Optimization Of Fermentation Conditions And Cloning And Expression Of Mannanase Gene From Bacillus Subtilis

β-mannanase belongs to the hemicellulase class and can randomly hydrolyze glucomannan,mannan,galactomannan and other polysaccharides containingβ-1,4-D-mannosidic bonds to release mannose or functional oligosaccharides from these sugars.β-Mannanase is a rich source,mostly found in plants and animals and microorganisms,and has important applications in feed,paper,pharmaceutical,petroleum,textile and laundry industries,with a broad application prospect.1.Optimization of fermentation conditions:In this study,the optimal fermentation conditions for mannanase production from Bacillus subtilis J were determined using a single-factor test and a response surface test design.The study showed that the optimal fermentation conditions were 28 g/L konjac flour,21 g/L tryptone,6 of K₂HPO₄,1 g/L Mg SO₄·7H₂O,31℃,p H8.5,1%inoculum,50 m L/250m L loading and 24 h fermentation period.The production ofβ-mannanase using the optimized conditions resulted in an enzyme activity of 84.38 U/m L,which was 3.36times higher than that of initial fermentation conditions.By optimizing the fermentation conditions,the yield ofβ-mannanase was substantially increased,which provided data reference for its industrial production.2.Genome sequencing and cloning and expression of mannanase gene:By sequencing the whole genome of Bacillus subtilis J,it was found that the genome size is 4154006 bp,GC content is 43.41%,encoding a total of 4334 genes.Based on the functional gene annotation information of strain J,aβ-mannanase gene was identified from its genome with a size of 1095 bp,encoding 364 amino acids,and its predicted molecular weight of 41.06 k Da with a theoretical p I of 5.88,containing a signal peptides with 28 amino acids and its protein was predicted to belong to the GH26family using the Pfam database.Acorrding to the sequence ofβ-mannanase gene,specific primers were designed and the partial sequence of this gene（960 bp）was amplified.The product was successively ligated with p MD19-T and p ET-28a（+）,and finally the recombinant expression vector p ET-28a-man was transformed into E.coli BL21（DE3）.The gene was induced by IPTG at low temperature and it successfully expressed in E.coli in the form of inclusion bodies after detecting by SDS-PAGE.3.Characterization of Recombinantβ-mannanase and identification of enzymatic products:Through urea denaturation,nickel column affinity chromatography and dialysis renaturation of the inclusion body,the recombinant protein with high purity was obtained and its molecular weight was about 36 k Da.The study of enzymatic properties showed that the optimum reaction temperature and p H ofβ-mannanase（Man）were 50℃and 6.0;the enzyme was relatively stable at temperatures below 55℃and p H6.0-8.0;the metal ions Ni⁺and Co²⁺had activating effects on the enzyme,and Mn²⁺,Mg²⁺,Zn²⁺,Cr³⁺and Ca²⁺had inhibiting effects on the enzyme.Mn²⁺had the strongest inhibitory effect and reduced the relative enzyme activity to 65.80%.Chemical reagents Tween 20,Tween 80 and Triton 100 all had higher activation effect onβ-mannanase,the relative enzyme activity could reach up to about 160%,while isopropyl alcohol,EDTA,SDS,PMSF and methanol had stronger inhibition effect onβ-mannanase.SDS had the strongest inhibition effect onβ-mannanase and the relative enzyme activity was almost 0.The V_maxand K_mof the enzyme were 208.33μmol/（min·m L）and 7.54 mg/m L,respectively,when locust bean gum was used as the substrate.Thin layer chromatography and mass spectrometry analysis showed that the enzyme hydrolyzed both locust bean gum and konjac powder in trace amounts of monosaccharide and oligosaccharides consisting of two to nine sugars,but the enzymatic products of locust bean gum were mainly disaccharide to pentasaccharide,while the enzymatic products of konjac flour were mainly disaccharide to heptosaccharide.In this study,heterologous expression of theβ-mannanase gene was achieved,providing a theoretical study for the industrial production of mannan oligosaccharides catalyzed by this enzyme.