Biochemical Characterization Of Hemicellulose Degradation Enzyme From Caldicellulosiruptor Sp. F32 And Screening Of Ligninolytic Bacteria

China has abundant biomass resources.Among them in terrestrial,cellulose,hemi-cellulose and lignin are major compositions.Cellulose is a linear homopolymer of glucose(C₆H₁₂O₆)units linked together in the form of D-anhydroglucopyranose units through?-?1,4?-glycosidic bonds.The structure of cellulose is relatively simple leading to its deg-radation process.Hemicellulose and lignin have a variety of chemical structural mono-mers composed of different chemical bonds,so the degradation process is more compli-cated,and the research progress is not satisfied till now.In this paper,we will focus on the study of?-1,3-1,4 glucan and lignin-degrading bacteria.?-1,3-1,4 glucan is widely distributed in grass species,and mainly composed of?-1,3 and?-1,4 glucuronic acid bonds.To completely hydrolyze it,it is necessary to use?-1,3-1,4 glucanase and?-glucosidase together.In this paper,two different?-1,3-1,4 glu-canases?F32EG5 and Lam16A-GH?from the extreme thermophilic bacteria Caldicellu-losiruptor sp.F32,and a?-glucosidase BlgA were heterologous to E.coli,and the recom-binant proteins of these three enzymes were obtained.The synergistic effect and end-product inhibition were characterized,and effect of F32EG5 in the hydrolysis of wheat bran were explored.At the same time,a series of Lam16A proteins containing dif-ferent number of substrate binding domains were characterized in order to obtain a better choice with higher substrate affinity.In addition,a lignin-degrading bacterium Pantoea,which may have a Fenton chemistry assisted mechanism,was isolated from the digestive gland of the Anoplophora glabripennis.Both of F32EG5 and Lam16A-GH are bifunctional glycoside hydrolase.Compared with the typical?-1,3 or?-1,4 glucanase,they have novel enzymatic cleavage sites.It can increase the proportion of oligosaccharides in the component when combined them to-gether to hydrolyze?-1,3-1,4 glucan.It showed a significant change in end-product when BlgA were added compared with F32EG5 or Lam16A-GH alone.The addition of?-glucosidase significantly increases fermentable monosaccharides.In addition,F32EG5and Lam16A-GH showed some tolerance to the common?-glucanase feedback inhibitors glucose and cellobiose,which was characterized by the addition of 1%w/v?10 g/L?glu-cose and cellobiose.The residual enzyme activity of BlgA was more than 60%against pNP-G at the 500 mmol/L concentration of end products commonly found in biotrans-formation processes such as arabinose and xylose added in.This property reveals its ap-plication potential as a credible member in enzyme cocktail.The full-length Lam16A which contains six substrate binding domains,has a seven-fold catalytic efficiency than Lam16A-GH,providing an additional option for the application of?-1,3-1,4 glucanase.The ligninolytic bacteria Pantoea isolated from the digestive glands of Anoplophora glabripennis is a kind of Gram-negative Enterobacter,which could be cultured with lig-nin alkaline as a sole carbon source and may have Fenton chemistry assisted mechanism.This lignin degradation mechanism could provide new resources for discovering lignino-lytic enzymes.And it is feasible to be used in biomass pretreatment process to increase the production of methane gas by biomass fermentation.