Thermal Stability Improvement And The Application Of Lactate Oxidase On Silage Treatment Of Straw Waste

As a country with large-scale agriculture,China has an astonished straw mass yield.Due to the lack of effective solutions,the straw excess problem with regional,seasonal and structural characters has appeared.Straw burning in the rural areas even induced heavy air pollution.In addition,without effective recycle of biomass,the soil in our country is becoming hard and the soil fertility is getting worse.Produce feed with straw is beneficial to improve straw utilization and incrase economic benefit.During the process,the silage treatment is an important part.The silage can improve the feed intake and promote digestibility in ruminant animals;however,the side-product,the large amount of lactic acid,is adverse for ruminant health.Lactate oxidase is the enzyme that converts lactic acid into pyruvic acid,which can be further utilized by most rumen microbes.This process consumes oxygen in the meantime,which will benefit to form an anaerobic rumen environment.In this study,we research the ruminal acidosis and lactate oxidase action mechanism with molecular biology.The results are shown as follow:?1?A lactate-utilizing microorganism,Enterococcus gallinarum,was isolated from rumen,and the lactate oxidase?EgLOD?was cloned from this strain.The deduced EgLOD shares the highest sequence identity of 64.8%with the lactate oxidase from Streptococcus faecalis.?2?The lactate oxidase?EgLOD?expressed in Escherichia coli?BL21?.The optimal pH and temperature for the purified EgLOD were 6.0?7.0 and 25?35oC.The K_m and V_max values for EgLOD were 0.74 mM and 50.20?mol min^?1mg^?1.During the optimized fermentation process,addition of 0.1?1.0 g/L riboflavin increased the EgLOD activity by approximately two folds.?3?We use bioinformatics methods to improve the thermal stability of lactate oxidase with no damage to enzyme activity.After incubated under 60?for 30 min,the mutants D250N and D281N can maintain more than 50%of the enzyme activity,and the wild type EgLOD only remained 20%enzyme activity.?4?After pretreatment with the EgLOD,the lactate conversion rate of corn straw silage reached up to 65%.The results indicate that the EgLOD has great application potentials to improve ruminant health against acidosis.