Study On Preparation Procedure Of LMW-HA By Enzymatic Hydrolysis Based On The Molecular Weight Monitering With NIRS

Hyaluronic acid(HA)is one kind of unbranched glycosaminoglycans(GAGs)of high molecular weight.consisting of N-acetylglucosamine and D-glucuronic acid glucuronide disaccharide repeat units via ?-(1?4)and ?-(1?3)glycosidic bonds.It has a relative molecular weight(Mt)ranging from 1×105 to 1×107.Studies have shown that the biological activity of hyaluronic acid is molecularly dependent,and the hyaluronic acids of different molecular weights tend to have different biological activities.Hyaluronic acid can be degraded to produce low molecular weight hyaluronic acid(LMW-HA,which is generally considered to have a molecular weight of less than 50 k).LMW-Has have many biological activities such as anti-tumor,anti-oxidation,osteogenesis and angiogenesis promotion,wound healing acceleration and immune regulation.Its application prospects are very broad.Currently,the methods for LMW-HA include physical degradation,chemical degradation,enzymatic hydrolysis,and biological fermentation.Among them,the enzymatic degradation method has a lot of advantages,such as high specificity,mild reaction conditions,less by-products,no damage to HA chain structure,etc.Studies have confirmed that chondroitin sulfate ABC lyase I(ChS ABC lyase I)is an endonuclease that can degrade some kinds of GAGs,including HA.At present,the enzyme can be obtained in large quantity by genetic engineering,but there is no report on a specific method for preparing LMW-HA by degrading hyaluronic acid the engineered chondroitinase ABC I.Therefore,the main research content and research purpose of this subject is to study the relationship between enzymatic hydrolysis conditions and molecular weight of LMW-HAs and establish an effectively controllable method for the preparation of any specific molecular weight LMW-HAs by enzymatic hydrolysis of chondroitinase ABC I,so as to lay the basis for providing LMW-HAs products to the market.The research contents and results are as follows:(1)Preparation of chondroitin sulfate enzyme ABC I and study on its enzymatic propertiesIn this study,a sufficient amount of degradable hyaluronic acid chondroitinase ABC I was prepared,and the enzyme activity and some enzymatic properties were determined.The experimental results showed that the optimum pH of the reaction of chondroitinase ABC I was 7.0,and the optimum temperature range of the reaction was 20-30?.The enzyme showed very good biological activities for the degradation of some GAGs such as chondroitin sulfate,dermatan sulfate,hyaluronic acid,etc.,but it showed little degradation activity to heparin and chitosan.In addition to Ba2+ and Ca2+,most of the metal ions showed a significant inhibitory effect on the activity of chondroitinase ABC I.(2)Preparation of LMW-HAs and study on the enzymatic hydrolysis conditionsIn this study,the relationship between enzymatic hydrolysis conditions and molecular weight of LMW-HAs was studied,and a method for preparing LMW-HAs was established.The effects of enzyme concentration,reaction time and substrate concentration on the enzymatic hydrolysis were investigated.Then orthogonal experiments were designed and carried out.According to the analysis of the range difference(R),the effects of the above three factors on the hydrolysis of HA with chondroitinase ABC I were as follows:substrate concentration,enzyme concentration,and reaction time.In addition,according to the average value k1,k2 and k3 of the index corresponding to each level of each factor,the optimal level of each factor can be determined as A3B3C1.That is,the reaction condition is 6×104 U/L of enzyme concentration,4 h of reaction time,and 1 mg/mL of substrate concentration.(3)Establishment of a method for determining the molecular weight of HA in solution by near infrared spectroscopy(NIRS)In order to use NIRS in the LMW-HA preparation process,a method model for determining the molecular weight of HA in solution by NIRS was established and optimized.the optimal preprocessing method was determined as second-order derivative SG25 point smoothing.The RMSEC,RMSEP,Rc2,and Rp2 values of the best SVM model were 3.39×103,5.79×104,0.999,and 0.847,respectively.So,a stable and reliable molecular weight quantitative analysis model of HA was established.The predicting capability of the established HA model was also tested and the results showed that there was no significant difference between the data measured by the reference method and the data predicted by the NIRS method,and the predicting capability of the model was good.(4)Design of NIRS application in the preparation process of LMW-HAsThe application of NIRS in industrial production was simulated and the predicting capability of the established NIRS model for HA molecular weight determination in solution was further validated.The feasibility of the application of NIRS in controlling the molecular weight of LMW-HAs in the production process was analyzed,and the preliminary process design was completed.The innovations of this paper include:(1)Preparation of LMW-HA by enzymatic hydrolysis of chondroitinase ABC I was systematically studied for the first time.(2)A method model for determining the molecular weight of HA in solution by NIRS was established and optimized.(3)The preparation process of LMW-HAs using NIRS to inspect the molecular weight was designed and the feasibility was analyzed for the first time.Design of NIRS application in the preparation process of LMW-HA.