Separation And Purification Of Lipoxygenase From Soybean Whey Wastewater And Its Application

In this paper,the resource utilization of lipoxygenase(LOX)in soybean whey wastewater was taken as a research target,and a technical solution for simple operation,economic feasibility and large-scale production of LOX was explored.In addition,LOX was used as a new enzyme preparation instead of chemical improver in flour products processing to study its role in food processing.The main research contents were as follows:(1)Optimization of process conditions for precipitation separation and purification system.By comparing the effects of isoelectric point separation precipitation,PEG6000separation precipitation and sodium polyacrylate flocculation precipitation on the extraction of LOX from soybean whey wastewater,it is concluded that the isoelectric point fractional precipitation technology had better effect on LOX precipitation.Then the LOX precipitation extract was subjected to ultrafiltration concentration optimization experiment.The optimal ultrafiltration conditions were as follows:operating pressure200KDa,and the precipitation extract was concentrated 10 times with 30KDa ultrafiltration membrane and secondary ultrafiltration method.The unit enzyme activity of the solution was 19713.092 U/m L.Finally,the ultrafiltration concentrate was further purified by Sephadex G-75 gel chromatography.The results of SDS-PAGE gel electrophoresis showed that the final purity and enzyme recovery of LOX extract were90.43%and 40.29%,respectively,the LOX specific activity was 25546.08 U/mg,and the purification factor increased by 25.44 times.(2)Optimization of process conditions for PEG6000-(NH₄)₂SO₄ aqueous two-phase purification system.The extraction conditions of LO X in soybean whey wastewater by PEG6000-(NH₄)₂SO₄ aqueous two-phase extraction system were optimized by single factor experiment and Box-Benhnken Center combined response surface design experiment.The optimal extraction conditions were as follows:PEG6000 mass fraction was 19.2%,(NH₄)₂SO₄ mass fraction was 10.5%,wastewater addition amount of 20%,p H 6.0,and under these conditions,the LOX extraction rate and purification factor were 85.45%and 4.06,respectively.Then the ATPS extract was concentrated and purified by ultrafiltration and gel filtration chromatography.The purification process of LO X was analyzed by SDS-PAGE gel electrophoresis.In the final purified solution,the LOX specific activity as high as 21223.75U/mg,the purification factor was increased by 21.39 times.The purity and enzyme activity recovery were 70.03%and 38.30%,respectively.(3)Optimization of process conditions for[Bmim]Br-K₂HPO₄ aqueous two-phase purification system.The extraction conditions of LOX in soybean whey wastewater by[Bmim]Br-K₂HPO₄ aqueous two-phase extraction system were optimized by single factor experiment and Box-Benhnken Center combined response surface design experiment.The optimal extraction conditions were as follows:[Bmim]Br mass fraction was 32.37%,K₂HPO₄ mass fraction was 25.84%,extraction time was 120min,p H 6.0,and under these conditions,the LOX extraction rate and purification factor were 91.21%and 2.57,respectively.Then the ILATPS extract was concentrated and purified by ultrafiltration and gel filtration chromatography.The LOX purification process was also analyzed by ultrafiltration,gel filtration chromatography and SDS-PAGE gel electrophoresis.The LOX specific activity in the purified solution was as high as18055.24 U/mg,the purification factor was increased by 17.03 times,and the purity and enzyme recovery were 68.55%and 18.05%,respectively.(4)By comparing the above three separation and purification methods for the extraction of LO X in soybean whey wastewater,it can be seen that the extract obtained by the isoelectric point grading precipitation had the highest purity(90.43%),the enzyme activity recovery rate was 40.29%,and the LOX specific activity was also higher than the other two extraction methods,and the scale of the isoelectric point grading precipitation system was easy to enlarge,saving the enterprise cost,and was more suitable for the industrial production of LOX.(5)The purified LOX was applied as a food improver to noodle processing to study the effect of LOX on the processing characteristics of the noodle.Combined with the results of LOX cooking characteristics and texture characteristics,it can be concluded that when the LOX addition amount was 0.6%,the best cooking time of noodles was shortest,the broken rate and dry matter loss rate reached the minimum,the water absorption rate of noodles rised.While the hardness,elasticity,adhesiveness and chewiness of the noodles increased to 7861.318,0.903,4525.252 and 3719.072,respectively,and the adhesiveness of the noodles decreased to-102.62.In addition,the addition of LOX had little effect on the cohesiveness and resilience of noodles.It can be seen from the results of sensory quality measurement of noodles that when the amount of LOX added in the noodles was in the range of 0.6%-0.8%,it was easily accepted by consumers.