Hydrolysis of ionic liquid-treated substrate with an Iocasia fonsfrigidae strain SP3-1 endoglucanase.

Recently, we reported the discovery of a novel endoglucanase of the glycoside hydrolase family 12 (GH12), designated IfCelS12A, from the haloalkaliphilic anaerobic bacterium Iocasia fonsfrigidae strain SP3-1, which was isolated from a hypersaline pond in the Samut Sakhon province of Thailand (ca. 2017). IfCelS12A exhibits high substrate specificity on carboxymethyl cellulose and amorphous cellulose but low substrate specificity on b-1,3;1,4-glucan. Unlike some endoglucanases of the GH12 family, IfCelS12A does not exhibit hydrolytic activity on crystalline cellulose (i.e., Avicel™). High-Pressure Liquid Chromatography (HPLC) and Thin Layer Chromatography (TLC) analyses of products resulting from IfCelS12-mediated hydrolysis indicate mode of action for this enzyme. Notably, IfCelS12A preferentially hydrolyzes cellotetraoses, cellopentaoses, and cellohexaoses with negligible activity on cellobiose or cellotriose. Kinetic analysis with cellopentaose and barely b-D-glucan as cellulosic substrates were conducted. On cellopentaose, IfCelS12A demonstrates a 16-fold increase in activity (KM = 0.27 mM; kcat = 0.36 s-1; kcat/KM = 1.34 mM-1 s-1) compared to the enzymatic hydrolysis of barley b-D-glucan (KM: 0.04 mM, kcat: 0.51 s-1, kcat/KM = 0.08 mM-1 s-1). Moreover, IfCelS12A enzymatic efficacy is stable in hypersaline sodium chlorids (NaCl) solutions (up to 10% NaCl). Specifically, IfCel12A retains notable activity after 24 h at 2M NaCl (10% saline solution). IfCelS12A used as a cocktail component with other cellulolytic enzymes and in conjunction with mobile sequestration platform technology offers additional options for deconstruction of ionic liquid-pretreated cellulosic feedstock. KEY POINTS: • IfCelS12A from an anaerobic alkaliphile Iocasia fronsfrigidae shows salt tolerance • IfCelS12A in cocktails with other enzymes efficiently degrades cellulosic biomass • IfCelS12A used with mobile enzyme sequestration platforms enhances hydrolysis.

Efficient production of polyhydroxybutyrate using lignocellulosic biomass derived from oil palm trunks by the inhibitor-tolerant strain Burkholderia ambifaria E5-3.

Lignocellulosic biomass is a valuable, renewable substrate for the synthesis of polyhydroxybutyrate (PHB), an ecofriendly biopolymer. In this study, bacterial strain E5-3 was isolated from soil in Japan; it was identified as Burkholderia ambifaria strain E5-3 by 16 S rRNA gene sequencing. The strain showed optimal growth at 37 °C with an initial pH of 9. It demonstrated diverse metabolic ability, processing a broad range of carbon substrates, including xylose, glucose, sucrose, glycerol, cellobiose, and, notably, palm oil. Palm oil induced the highest cellular growth, with a PHB content of 65% wt. The strain exhibited inherent tolerance to potential fermentation inhibitors derived from lignocellulosic hydrolysate, withstanding 3 g/L 5-hydroxymethylfurfural and 1.25 g/L acetic acid. Employing a fed-batch fermentation strategy with a combination of glucose, xylose, and cellobiose resulted in PHB production 2.7-times that in traditional batch fermentation. The use of oil palm trunk hydrolysate, without inhibitor pretreatment, in a fed-batch fermentation setup led to significant cell growth with a PHB content of 45% wt, equivalent to 10 g/L. The physicochemical attributes of xylose-derived PHB produced by strain E5-3 included a molecular weight of 722 kDa, a number-average molecular weight of 191 kDa, and a polydispersity index of 3.78. The amorphous structure of this PHB displayed a glass transition temperature of 4.59 °C, while its crystalline counterpart had a melting point of 171.03 °C. This research highlights the potential of lignocellulosic feedstocks, especially oil palm trunk hydrolysate, for PHB production through fed-batch fermentation by B. ambifaria strain E5-3, which has high inhibitor tolerance.

