Industrial Development Of A Thermo-tolerant Superoxide Dismutase And Its Functions Of Alleviating The Mycotoxins Toxicity To Animals

The application of growth-promoting antibiotics in feed has been rapidly popularized since American scientists found that adding aureomycin to feed could promote the growth of edible animals in 1950.The growth-promoting antibiotics can increase animal growth rate,improve feed utilization efficiency,and reduce animal mortality.However,the abuse of antibiotics in feed increases the resistance of pathogens,which would further increase the health risk of humans and animals.Currently,many countries,including China,have begun to ban the use of growth-promoting antibiotics in feedstuffs.Therefore,it is extremely important to develop alternatives to growth-promoting antibiotics.Previous studies have indicated that the growth-promoting effect of such antibiotics is derived from the bacteriostatic effect and related to the inhibition of inflammation caused by ROS and pro-inflammatory cytokines.Due to contamination of raw materials,mycotoxins are often present in animal feeds.Feeding such feeds would lead to excessive ROS production,oxidative damage,and inflammatory reactions in animals,which would further delay animal growth and cause disease in severe cases.Superoxide dismutase(SOD)is a metal enzyme that can catalyze the disproportionation of superoxide anion radicals.As an antioxidant,SOD can alleviate the oxidative stress reaction caused by excessive ROS in animal cells,inhibit inflammatory damage,and promote animal growth.It is a potential antioxidant enzyme preparation product to replace antibiotics.At present,most commercial SOD products are extracted from animals and plants,but the low contents,low extraction yields,and poor stabilities have always been the limiting factors for large-scale production.Therefore,this research aims to develop a low-cost SOD product that can be used as feed additives and can be industrialized on a large scale.The following works have been carried out in this work:(1)The SOD from Alicyclobacillus sp.HJ(AaSOD)was heterologously expressed in Escherichia coli BL21(DE3),and the enzymatic properties of recombinant AaSOD were characterized detailedly.The results showed that AaSOD prepared in this study has acidophilic and thermostable properties.The highest activity was obtained at pH 4.0,and the enzyme activity remained above 90%when treated at 70℃ for 1 h,and 50%after treatment at 80℃ for 1 h.The melting temperature(Tm)of AaSOD was determined to be 89.54℃ by differential scanning calorimetry.These results indicate that AaSOD has outstanding thermal stability,which endows it with the feasibility of being prepared with regular spray drying protocol in the industrial production process.The SOD prepared in this study has a high retention rate and low production cost,and it is suitable for large-scale production.(2)Through protein homology modeling,the three-dimensional structure of AaSOD was predicted,and the interaction between amino acid residues in the catalytic center and metal ions was analyzed.The point-saturation mutant libraries of Gln149 and Tyr35 in AaSOD were constructed,respectively.The activity of each mutant,when combined with Fe2+or Mn2+,was measured and compared,and 4 mutants with reversed metal ion preference and 4 mutants with significantly improved activity when using Mn2+ were obtained.The largest enhancement of activity was obtained with the Y35F mutant.The specific enzyme activity reached 1504.5 U/mg when combined with Mn2+,which was 313%higher than the wild-type enzyme.It could be expected that this mutant of AaSOD has excellent potential for practical applications.(3)After corresponding codon optimizations,the AaSOD coding gene was expressed in E.coli,Bacillus subtilis,and Saccharomyces cerevisiae,respectively.The B.subtilis was determined to be the host strain suitable for industrial production of AaSOD.Subsequently,a series of factors such as fermentation medium,fermentation process,and post-treatment process were optimized and designed for the constructed B.subtilis.Finally,the process for SOD production was implemented on a 30 m3 fermenter,and the AaSOD enzyme activity reached 6,802 U/mL.After centrifugation,membrane filtration,concentration,and spray drying of the fermentation broth,a product with an enzyme activity of 63,750 U/g was obtained,forming a large-scale SOD production process.The production cost of SOD by this process is low,and the product quality meets the requirements of feed enzyme preparation in the aquaculture industry,and so,the process we established is suitable for commercial production.(4)To evaluate the oxidative stress alleviating effects of exogenous SOD prepared in this study,several animal models,including zebrafish deoxynivalenol(DON)infection model,mice deoxynivalenol(DON)infection model,and mice oxidative stress liver inflammation model were established.The results showed that exogenous SOD could significantly inhibit the ROS generation in zebrafish.Similarly,providing exogenous SOD to DON mice can significantly restore their physical conditions,including recovery of body weight and increase the activities of SOD,CAT,GSH,and GSH-PX.Moreover,SOD also improved the physiological function of the liver and alleviated liver inflammation,which was manifested by the significant decrease in the expression of ALT and AST and inhibition of IL-1β and IL-6 mRNA expression.Transcriptomic results supported that SOD intake could significantly inhibit the expression of RAB5IF,hnRNPAB,and EndoG expression and promote the Ifit1 expression,demonstrating that the effect of SOD on alleviating liver inflammation is related to the regulation of liver cell apoptosis.In brief,a high temperature and acid-resistant AaSOD enzyme suitable for industrial production was screened by excavating stress-resistant microorganisms in this study.After being heterologous expressed in E.coli and purified,the AaSOD was characterized.Its enzymatic properties and mutation characteristics of key sites were studied.Moreover,various engineering model strains were used to express AaSOD,and the best host strain and optimized fermentation conditions were established.On this basis,a large-scale SOD production process route was also established using a 30 m3 fermenter,which laid the foundation for the commercial production of SOD.In addition,this study demonstrated that feeding exogenous SOD to animals can alleviate the oxidative stress and inflammatory response induced by DON,restore the antioxidant system of damaged animals,and improve the physiological function of the liver.These results supported that adding exogenous SOD to feed can resist DON and inhibit animal oxidative stress and inflammatory responses.Finally,the commercial application of the SOD prepared in this study will generate enormous social and economic benefits.