Screening Of Agar-degrading Bacteria And Characterization Of Agarase

Agarase can hydrolyze agar to produce low-polymerization agar oligosaccharides with various physiological activities such as antibacterial and anti-inflammatory effect.Agarase is mainly used in the production of agar oligosaccharides,preparation of seaweed protoplasts,and recovery of DNA from agarose gels.In this thesis,agar-degrading strains derived from non-marine environment were obtained by bacteria screening,and the agarase gene of the strain was cloned by molecular biological means and was heterologously expressed,its enzymatic properties were analyzed.It has certain theoretical value and application prospects.The main results of the paper includes:?1?Three strains with agarase activity were isolated from slime mold complex through the primary screening.After re-screening by shake flask fermentation,a strain P1 with stronger enzyme production capacity was obtained with an initial enzyme activity of 10.08.U·mL^-1;after 16S r RNA gene sequence analysis,we constructed a phylogenetic tree and found that strain P1 belongs to the Paenibacillus,and named it Paenibacillus odorifer P1.Physiological and biochemical experiments show that P.odorifer P1 has the ability to fix nitrogen and can use starch as substrate,while it can not hydrolyze gelatin and cellulose,its optimal enzyme-producing fermentation time is 40 h.?2?The single-factor experiment was used to optimize the fermentation medium components,and the final fermentation medium for P.odorifer P1 was determined as:Agar 3g·L^-1,peptone 2 g·L^-1,K₂HPO₄·3H₂O 1 g·L^-1,NaCl 0.3 g·L^-1,MgSO₄·7H₂O 0.05g·L^-1,FeSO₄·3H₂O 0.02 g·L^-1,CaCl₂ 0.04 g·L^-1.P.odorifer P1 was fermented in the optimized medium for 40 h,and the yield of agarase reached 34.7 U·mL^-1,which was 3.4 times the level of the enzyme productivity before optimization.?3?By analyzing the whole genome of the strain Paenibacillus odorifer DSM 15391,which is highly similar to the 16S rRNA gene sequence of P.odorifer P1,a gene sequence predicted to be agarase was obtained,and primers were designed based on it.Using the whole genome of P.odorifer P1 as the template,the DNA of AgaP1 sequence was successfully cloned.The AgaP1 gene was sequenced and bioinformatically analyzed.The evolutionary tree analysis of the amino acid sequence showed that AgaP1 belongs to the glycoside hydrolase 50 family in the agarase family.The heterologous expression vector of AgaP1 gene was constructed and expressed in E.coli.Using various expression optimization strategies such as optimizing IPTG concentration and adding molecular chaperone,the co-expression system of chaperone plasmid pKJE7 and recombinant vector was finally constructed to achieve soluble expression of recombinase.?4?Study the enzymatic properties of the recombinant enzyme AgaP1 using agarose as the substrate.The optimal reaction temperature of AgaP1 is 40°C and the optimal reaction p H is 7.0;AgaP1 has good thermal stability and p H stability.The enzyme activity was about 80%after treatment at 40°C for 1 hour,and the enzyme activity was above 45%after treatment for1 hour in the pH range of 5.0 to 8.0.Both Ca²⁺and K⁺can significantly promote the activity of recombinase.The recombina nt enzyme hydrolyzed agarose better than agar powder and low melting point agarose;the kinetic parameters of the recombinase were studied using agarose and low melting point agarose as substrates respectively.When agarose was used as the substrate,the V_max and K_m K_m of AgaP1 are 291.29 U·mg^-1 and 22.34 mg·mL^-1;when LMP was used as substrate,the V_max and K_m of AgaP1 are 43.48 U·mg^-1 and 5.03mg·mL^-1.The thin layer chromatography analysis of the hydrolysate of the recombinant enzyme showed that the agar oligosaccharide produced by the enzymolysis was new agarobiose.