Insulambacter thermoxylanivorax sp. nov., a thermophilic xylanolytic bacterium isolated from compost.

We isolated and analysed a Gram-negative, facultatively thermophilic, xylan-degrading bacterium that we designated as strain DA-C8T. The strain was isolated from compost from Ishigaki Island, Japan, by enrichment culturing using beech wood xylan as the sole carbon source. The strain showed high xylan degradation ability under anaerobic growth conditions. The isolate grew at 37-60 °C (optimum, 55 °C) and pH 4.0-11.0 (optimum, pH 9.0). As well as xylan, strain DA-C8T could use polysaccharides such as arabinoxylan and galactan as carbon sources. Comparison of 16S rRNA gene sequences indicated that strain DA-C8T was most closely related to Paenibacillus cisolokensis LC2-13AT (93.9 %) and Paenibacillus chitinolyticus HSCC596 (93.5 %). In phylogenetic analysis, strain DA-C8T belonged to the same lineage as Xylanibacillus composti K13T (92.5 %), but there was less statistical support for branching (70 %). Digital DNA-DNA hybridization, average nucleotide identity values and average amino acid sequence identity between strain DA-C8T and P. cisolokensis LC2-13AT were 21.8, 68.3 and 58.2 %, respectively. Those between strain DA-C8T and X. composti K13 were 23.7, 67.7 and 57.6 %, respectively. The whole-genome DNA G+C content of strain DA-C8T was 52.3 mol%. The major cellular fatty acids were C16 : 0 (42.9 %), anteiso-C15 : 0 (20.0 %) and anteiso-C17 : 0 (16.7 %), the major quinone was menaquinone 7, and the major polar lipids were unidentified glycolipids. On the basis of phenotypic, chemotaxonomic and phylogenetic evidence, a novel genus is proposed-Insulambacter gen. nov.-for the novel species Insulambacter thermoxylanivorax sp. nov. The type strain is DA-C8T (=JCM 34211T=DSM 111723T).

A Novel D-Psicose 3-Epimerase from Halophilic, Anaerobic Iocasia fonsfrigidae and Its Application in Coconut Water.

D-Psicose is a rare, low-calorie sugar that is found in limited quantities in national products. Recently, D-psicose has gained considerable attention due to its potential applications in the food, nutraceutical, and pharmaceutical industries. In this study, a novel D-psicose 3-epimerase (a group of ketose 3-epimerase) from an extremely halophilic, anaerobic bacterium, Iocasia fonsfrigidae strain SP3-1 (IfDPEase), was cloned, expressed in Escherichia coli, and characterized. Unlike other ketose 3-epimerase members, IfDPEase shows reversible epimerization only for D-fructose and D-psicose at the C-3 position but not for D-tagatose, most likely because the Gly218 and Cys6 at the substrate-binding subsites of IfDPEase, which are involved in interactions at the O-1 and O-6 positions of D-fructose, respectively, differ from those of other 3-epimerases. Under optimum conditions (5 µM IfDPEase, 1 mM Mn2+, 50 °C, and pH 7.5), 36.1% of D-psicose was obtained from 10 mg/mL D-fructose. The IfDPEase is highly active against D-fructose under NaCl concentrations of up to 500 mM, possibly due to the excessive negative charges of acidic amino acid residues (aspartic and glutamic acids), which are localized on the surface of the halophilic enzyme. These negative charges may protect the enzyme from Na+ ions from the environment and result in the lowest pI value compared to those of other 3-epimerase members. Moreover, without adjusting any ingredients, IfDPEase could improve coconut water quality by converting D-fructose into D-psicose with a yield of 26.8%. Therefore, IfDPEase is an attractive alternative to enhancing the quality of fructose-containing foods.

Cellulomonas palmilyticum sp. nov., from earthworm soil biofertilizer with the potential to degrade oil palm empty fruit bunch.

