Study On Key Nitrogen Source Factors And Enzymatic Degradation Conditions Of Hyaluronic Acid Fermentation

Hyaluronic acid（HA）is a kind of linear mucopolysaccharide,which is composed of D-glucuronic acid and D-n-acetylglucosamine disaccharide units in the condition of alternating connection ofβ-1,3 andβ-1,4 glucoside bonds.Due to the different degree of disaccharide unit extension,hyaluronic acid has three molecular weight types,low,medium and high,and corresponding to three different physiological functions.Because of its unique physiological function and physicochemical properties,hyaluronic acid has been widely used in food,cosmetics and medical fields.At present,the main production method of hyaluronic acid is still the fermentation method of Streptococcus zooepidemicus,but the production of hyaluronic acid by Streptococcus zooepidemicus has the technical problem of high fermentation cost,so it is necessary to optimize the fermentation process of Streptococcus zooepidemicus.In addition,the high molecular weight of the hyaluronic acid produced by Streptococcus zooepidemicus greatly restricts its application.Therefore,the hydrolysis of high molecular weight hyaluronic acid is also the focus of research and development of hyaluronic acid.Firstly,in this study,Streptococcus equi subsp.zooepidemicus WTF 001 was used as the original fermentation strain.In order to further improve the yield of hyaluronic acid and reduce the fermentation cost,mutagenesis screening and optimization of culture medium and fermentation conditions were carried out respectively.Secondly,in order to solve the problem of high molecular weight of hyaluronic acid produced by the fermentation of S.equi subsp.zooepidimicus,high molecular weight hyaluronic acid was hydrolyzed by enzymolysis.In this study,the safe strain Pichia pastoris was used as the host bacterium to heterogenetically express Leech HAase（LHAase）from leech,and the enzymological properties,hydrolysis properties and fermentation optimization of LHAase were studied.The main work of this thesis is as follows:（1）ARTP plasma mutagenesis was used to mutate S.equi subsp.zooepidimicus（S.equi subsp.zooepidimicus WTF 001）.A mutagenic strain,S.equi subsp.zooepidemicus WTF 101 CCTCC,with high hyaluronic acid yield,was finally obtained through repeated screening with plate screening and flask re-screening.The yield of the mutagenic strain increased from 0.34±0.01 g/L to 0.55±0.02 g/L,and the yield increased by 61.67%,compared with the fermentation results of the original strain in shaking flask for 24 hours.（2）The composition formula of fermentation medium was optimized by combining single factor experiment and orthogonal experiment.The optimal composition formula of fermentation medium was:glucose 72 g/L,peptone 17.5 g/L,yeast extract 10 g/L,MgSO₄·7H₂O 2 g/L,K₂HPO₄·3H₂O 2 g/L.A 5-L multi-tandem fermenter was used to optimize the fermentation control parameters for the production of hyaluronic acid by S.equi subsp.zooepidimicus WTF101.The optimal fermentation control parameters were determined by single factor experiment as follows:pH=7.0,inocu Lation volume was 15%,fermentation temperature was 37℃,fermenter pressure was 0.05MPa,initial ventilation volume was1 vvm,initial propeller speed was 200 rpm,initial glucose concentration was 4%.The carbon source glucose was added in batch flow to maintain the glucose concentration in the fermenter between 15-25 g/L.The OD value of dissolved oxygen in fermenter was maintained between 20%-50%by increasing ventilation ratio and stirring speed.Combined with the above mutated strain and optimized fermentation conditions,the yield of hyaluronic acid reached 12.2±0.2 g/L,the product conversion rate is 16.94%,the viscosity reached 75000 m Pa·s,and the yield of lactic acid reached 45 g/L within20-24 hours of fermentation cycle,which was 62.67%higher than that of hyaluronic acid yield of 7.5±0.2 g/L under unoptimized conditions.Through single factor experiment and orthogonal experiment,the types and contents of key nitrogen source factors affecting the yield of hyaluronic acid in fermentation medium were determined as follows:Vitamin B3 0.1 g/L,inosine 0.03 g/L,lysine 0.7 g/L,arginine 0.7 g/L,threonine 0.9 g/L,alanine 1.4 g/L,histidine 0.03 g/L,proline 0.3 g/L.（3）In this study,hyaluronidase genes derived from leeches（belonging to the hyaluronic acid-3-glycosyl hydrolase family,EC 3.2.1.36,LHAase）were heterologous expressed in Pichia pastoris GS115.The binding vector is pPIC9k,the signal peptide isα-signal peptide,and the promoter is AOX1.The recombinant strain P.pastoris GS115/p PIC9k-LHAase-His⁺-Mut⁺-4 was obtained by histidine defect type screening and genetic high resistance screening.The LHAase enzyme activity of P.pastoris/pPIC9k-LHAase-4 was hetero-expressed in a 5-L fermentation tank.Under the condition of constant flow of methanol,the enzyme activity of LHAase reached 2.0×10⁵U/mL for 96 h.the fermentation conditions of P.pastoris GS115/p PIC9k-LHAase-His⁺-Mut⁺were optimized in a 5-L fermentor.The optimal inoculation OD_{600 nm}was 50,the optimal mean concentration of methanol（inducer）was 38.66 g/L,and the optimal solution oxygen feedback feed was 30%.The enzyme activity reached 7.11×10⁵U/m L for 96hours of high density fermentation,which was 255.5%higher than that before the fermentation conditions were optimized.The hydrolysis of LHAase was studied.Under the optimal conditions of pH=5.5and temperature 37℃,1×10⁵U LHAase could hydrolyze 0.1 g of hyaluronic acid within 15 min.The average molecular weight of hyaluronic acid decreased from2.0×10⁶Da to 2408 Da.