Heterologous Expression Of Levansucrase From Bacillus And Enzymatic Synthesis Of Levan

Levan is a fructose polymer synthesized by levansucrase.Levan is known to exert various pharmacological effects,such as antioxidant,anti-inflammatory,anticancer,antiviral,immunomodulatory and hypoglycemic effects,and has a very broad application prospects in the fields of food and biomedicine.However,at present,the large-scale industrial production technology of polysaccharides in China is not mature,which hinds the application of polysaccharides in food and biomedicine.The purpose of this study is to express the recombinant levansucrase efficiently by genetic engineering technology,to achieve the cell-free enzymatic synthesis of levan using recombinant levansucrase,and to characterize the enzymatically synthesized levan.This study will provide the basis for large-scale industrial production and application of levan.In the currrent study,a Bacillus amyloliquefaciens strain was isolated from honey and termed Bacillus amyloliquefaciens LH019.The levansucrase gene sac B was detected in this strain,and its size was 1422 bp by sequencing,indicating that the strain had the possibility of producing levan.On LB-sucrose medium,the strain produced a transparent gelatinous material.Bioinformatics analysis showed that the levansucrase of this Bacillus amyloliquefaciens was a secreted protein containing a signal peptide.The cleavage site of this signal peptide was located between amino acids 29-30.This enzyme did not have a transmembrane region.It could be inferred that the signal peptide of levansucrase was responsible for protein transport.A mature levansucrase could be generated by removal of the secretory signal peptide,therefore this levansucrase belonged to an extracellular protease.sac B gene was amplified from Bacillus amyloliquefaciens LH019 strain by PCR,and then sac B gene was subcloned into the plasmid p ET-32A-ACMA-ZZ to construct sac B expression vector p ET-32a-sac B-acm A-ZZ.This expression vector was transformed into Escherichia coli BL21(DE3),and then a recombinant expression strain was successfully obtained.After the optimization of the expression conditions,the best expression efficiency was abtained when OD 600 of the culture of the recombinant strain was 0.3,IPTG concentration was 0.25 m M,and the culture temperature was 16°C.Due to the introduction of the acm A purification tag during the construction of the recombinant plasmid,bacterial enhancer matrix(BEM)peptidoglycan was used as the purification matrix to achieve one-step immobilization and purification of recombinant levansucrase.This study determined that the optimal reaction conditions of recombinant levansucrase were 40?and p H 5.4.5 m M Ca²⁺,Ba²⁺,K⁺,Ni²⁺,Mn²⁺and 50 m M Ca²⁺,K⁺had a positive effect on the catalytic activity of the recombinant levansucrase,while the other metal ions at 50 m M showed a significant inhibitory effect.It suggested that these metal ions at a high concentration may inhibit the activity of recombinant levan sucrase.5 m M and 50 m M Cu²⁺,Mg²⁺and Zn²⁺ions inhibited enzyme activity significantly.K_mand V_maxof recombinant levansucrase were 18.59m M and 0.324?mol/L?s,respectively.In the current study,the cell-free enzymatic synthesis of levan was achieved using recombinant levansucrase.The levan purification procedure was simple,and could prepare the levan with high purity and high homogeneity.The dried levan synthesized by this method was white floc with loose texture.It was observed under the electron microscope that the polysaccharide was in the form of irregular shiny flakes,and the surface had obvious porous and branched structure.This study characterzed that the average relative molecular mass of enzymatically synthesized levans was 126953.1 Da.The levans were mainly composed of fructose in?-D-furan configuration with a large number of branched structures by ft-IR and NMR analysis.The main chain was connected by?-(2,6)glycosidic bonds,the end of the main chain was a single D-glucosyl residue,and the branched chain was connected with the main chain via?-(2,1)glycosidic bonds.In summary,Bacillus amyloliquefaciens LH019 isolated from honey encoded sac B gene and was able to produce levan.sac B gene of the strain was cloned into E.coli by genetic engineering technology,and the high-efficient expression of levansucrase was achieved.The cell-free enzymatic synthesis of levan was establised using recombinant levansucrase.The levan synthesized by enzymatic method had physicochemical properties of natural levan.This study provided a basis for the production and application of levan.