Insects living on wood and plants harbor a large variety of bacterial flora in their guts for degrading biomass. We isolated a Paenibacillus strain, designated ICGEB2008, from the gut of a cotton bollworm on the basis of its ability to secrete a variety of plant-hydrolyzing enzymes. In this study, we cloned, expressed, and characterized two enzymes, -1,4-endoglucanase (Endo5A) and -1,4-endoxylanase (Xyl11D), from the ICGEB2008 strain and synthesized recombinant bifunctional enzymes based on Endo5A and Xyl11D. The gene encoding Endo5A was obtained from the genome of the ICGEB2008 strain by shotgun cloning. The gene encoding Xyl11D was obtained using primers for conserved xylanase sequences, which were identified by aligning xylanase sequences in other species of Paenibacillus. Endo5A and Xyl11D were overexpressed in Escherichia coli, and their optimal activities were characterized. Both Endo5A and Xyl11D exhibited maximum specific activity at 50°C and pH 6 to 7. To take advantage of this feature, we constructed four bifunctional chimeric models of Endo5A and Xyl11D by fusing the encoding genes either end to end or through a glycine-serine (GS) linker. We predicted three-dimensional structures of the four models using the I-TASSER server and analyzed their secondary structures using circular dichroism (CD) spectroscopy. The chimeric model Endo5A-GS-Xyl11D, in which a linker separated the two enzymes, yielded the highest C-score on the I-TASSER server, exhibited secondary structure properties closest to the native enzymes, and demonstrated 1.6-fold and 2.3-fold higher enzyme activity than Endo5A and Xyl11D, respectively. This bifunctional enzyme could be effective for hydrolyzing plant biomass owing to its broad substrate range.Cellulose and hemicellulose constitute ϳ70 to 80% of wood and agricultural biomass and can serve as an abundant and inexpensive source of fermentable sugar for producing various chemicals and biofuels (34,35). Three enzymes hydrolyze the -1,4 glycosidic linkages that are present in cellulose. -1,4-Endoglucanases cleave within the chain, cellobiohydrolases cleave at either end of the chain, and -glucosidases break oligomers into monomers (22). Another class of enzymes, which includes xylanase and xylosidase, hydrolyzes -1,4-xylan into xylose and is involved in the breakdown of hemicellulose (2).Although a process to convert lignocellulosic biomass into ethanol has been developed by various research groups, costeffective production of lignocellulosic ethanol is still a major issue, largely because of the high cost associated with the enzymes (20,22,34). Fungi such as Trichoderma reesei and Aspergillus niger are common sources of plant-hydrolyzing enzymes (6, 23, 32); however, these enzymes require acidic pH (30) and have a short half-life at high temperatures (12) and a low specific activity (10), which are bottlenecks to using fungal enzymes. Bacterial plant-hydrolyzing enzymes have been reported to work at a wide range of pHs and temperatures (10, 21) and, therefore, are potential cata...
bIdentification and design of new cellulolytic enzymes with higher catalytic efficiency are a key factor in reducing the production cost of lignocellulosic bioalcohol. We report here identification of a novel -glucosidase (Gluc1C) from Paenibacillus sp. strain MTCC 5639 and construction of bifunctional chimeric proteins based on Gluc1C and Endo5A, a -1,4-endoglucanase isolated from MTCC 5639 earlier. The 448-amino-acid-long Gluc1C contained a GH superfamily 1 domain and hydrolyzed cellodextrin up to a five-sugar chain length, with highest efficiency toward cellobiose. Addition of Gluc1C improved the ability of Endo5A to release the reducing sugars from carboxymethyl cellulose. We therefore constructed six bifunctional chimeric proteins based on Endo5A and Gluc1C varying in the positions and sizes of linkers. One of the constructs, EG5, consisting of Endo5A-(G 4 S) 3 -Gluc1C, demonstrated 3.2-and 2-fold higher molar specific activities for -glucosidase and endoglucanase, respectively, than Gluc1C and Endo5A alone. EG5 also showed 2-fold higher catalytic efficiency than individual recombinant enzymes. The thermal denaturation monitored by circular dichroism (CD) spectroscopy demonstrated that the fusion of Gluc1C with Endo5A resulted in increased thermostability of both domains by 5°C and 9°C, respectively. Comparative hydrolysis experiments done on alkali-treated rice straw and CMC indicated 2-fold higher release of product by EG5 than that by the physical mixture of Endo5A and Gluc1C, providing a rationale for channeling of intermediates. Addition of EG5 to a commercial enzyme preparation significantly enhanced release of reducing sugars from pretreated biomass, indicating its commercial applicability.
Arthropods living on plants are able to digest plant biomass with the help of microbial flora in their guts. This study considered three arthropods from different niches - termites, pill-bugs and yellow stem-borers - and screened their guts for cellulase producing microbes. Among 42 unique cellulase-producing strains, 50% belonged to Bacillaceae, 26% belonged to Enterobacteriaceae, 17% belonged to Microbacteriaceae, 5% belonged to Paenibacillaceae and 2% belonged to Promicromonosporaceae. The distribution of microbial families in the three arthropod guts reflected differences in their food consumption habits. Most of the carboxymethylcellulase positive strains also hydrolysed other amorphous substrates such as xylan, locust bean gum and β-D-glucan. Two strains, A11 and A21, demonstrated significant activity towards Avicel and p-nitrophenyl-β-D-cellobiose, indicating that they express cellobiohydrolase. These results provide insight into the co-existence of symbionts in the guts of arthropods and their possible exploitation for the production of fuels and chemicals derived from plant biomass.
We report here the production of pure (R,R)-2,3-butanediol (2,3-BDO) isomer by the non-pathogenic Paenibacillus polymyxa ICGEB2008 using lignocellulosic hydrolysate as substrate. Experimental design based on Plackett-Burman resulted in identification of Mn and K as most crucial salt elements along with the yeast extract for 2,3-BDO production. Further experiments using Box-Behnken design indicated that both KCl and yeast extract together had major impact on 2,3-BDO production. Optimized medium resulted in 2,3-BDO production with 2.3-fold higher maximum volumetric productivity (2.01 g/L/h) and similar yield (0.33 g/g sugar) as compared to rich yeast extract-peptone-dextrose medium in the bioreactor studies. Considering that the balance substrate was channeled towards ethanol, carbon recovery was close to theoretical yield between the two solvents, i.e., 2,3-BDO and ethanol. Biomass hydrolysate and corn-steep liquor was used further to produce 2,3-BDO without impacting its yield. In addition, 2,3-BDO was also produced via simultaneous saccharification and fermentation, signifying robustness of the strain.
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