Enzymatic Action Of Bacillus Subtillis Strains On Hemicellulose And Production Of Acidic Xylooligosaccharides

Bacillus subtilis has been applied in primary research of biology technology and industrial production more and more as its refined method of separation and culture, advantage of known genetic background and extracellular secretion, no pathogenicity as well. Bacillus subtilis subsp. subtilis str. 168(Bacillus subtilis 168) not only secrete Endo-1,4-β-xylanase A(Xyn A) and Glucurono xylanase C(Xyn C), but also Arabinoxylan arabinofuranohydrolase GH43(Axh43). The enzymatic actions of Xyn A and Xyn C secreted by Bacillus subtilis 168, individually and in combination, were demonstrated on sweetgum xylans(Methylglucuronoxylans, Me GXn). Through evaluating the growth condition and hydrolysis products of Bacillus subtilis derivate strains on the same xylans, the appropriate strain was achieved for production of acidic xylooligosaccharides(U-XOS). The enzymatic actions of Xyn A, Xyn C and Axh43, individually and in combination, were demonstrated on sweet sorghum xylans(Methylglucuronoarabinoxylans,Me GAXn) with more complicated structure. The selected strain was applied further to Me GAXn, the hydrolysis products was determinated. The main results and conclusions were showed as below:1. The enzymatic actions of Xyn A and Xyn C on sweetgum Me GXnThrough evaluating the enzymatic actions of Xyn A and Xyn C on Me GXn, individually and in combination, showed the difference of their binding specificities. The U-XOS with different degree of polymeriation(DP) were generated. TLC, MALDI-TOF-MS and 1H NMR analysis of hydrolysis products showed the low binding specificity of Xyn A on the substrate, resulting the various hydrolysis products. Neutral XOS(xylobiose and xylotriose mainly) and U-XOS(Methylglucuronoxylotetraose, Me GX4 mainly) were both exsited in the culture. 1H NMR analysis defined the generated Me GX4 with a single 4-O-methyl-glucuronic acid α-1,2-linked each penultimate to the nonreducing terminal xylose, showing the ability of binding the specific sites of Xyn A on the xylan with the presence of methylglucuronate(Me G). Xyn C, glucuronoxylan-specific Xylanase, generated Me GX7 mainly, each with a single Me G linked α-1,2 to the xylose penultimate to the xylose at the reducing terminus, no neutral XOS exsited in the culture also proofed the ability of the high binding specificity of Xyn C. In combination of Xyn A and Xyn C on Me GXn, Me GX3 was accumulated in which the internal xylose is substituted with Me G.2. The selection of mutant strain for production of U-XOSThe growth courves of each B. subtilis derived strains on sweetgum Me GXn were drawed and total sugar in the culture were measured. Depended on MALDI-TOF-MS and 1H NMR analysis on hydrolysis products, the culture of MR42 strain(168△xyn C-Km) contained 43% total carbohydrate. The main hydrolysis products is Me GX4 with a single 4-O-methyl-glucuronic acid α-1,2-linked each penultimate to the nonreducing terminal xylose, which was the same as those generated in Xyn A hydrolasate. The culture of MR44 strain(168△xyn A-Spc) contained 75% total carbohydrate. The main hydrolysis products is Me GX7 with a single 4-O-methyl-glucuronic acid α-1,2-linked each penultimate to the reducing terminal xylose. MR44 strain may be more suitable for production of U-XOS considering its yield and defined hydrolysis products.3. The enzymatic actions of Xyn A, Xyn C and Axh43 on sweet sorghum Me GAXnThe enzymatic actions of Xyn A, Xyn C and Axh 43, individually and in combination, were evaluated on sweet sorghum methylglucuronoarabinoxylans(Me GAXn). TLC and 1H NMR analysis showed Axh 43 is able to release the arabinose branched on xylan directly without hydrolysing the xylan main chain, which comfirmed Axh 43 is arabinose-substituted xylans specific hydrolase. In combination of Axh 43 and Xyn A, release of arabinose from Me GAXn by Axh43 prior to xylanolytic digestion by Xyn A, forming Me GX4 and arabniose as main products. In combination of Axh 43 and Xyn C, the action model was the same as it with Xyn A, while generating the U-XOS with larger DP of 12.4. The products generated by MR44 strain on Me GAXnThe growth courve of B. subtilis strain MR44 on sweet sorghum Me GAXn were drawed and total sugar in the culture were measured. The culture of MR44 contained 70% total carbohydrate, and the released arabinoses were assimilated by MR44 itself. 1H-NMR analysis of U-XOS purified by anion exchange chromatography QAE Q25 column structurally defined the U-XOS with U-XOS with an average degree of polymerizationof xylose units(DP) of 12 and a single 4-O-methyl-glucuronic acid α-1,2-linked on average to one of 12 xylose residues each penultimate to the reducing terminal xylose.This study demonstrated the enzymatic actions and hydrolysis products of Xyn A, Xyn C and Axh 43 were helpful to their applications for catalyzing defined products from biomass. The derived Bacillus subtilis strain which is able to produce the U-XOS with defined DP may provide the new thought for high-valued bioconverstion of biomass.