboratory;After reviewing the literatures,we chose a batch of signal peptides（SP）conducive to the secretion and expression of amylase:SPApre,SPGgt,SPAmy E,SPNpre,SPYoj L,SPNpr B,SPBgls,SPepr.Based on the recombinant expression vector p P43NMK-SP_STP01-MFA_PS,the signal peptides in the vector were replaced by the above signal peptides to construct the signal peptide recombinant expression vectors.After fermentation,the vector p P43NMK-SP_Bgls-MFA_PS showed the highest enzyme activity,which was 1.27 times higher than that before the signal peptide was replaced（For ease of writing,vector and signal peptide names are omitted subsequently）.Secondly,by replacing and truncating the CBM of MFA_PS,and the replacement of CBM nucleotides were derived from CBM_{Sd MFA}（CBM of MFA from Saccharophagus degradans）and CBM_{Ro GBE}（CBM of 1,4-α-Glucan branching enzyme from Rhodothermus obamensis）constructed in our laboratory,mutants of MFA_PS-CBM_{Sd MFA},MFA_PS-CBM_{Ro GBE} and MFA_PS-ΔCBM were constructed.The enzyme activity,substrate conversion and maltotetraose conversion of the MFA_PS-ΔCBM reached310.00U/m L,79.80%and 56.36%,respectively,which were increased by 35.00%,11.10%and 14.20%compared with the wild type.Alpha Fold2 was used to simulate the protein structure of the enzyme,and Auto Dock was used to obtain the docking results of the enzyme and substrate.After analyzing the results,hydrolysis characteristics of the mutant were obtained:（1）Removal of CBM did not decrease the product specificity of MFA_PS;（2）For substrates with shorter chain lengths,MFA_PS could still maintain a high substrate conversion rate in the absence of CBM;（3）The product specificity of MFA_PS was related to two aspects:one is the forces generated by the enzyme on the ends of the substrate,and the other is the forces provided by amino acids near the catalytic site.The former affected the mode of enzyme action on the substrates,while the latter affected the specificity and efficiency of the enzyme in catalyzing the substrates.Thirdly,the shaking flask fermentation optimization of MFA_PS-ΔCBM was carried out from the aspects of nutrient composition adjustment of fermentation medium,two-stage temperature control strategy and initial p H of reaction.The optimal nutrient composition of fermentation medium was as follows:corn starch 10 g/L,soybean peptone 6 g/L,Mg²⁺0.5 mmol/L,yeast powder 24 g/L,KH₂PO₄ 2.3135 g/L,K₂HPO₄-3H₂O 16.4318 g/L;the two-stage temperature control fermentation strategy was as follows:fermentation at 37℃for 48 h,then transferred to 30℃for 12 h;initial p H was 7.5.The enzyme activity after optimization was 441.00 U/m L,which increased by 42.30%compared with before optimization,and the fermentation time was reduced by 16.67%.5 L fermenter was used to expand the culture,and the expanded system was2.5 L.Soybean peptone and yeast powder were replaced from experimental grade to industrial grade,and other conditions were expanded according to the system.Based on the two-stage temperature control strategy and dissolved oxygen,the optimized fermentation scheme was as follows:dissolved oxygen was 30%,fermentation at 37℃for 18 h,then transferred to 30℃for 12 h,the final enzyme activity reached 483 U/m L,fermentation time was 30 h,which was 9.53%higher and 50%lower than shaking flask fermentation,respectively.Finally,the preparation of enzymatic hydrolysis products of MFA_PS-ΔCBM was studied.The optimum enzymatic hydrolysis conditions of MFA_PS-ΔCBM were optimized from the aspects of reaction temperature,substrate concentration,reaction p H,enzyme dosage,rotation speed and reaction time.The optimum conditions were as follows:temperature 50℃,p H 7.0,substrate concentration 30%（w/w）,20 U/g substrate-based MFA_PS-ΔCBM was added to the system,rotation speed 500 rpm and reacted 20 h.Under these conditions,the conversion rate of G4 was 58%.After the system was expanded 2000 times,the G4 conversion rate was 54.42%.The reaction liquid after enzymatic hydrolysis was pretreated and the SMB was used for secondary separation.Potassium K310 ion exchange resin was used as stationary phase and water was used as eluting agent.The first separation cycle time was 1230 s,and the second separation cycle time was 1840 s.The flow rates of water and material were 30 and 20m L/min,respectively.The purity of G4 was 83.83%and the recovery rate was about66.31%.