Production Of Glucanases By Salecan-degrading Bacteria

β-Glucanases hydrolyze the P-glucans existing in the cell wall of cereals and fungi, showing great value of applications in poultry feeding and brewing industry. Salecan is a novel water-soluble linear β-1,3-glucan produced by Agrobacterium sp. ZX09and processes many kinds of biological activities. The high molecular weight of salecan （2×106Da） is related to the high viscosity of salecan solutions and leads to solubility problems which restrict the further biological research and pharmaceutical applications. We were trying to screen novel β-1,3-glucanase-producing bacteria by empolying salecan as the substrate, meanwhile we could obtain the source of efficient salecan hydrolying enzyme. It would be very important of making studies on novel glucanases isolation and characterization, novel β-1,3-glucan oligosaccharides preparation and research.A novel salecan-degrading bacterial strain S09was isolated by observing the decrease of viscosity and formation of clear zone on the salecan-CTAB agar. S09was identified as gram-negative, rod-shaped, aerobic bacteria, and cabable of degrading different polysacchairdes including starch, pachyman, laminarin, oat meal, yeast β-glucan, xanthan gum and salecan, while could not utilize glucose, sucrose, lactose, cellulose and xylan. S09was salt tolerant and could grow in a medium with7%NaCl.16S rDNA sequence analysis indicated that S09is related to the genus Paenibacillus（Paenibacillus sp. S09）. S09could secretβ-1,3-1.4-glucanase, β-1,3-glucanase and salecan hydrolase.The medium optimization for salecan hydrolase production by S09was performed by single-factor experiment and response surface methodology. The optimized Hty medium was: salecan7.0g, urea0.7g, KH2PO40.5g, CaCl20.1g, MgSO4·7H2O0.15g, FeSO4·7H2O0.015g, ZnCl20.0025g, water1000ml, pH7.9. The enzyme activity reached up to15.2U/ml in the supernatant of Hty medium when inoculated with4%（v/v） of the seed culture and then cultivated at35℃for30h. The viscosity of salecan solutions （100mL,1%, w/v） decreased rapidly to the level of water, and amount of reducing sugars were released at the same time, when incubated with the crude salecan hydrolase （1ml） for10min.A new β-1,3-1,4-glucanase gene designated as plicA was screened from the genomic library of Paenibacillus sp. S09. The open reading frame of the gene plicA contained717bp coding for238amino acids with a predicted N-terminal signal peptide （residues1-26）. plicA gene fragment without the signal peptide was subcloned into pET29a, expressed in E. coli BL21（DE3） and purified by Ni2+affinity chromatography. The recombinant PlicA exhibited optimum activity at55℃and pH6.2. The specific activity toward barley β-glucan reached7,055U/mg. The Km and Vmax values on barley β-glucan were3.7mg/ml and3333.3μmol min-1mg-1, respectively. The enzyme exhibited acid-and alkali-tolerant properties between pH3.5-12.0. PlicA was salt-tolerant, with more than90%activity retained after being incubated in4M NaCl, and salt-activated, with activity rising to1.5-fold in0.5M NaCl. This is the first report on the characterization of an acid-, alkali-and salt-tolerant bacterial β-1,3-1,4-glucanase with high catalytic efficiency.A new β-1,3-glucanase gene designated as pglA was cloned from Paenibacillus sp. S09by degenerate PCR, I-PCR and SEFA-PCR. The open reading frame of the gene plicA contained2631bp coding for876amino acids with a predicted N-terminal signal peptide （residues1-38）. PglA is a multidomain enzyme, containing an N-terminal leader region, a GH16catalytic domain and a C-terminal domain with an Ig-like fold. Five truncated mutants were designed:rPglAâ–³C, rPglAâ–³N, rPglA-CD, rPglA-N, and rPglA-C. The full-length PglA and truncated enzymes rPglAâ–³C, rPglAâ–³N and rPglA-CD exhibited optimum temperature at60℃,60℃,50℃,60℃; optimum pH at5.5,6.5,5.5,6.0, respectively. Deletion of the C-terminus increased the optimal temperature and thermal tolerance of the enzyme. PglA specifically hydrolyzed β-1,3-glucan. The N-and C-terminal domains enhanced the catalytic activity toward insoluble substrates, the substrate affinity and catalytic efficiency of the catalytic domain. Both the N-and C-terminal domains exerted strong binding activity toward insoluble β-1,3-glucans, and could be classified into the CBM families. It was revealed that the C-terminal region influences enzymatic activity and stability.A novel salecan hydrolase designated as SalA was purified from the culture supernatant of Paenibacillus sp. S09by a combination of ammonium sulfate precipitation, ion-exchange and gel filtration chromatography. SalA was a glycoprotein with a molecular mass of170kDa and a carbohydrate content of18.5%（w/w）. Seven peptide sequences were obtained by MS/MS and de novo sequencing. The salA gene was cloned by degenerate PCR and SEFA-PCR. The open reading frame of the gene salA contained4374bp coding for1457amino acids with a predicted N-terminal signal peptide （residues1-32）. SalA is a multidomain enzyme, which include, from N to C terminus, a catalytic module of Beta_helix, a carbohydrate-binding module of family CBM₄₉, a analogue of coagulation factor Fa5/8C, a bacterial Ig-like fold Big₂, and two uncharacterized sugar-binding domain. SalA specifically hydrolyzed salecan with special activity as251.1U/mg and exhibited optimum activity at55℃and pH6.2. SalA was thermostable below65℃, as well as acid-and alkali-tolerant between pH3.0-10.0. The Km and Vmax values on salecan were11.1mg/ml and 833.3μmol min-1mg-1, respectively. SalA was the first isolated and characterized salecan hydrolase.The final enzymatic hydrolysate of salecan by SalA was salecan oligosaccharide SO8with homogeneous molecular weight and structure. SO8was characterized to be1484Da by ESI-MS.1H and13C NMR spectroscopy analysis showed that the backbone structure of SO8was α-D-Glcp-（1â†'3）-[β-D-Glcp-（1â†'3）-β-D-Glcp-（1â†'3）]3-D-Glcp with a methyl group and a carboxypropyl group at both terminal glucose residues, respectively. SalA was considered as a novel α-1,3-glucanase acting specifically on the α-1,3-glycosidic linkages between two α-glucose residues. SO8had antioxidant activity in vitro as measured by the DPPH radical scavenging tests. SO8increased spleen indices and numbers of leukocytes in peripheral blood to the normal levels, indicating that SO8has immunomodulating activities and could restore the immunosuppression induced by Cy-treatment in mice.The novel strain Paenibacillus sp. S09capable of degrading salecan was a novel glucanase-producing bacteria. The β-1,3-1,4-glucanase PlicA,β-1,3-glucanase PglA and salecan hydrolase SalA from S09had excellent enzymatic properties and could be applied in industries such as feeding, brewing and pharmaceuticals.