Oil palm empty fruit bunch (OPEFB) is lignocellulosic waste from the palm oil industry in Southeast Asia. It is difficult to degrade because of its complex matrix and recalcitrant structure. To decompose OPEFB, highly efficient micro-organisms and robust enzymatic systems are required. A bacterium with high degradation ability against untreated OPEFB was isolated from earthworm soil biofertilizer and designated as strain EW123T. Cells were Gram-stain-positive, rod-shaped and catalase-positive. In tests, the strain was negative for mycelium formation, motility, nitrate reductase and urease. The 16S rRNA gene analysis of the isolate showed 98.21 % similarity to Cellulomonas uda NBRC 3747T, whereas similarity to other species was below 98 %. The genome of strain EW123T was 3 834 009 bp long, with 73.97 mol% G+C content. Polar lipid analysis of strain EW123T indicated phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol and aminophospholipid as the lipid components of the cell wall. The major cellular fatty acid was anteiso-C15 : 0 (41.26 %) and the isomer of 2,6-diaminopimelic acid (DAP) was meso-DAP. The average nucleotide identity value between the genome sequences of EW123T and C. uda NBRC 3747T was 88.6 %. In addition, the digital DNA-DNA hybridization and genome average amino acid between those strains were 36.1 and 89.68 %, respectively. The ORF number (186) of carbohydrate-active enzymes, including cellulases, xylanases, mannanase, lipase and lignin-degrading enzymes, was higher than those of related strains. These results indicate that the polyphasic characteristics of EW123T differ from those of other related species in the genus Cellulomonas. We therefore propose a novel species of the genus Cellulomonas, namely Cellulomonas palmilyticum sp. nov. (type strain TBRC 11805T=NBRC 114552T), with the ability to effectively degrade untreated OPEFB.

Biological cellulose saccharification using a coculture of Clostridium thermocellum and Thermobrachium celere strain A9.

An anaerobic thermophilic bacterial strain, A9 (NITE P-03545), that secretes ß-glucosidase was newly isolated from wastewater sediments by screening using esculin. The 16S rRNA gene sequence of strain A9 had 100% identity with that of Thermobrachium celere type strain JW/YL-NZ35. The complete genome sequence of strain A9 showed 98.4% average nucleotide identity with strain JW/YL-NZ35. However, strain A9 had different physiological properties from strain JW/YL-NZ35, which cannot secrete ß-glucosidases or grow on cellobiose as the sole carbon source. The key ß-glucosidase gene (TcBG1) of strain A9, which belongs to glycoside hydrolase family 1, was characterized. Recombinant ß-glucosidase (rTcBG1) hydrolyzed cellooligosaccharides to glucose effectively. Furthermore, rTcBG1 showed high thermostability (at 60°C for 2 days) and high glucose tolerance (IC50 = 0.75 M glucose), suggesting that rTcBG1 could be used for biological cellulose saccharification in cocultures with Clostridium thermocellum. High cellulose degradation was observed when strain A9 was cocultured with C. thermocellum in a medium containing 50 g/l crystalline cellulose, and glucose accumulation in the culture supernatant reached 35.2 g/l. In contrast, neither a monoculture of C. thermocellum nor coculture of C. thermocellum with strain JW/YL-NZ35 realized efficient cellulose degradation or high glucose accumulation. These results show that the ß-glucosidase secreted by strain A9 degrades cellulose effectively in combination with C. thermocellum cellulosomes and has the potential to be used in a new biological cellulose saccharification process that does not require supplementation with ß-glucosidases. KEY POINTS: • Strain A9 can secrete a thermostable ß-glucosidase that has high glucose tolerance • A coculture of strain A9 and C. thermocellum showed high cellulose degradation • Strain A9 achieves biological saccharification without addition of ß-glucosidase.

A Novel Multifunctional Arabinofuranosidase/Endoxylanase/ß-Xylosidase GH43 Enzyme from Paenibacillus curdlanolyticus B-6 and Its Synergistic Action To Produce Arabinose and Xylose from Cereal Arabinoxylan.

