Preparation Of Agarooligosaccharides By Enzymatic Hydrolysis And Evaluation Of Their Antibacterial Activity

Research background and purpose:Agar oligosaccharides are functional oligosaccharides obtained by chemical or enzymatic degradation of agar.They can be divided into agarooligosaccharides and neooligosaccharides according to the difference of reduction end groups.Among them,agarooligosaccharides are considered to have a good development potential and application prospect because of their small molecular weight,good water solubility,easy digestion and absorption by the body,and have various biological activities such as antioxidant and anti-tumor.At present,there are two main ways to produce agarooligosaccharides:acid hydrolysis process and enzymatic process.Compared with the two methods,enzymatic method has the advantages of mild reaction conditions and strong specificity of substrate.Therefore,enzyme preparation of agarooligosaccharides,as a green and efficient way,has gradually become a hot topic in the field of preparation of agarooligosaccharides.In this study,novel agarase genes were extracted from marine bacterial genomes,expressed in prokaryotes,isolated and purified enzyme proteins.The agarooligosaccharides were prepared by enzymolysis and the bacteriostatic activity of agarooligosaccharides was studied.This study has important theoretical value and practical significance for enriching the resource base of Agar oligosaccharides and developing the biological activity of agarooligosaccharides.Materials and Methods:Firstly,the structure and function of recombinant agarase were analyzed by bioinformatics method,and its enzymatic properties were studied on the basis of enzyme separation and purification.Secondly,the optimum process for the preparation of agarooligosaccharides by enzymolysis was established to optimize the separation and purification process of agarooligosaccharides.Finally,the antibacterial activity of the agarooligosaccharides prepared by enzymolysis was evaluated and transcriptome analysis was performed.Result:1.A new type of agarase Agab was excavated from Agarilytica rhodophyticola.Bioinformatics analysis showed that the agarase had good hydrophilicity,no transmembrane region,and there was a signal peptide between the 29th amino acid and the 30th amino acid,and contained the carbohydrate binding module CBM2 of the conservative domain of glycoside hydrolase GH96 family.2.The molecular weight of agarase Agab is about 86 kDa,Agab.The optimum temperature is 40℃and the optimum pH is 7.the agarase has good thermal and pH stability.Cu²⁺,Li⁺,Zn²⁺,Cr²⁺and Fe³⁺had strong inhibitory effect on Agab,while Ba²⁺could promote the activity of Agab.Ca²⁺is a non-essential activator of Agab.When the concentration of DTT was 5 mM and the concentration of TCEP was 5 mM,the concentration of Vc was 1 mM,the activity of Agab was the highest.The results of enzyme kinetic parameters showed that Agab had the highest affinity to the substrate containing Ca²⁺and DTT.TLC results showed that Agab belonged to endonucleaseα-agarase.3.The results of single factor experiment on the preparation of AOSs by enzymatic hydrolysis showed that the optimum concentration of DTT,enzyme and substrate were6 mM,0.80 U/mL and 0.30%,respectively.The response surface optimization was carried out on the basis of single factor experiment.The results showed that the influence degree of each factor on reducing sugar content was substrate concentration>DTT concentration>enzyme content.The single factor experiment of separation and purification of AOSs by activated carbon showed that the best adsorption time was 70 min,the best elution ethanol concentration was 30%,and the best flow rate was CW=7.The hydrolyzed products were analyzed by TLC,MS and NMR.The results showed that AOSs with high purity were isolated by activated carbon chromatography,and the main products were agarotetraose and agarohexaose.4.Agarotetraose and agarohexaose significantly inhibited the growth of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus（MRSA）.The differentially expressed genes produced by MRSA treated by AOSs focus on affecting the ribosome structural components of MRSA to inhibit the growth of MRSA.Conclusion:1.Agab belongs toα-agarase.The molecular weight of agarase Agab is about 86kDa.The optimum temperature and pH of agarase is 40℃and 7.the agarase has good temperature and pH stability.2.The main products of enzymatic hydrolysis are agarotetraose and agarohexaose,which can be separated and prepared by activated carbon chromatography column.3.Agarotetraose and agarohexaose have antibacterial activity,which mainly affect the metabolism of the ribosome pathway,disturbing the protein synthesis process of MRSA and then inhibiting MRSA.