Gene Excavation And Molecular Engineering Of Enzyme To The Removal Of Anti-Nutrients From Mixed Meals

Rapeseed meal and cottonseed meal are common unconventional feed ingredients,which have high application potential in livestock diets.However,the existence of anti-nutritional factors such as sinapic acid,gossypol and glucosinolates severely limits their application as feed ingredients.At present,there are many disadvantages in detoxification methods of rapeseed meal and cottonseed meal,but enzymatic degradation has great potential.It is of great significance for the research and development of new enzyme preparation and the application of miscellaneous meal in feed to screen and study the mechanism of removing anti-nutritional factors related enzymes.In this study,sinapic acid and gossypol were degraded by fungal laccase.The fungal laccases from Talaromyces leycettanus JCM12802,Bispora sp.MEY-1,Trametes cinnabarina,Trametes versicolor,and Coprinopsis cinere were expressed in Pichia pastoris named Tllac,Bslac,Tvlac7,Tclac1 and Cclac9.The laccase activity of CcLac9 reached 310 U/mg with ABTS as substrate,and the optimum temperature was60°C.Therefore,CcLac9 is an ideal material for eliminating sinapic acid and gossypol.CcLac9 can remove sinapic acid and gossypol effectively.The optimal pH for the degradation of sinapic acid by CcLac9 was 5.0,and the K_m was 25μmol/L.At 30°C,50μg/m L sinapic acid could be completely degraded by 1.0 U/m L CcLac9 within 30 min.The MS analysis showed that the degraded product was a lactone dimer formed by the C-C coupling of two sinapic acid.Sinapic acid can enter the TPO enzyme activity center to inhibit the activity of TPO and affect the synthesis of thyroid hormones.Through the detection of the enzyme activity of chicken thyroid peroxidase（TPO）,it was found that its inhibitory intensity was stronger than that of potassium thiocyanate and oxazolidinone.It shows that the main anti-nutritional factor causing goiter in rapeseed meal is sinapic acid.After sinapic acid was degraded by CcLac9,the dimer form hindered its entry into TPO enzyme molecules,which weakened the inhibitory effect.The optimum pH for the degradation of gossypol by CcLac9 was 7.0,and 1.0 U/m L CcLac9 could completely degrade 10μg/m L gossypol within 2 h.Through mass spectrometry analysis,CcLac9 eliminated aldehyde groups and hydroxyl groups in gossypol molecules and achieved ring-opening degradation.Phloroglucinol can undergo autooxidation under alkaline conditions,while gossypol can combine with superoxide anion generated during the reaction to accelerate the autooxidation rate and increase the amount of autooxidation products.This phenomenon disappeared after gossypol was degraded by CcLac9,indicating that CcLac9 eliminated the active groups on gossypol molecules and weakened the biological activity of gossypol.It indicated that CcLac9 had great research value in gossypol degradation.In order to improve the catalytic activity of CcLac9 and make it better meet industrial needs.The catalytic activity of CcLac9 was improved by irrational design and semi-rational design.The correct expression of CcLac9 in E.coli was realized,and the directional evolution high throughput screening method was established with E.coli as the host,QPix 420 automatic cloning selection system and Biome K NXp automatic liquid transfer workstation.The three mutants with increased enzyme activity were V89L:426U/mg,S342T:364 U/mg,D165E/M253I/V268I/S372L/D447N:429 U/mg,respectively.In addition,molecular docking and virtual mutation of CcLac9 and ABTS were performed by Discovery Studio software.Site-directed mutagenesis was performed on amino acids near the substrate pocket of CcLac9,and finally two mutants with increased enzyme activity were obtained,namely,N285D:408 U/mg,F475W:674 U/mg.The effective mutants were combined by combined mutation,and finally the combined mutant N285D/F475W was obtained.Its enzyme activity was 220%higher than that of the wild type,reaching 695U/mg.In addition,its thermal stability had no obvious loss compared with that of the wild type,and it had good application potential.Nitrile derivatives derived from glucosinolates in rapeseed meal can cause liver and kidney damage in animals,which is the main pathogenic factor of glucosinolates.Nitrilase converts nitrile into corresponding nontoxic carboxylic acids and NH₃.Therefore,in this study,nitrilase was involved in the degradation of nitrile compounds derived from glucosinolates.A nitrilase BnNIT2 from Brassica napus was selected.The optimum temperature and pH of BnNIT2 were 45°C and 7.0,respectively.BnNIT2 was stable at 40°C.BnNIT2 could convert three main nitriles（3-hydroxy-4-pentenonitrile,3-butenonitrile and 4-pentenonitrile）derived from glucosinolates,and the specific activity of 4-pentenonitrile was the highest（58.6 U/mg）.Multiple sequence alignment and FoldX software were used to screen mutants that improved the thermal stability of BnNIT2.The results showed that the residual activity of mutant BnNIT2-H90M increased by14.5%after incubation at 50°C for 1 h.In order to verify the function of BnNIT2,thioglucoside was extracted from RSM and converted into nitrile in acetic acid buffer（pH 5.0）containing Fe²⁺.Then BnNIT2was used to degrade the converted nitriles,and finally about 80%of the nitriles were removed.In conclusion,nitrilase BnNIT2 can effectively degrade thioglucoside-derived nitriles.This is a new application of nitrile hydrolase and a new way to eliminate glucosinolate toxicity in RSM.In this paper,fungal laccase CcLac9 and plant nitrilase BnNIT2 were screened for their good degradation effects on sinapic acid,gossypol and glucosinolates.Through high performance liquid chromatography（HPLC）,mass spectrometry（MS）,TPO activity analysis,pyrogallol autoxidation and non-enzymatic conversion of glucosinolates,their degradation mechanisms were elaborated and the toxicity of the degraded products was analyzed.Meanwhile,the properties of CcLac9 and BnNIT2 were improved by irrational design and semi-rational design.It provides a new method for the removal of anti-nutritional factors in the mixed meal,a theoretical guidance for the mining of new enzyme preparations,and an important reference for the molecular improvement of laccase and nitrile hydrolase.