| Maltotetraohydrolase or maltotetraose amylase?EC 3.2.1.60?has exonuclease activity and belongs to the GH13 family of starch hydrolyzing enzymes.Maltotetraose is a linear malto-oligosaccharide composed of four?-D-type glucose groups linked by?-1,4 glycosidic bonds.It is a functional food with low sweetness,high viscosity and good moisturizing properties.It has the characteristics of easy digestion and absorption,low osmotic pressure,inhibiting intestinal spoilage bacteria,maintaining intestinal health,and promoting the body's absorption of Ca2+.It is mainly used in food and medical fields.In this study,the maltotetraose amylase derived from Pseudomonas saccharophila was first expressed in Bacillus subtilis WS11?food safety strain?.Followed by the recombinant maltotetraose amylase was separated and purified,then the enzymatic properties of the recombinant maltotetraose amylase were examined.The fermentation medium of maltotetraose amylase in shake flasks and 3-L fermentor experiments was then optimized to achieve high expression of maltotetraose amylase.Finally,the process conditions for the preparation of maltotetraose by recombinant maltotetraose amylase were optimized,and the application effect of recombinant enzyme in bread was investigated,which laid a foundation for industrial production.The main results are as follows:?1?The recombinant strain B.subtilis/pHY300PLK-g4 was constructed,and the extracellular enzyme activity of maltotetraose amylase reached a maximum of 147 U·mL-1after cultured in shake flask for 48 h at 33?.SDS-PAGE results showed that there was a protein band in each of 57 kDa?type I maltotetraose amylase?and 47 kDa?type II maltotetraose amylase?,and the results showed that maltotetraose amylase derived from Pseudomonas saccharophila was expressed in B.subtilis WS11 successfully.The recombinant enzyme was purified by a Mono Q anion exchange column,and then the enzymatic properties of the recombinant maltotetraose amylase were examined.The results showed that with the soluble starch as the substrate,the maximum reaction rate(Vmax),substrate affinity?Km?,catalytic constant(kcat)and catalytic efficiency(kcat/Km)of type I enzyme were 1739.24?mol·min-1·mg-1,0.13 mg·mL-1,1673.73 s-1 and 12434.84 mL·mg-1·s-1;the type II enzyme were 1390.67?mol·min-1·mg-1,0.42 mg·mL-1,1097.70 s-1 and 2638.70mL·mg-1·s-1.The optimum temperature for type I and type II enzymes was 55?,the optimum pH for type I enzyme was 7.5,and the optimum pH for type II enzyme was 7.0.?2?Fermentation optimization of recombinant bacteria,firstly the fermentation medium of maltotetraose amylase produced by recombinant strain was optimized at the shake flask level.The results showed that the optimal medium composition was:25 g·L-1 soybean meal and 25 g·L-1 industrial peptone,5 g·L-1 glycerol.Under these conditions,the enzyme activity of maltotetraose amylase was 236 U·mL-1 after cultured in shake flask for 48 h.Based on the shake flask fermentation experiments,the medium composition was optimized at the level of3-L fermentor and the cost was reduced.The results showed that at 37?,pH 7.0,12 g·L-1glucose,25 g·L-1 industrial peptone and 25 g·L-1 soybean meal as the initial medium,250g·L-1 glucose,180 g·L-1 industrial peptone and 70 g·L-1 soybean meal as the feed medium,by78 h,the enzyme activity of maltotetraose amylase reached a maximum of 1907 U·mL-1,which was 13 times greater than that seen in the original shake-flask experiments.Then,the growth and enzyme production of recombinant bacteria in 15-L fermenter were investigated.The enzyme activity reached 1219 U·mL-1 by 96 h.?3?The effects of reaction temperature,initial pH,enzyme dosage,substrate concentration and reaction time on the preparation of maltotetraose by recombinant maltotetraose amylase were investigated.The optimal conditions for the preparation of maltotetraose by type I enzyme were finally determined:temperature was 50?,initial pH was 7.5,enzyme amount was 30 U·g-1 maltodextrin?DE 5-7?,substrate concentration was150 g·L-1,the reaction time was 12 h,the maximum conversion rate can reach 73.2%;the optimal conditions for the preparation of maltotetraose by type II enzyme:temperature was50?,initial pH was 7.0,enzyme amount was 30 U·g-1 maltodextrin?DE 5-7?,the substrate concentration was 250 g·L-1,the reaction time was 12 h,and the maximum conversion rate can reach 73.0%,the conversion rates of the preparation of maltotetraose under the optimal conversion conditions of type I and type II enzymes are close.?4?The effect of recombinant maltotetraose amylase in bread was studied.Toast bread was made by high-gluten flour,eggs,milk,sugar,salt and butter.The texture profile analysis of the toast bread was determined,the results showed that the toast bread with the type I enzyme had the highest score.Then,the amount of enzyme of the type I enzyme was optimized and the change of texture profile analysis parameters of the bread during storage was examined,after 7 days of storage,the hardness?g?,adhesiveness?g?and springiness of the main parameters of the bread supplemented with 0.45 U·g-1 type I enzyme dough were6058.008,-83.419 and 1.082,while the control group was 8685.562,-268.709 and 0.805,indicated the recombinant maltotetraose amylase can extend the shelf life of the bread. |
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