| Cellulose is the most abundant polymer in nature,the main component of plant cell wall,which is one of the most popular research materials and the inexhaustible source of bioenergy at present.Cellulose can be converted into monosaccharide by cellulase,and its products can be converted to ethanol by microbial fermentation.This process need a series of cellulases,among which the β-glucosidase plays an important role and named to be a rate-limiting enzyme,which is the bottleneck of the natural cellulose materials by enzymatic degradation.In recent years,a large number of literatures have shown that higher termites,which account for 75%of the total number of termites,have a strong ability to degrade cellulose.In this paper,the fungus-growing termite Macrotermes barneyi was used as the research object.In previous experiment,the termites were collected in Leiyang City,Hunan Province(2015),and to measure the activity of P-glucosidase in their intestinal tract.A complete β-glucosidase sequence,named MbmgBGl,was derived from the midgut of M.barneyi,which belongs to the GHF1 family and has higher glucose tolerance.However,low enzyme activity and poor thermal stability has a limitation in food and industrial applications of β-glucosidase.Therefore,in order to obtain P-glucosidase with better enzymatic properties,we carried out the experiments as follows:1.Molecular modification of the P-glucosidase from midgut of M.barneyi.In previous studies of the M.barneyi transcriptome sequencing,we found an activeβ-glucosidase(MbmgBG1)gene,which belongs to GHF1 family.And it has higher glucose tolerance(maintaining more than 60%enzyme activity at concentration of 1.5 mol/L glucose),however,low enzyme activity and poor thermal stability has a limitation in food and industrial applications of β-glucosidase.Point mutants(F167L,T176C,E347I,R354K,N393G and V425M)were obtained by site-directed mutagenesis of non-conserved amino acids near conserved amino acids.Among them,the specific activities against pNPG of two mutants(F167L and R354K)were about 2-fold and 4-fold higher than that of MbmgBGl.Kcat/Km values were also higher than the wild-type,reflecting stronger affinity to the substrate and higher catalytic ability of mutants than MbmgBGl.When the glucose concentration was 1.5 mol/L,the enzyme activity of MbmgBGl was about 60%of original activity,while keeping 60%enzymatic activity,the glucose concentrations of F167L and R354K was 2.0 mol/L and 3.0 mol/L,respectively.These results lay a foundation for further studies on the catalytic efficiency of β-glucosidase.2.Cloning and analyse of β-glucosidase from Dysgonomonas macrotermitis.Higher termites have the highest ability to degrade cellulose in natural cellulose degradation model organism,and natural cellulose materials that can almost completely degrade themselves.β-glucosidase of termites themselves is far from fully explaining the cellulose materials.Therefore,the symbiotic microorganism in the intestinal tract of termites has strong cellulose degradation ability.The D.macrotermitis(the second dominant bacterium in the hindgut)was isolated from the hindgut of M.barneyi,which has cellulose activity.To study all the β-glucosidase gene in D.macrotermitis,total of 15 β-glucosidases were cloned and 6 of that were active.3.Cloning and Characterization of β-Glucosidase(DysbglE)from D.macrotermitis.One of the most active β-glucosidase genes was cloned and the nucleotide sequence of DysbglE was analyzed.The full-length of the DysbglE fragment was 2 286 bp,encoding 762 amino acids and one stop codon and molecular weight is 80.15 kDa,among which 22 signal peptides were removed.It is identified as homologous dimer and belongs to GHF3 family.This gene was heterologously expressed and purified in E.coli JM109.The optimum temperature and pH of recombinant DysbglE were 45℃and 5.5,respectively;The Km and Vmax,pNPG was used as substrate,of recombinant DysbglE were 1.4 mmol/L and 666.66 U/mg,respectively;and recombinant DysbglE has lower glucose tolerance and the IC50 values are 0.1 mol/L.4.Expression of D.macrotermitis β-glucosidase(DysbglE)gene in Bacillus subtilis WB800N and optimization of fermentation conditions.In the field of food industry,β-glucosidase is added as food additive to enhance fruit flavor,which requires the use of easy purification and security expression system.B.subtilis,a Gram-positive bacterium classified as "generally recognized as safe" by the United States Food and Drug Administration,and it has a naturally high secretory capacity and exports proteins directly into the extracellular medium.This research was to study the heterologous expression of β-glucosidase gene DysbglE in B.subtilis WB800N.And the fermentation conditions were optimized,which increased the expression of β-glucosidase by 4-5 times as compared with the original expression.In conclusion,a β-glucosidase gene MbmgBGl of M.barneyi was optimized,and a D.macrotermitis β-glucosidase gene was analyzed.Finally,heterologous expression of β-glucosidase gene in B.subtilis WB800N was succeed and the B.subtilis β-glucosidase secretion expression system was expected to be further optimized.This study deepens our understanding of the mechanism of termite cellulose degradation and provides a theoretical basis for the application of cellulose degradation enzymes from termite and its microbes. |