PcAxy43B is a modular protein comprising a catalytic domain of glycoside hydrolase family 43 (GH43), a family 6 carbohydrate-binding module (CBM6), and a family 36 carbohydrate-binding module (CBM36) and found to be a novel multifunctional xylanolytic enzyme from Paenibacillus curdlanolyticus B-6. This enzyme exhibited α-l-arabinofuranosidase, endoxylanase, and ß-d-xylosidase activities. The α-l-arabinofuranosidase activity of PcAxy43B revealed a new property of GH43, via the release of both long-chain cereal arabinoxylan and short-chain arabinoxylooligosaccharide (AXOS), as well as release from both the C(O)2 and C(O)3 positions of AXOS, which is different from what has been seen for other arabinofuranosidases. PcAxy43B liberated a series of xylooligosaccharides (XOSs) from birchwood xylan and xylohexaose, indicating that PcAxy43B exhibited endoxylanase activity. PcAxy43B produced xylose from xylobiose and reacted with p-nitrophenyl-ß-d-xylopyranoside as a result of ß-xylosidase activity. PcAxy43B effectively released arabinose together with XOSs and xylose from the highly arabinosyl-substituted rye arabinoxylan. Moreover, PcAxy43B showed significant synergistic action with the trifunctional endoxylanase/ß-xylosidase/α-l-arabinofuranosidase PcAxy43A and the endoxylanase Xyn10C from strain B-6, in which almost all products produced from rye arabinoxylan by these combined enzymes were arabinose and xylose. In addition, the presence of CBM36 was found to be necessary for the endoxylanase property of PcAxy43B. PcAxy43B is capable of hydrolyzing untreated cereal biomass, corn hull, and rice straw into XOSs and xylose. Hence, PcAxy43B, a significant accessory multifunctional xylanolytic enzyme, is a potential candidate for application in the saccharification of cereal biomass. IMPORTANCE Enzymatic saccharification of cereal biomass is a strategy for the production of fermented sugars from low-price raw materials. In the present study, PcAxy43B from P. curdlanolyticus B-6 was found to be a novel multifunctional α-l-arabinofuranosidase/endoxylanase/ß-d-xylosidase enzyme of glycoside hydrolase family 43. It is effective in releasing arabinose, xylose, and XOSs from the highly arabinosyl-substituted rye arabinoxylan, which is usually resistant to hydrolysis by xylanolytic enzymes. Moreover, almost all products produced from rye arabinoxylan by the combination of PcAxy43B with the trifunctional xylanolytic enzyme PcAxy43A and the endoxylanase Xyn10C from strain B-6 were arabinose and xylose, which can be used to produce several value-added products. In addition, PcAxy43B is capable of hydrolyzing untreated cereal biomass into XOSs and xylose. Thus, PcAxy43B is an important multifunctional xylanolytic enzyme with high potential in biotechnology.

Efficient biological pretreatment and bioconversion of corn cob by the sequential application of a Bacillus firmus K-1 cellulase-free xylanolytic enzyme and commercial cellulases.

We used agricultural residue, corn cob, with biorefinery and bioeconomy concepts. At short-time cultivation in corn cob (12 h), Bacillus firmus K-1 produced cellulase-free xylanolytic enzyme, with xylooligosaccharides (XOSs), X5 and X6, as the main products, which can be used in a variety of applications. The xylanolytic enzyme produced from B. firmus K-1 effectively degraded xylan in corn cob, which was examined by chemical composition, scanning electron microscope (SEM), and Fourier transform infrared spectroscopy (FTIR). After cultivation, the xylan contained in the corn cob residue was decreased (as biological pretreatment), causing morphological and structural changes, including creating porosity and increasing the surface area and the exposure of cellulose of pretreated corn cob. These results lead to an improvement of cellulose access by cellulases. Commercially available cellulases, Accellerase® 1500 and Cellic® CTec2, yielded significantly higher glucose concentrations from pretreated corn cob compared to untreated corn cob. After saccharification, the lignin-rich corn cob residue can be used as a raw material for other purposes. Moreover, the B. firmus cells, with a low risk to human health, can be used in some applications. This study presents an efficient method for producing high-value-added products from agricultural residue (corn cob) through biological processes which are environmentally friendly and economically viable. KEY POINTS: • High-value-added products were efficiently produced from corn cob by B. firmus K-1. • After biological pretreatment by B. firmus K-1, cellulase can better reach cellulose. • XOSs and cellulose-derived glucose were the main products from corn cob.

A novel amylolytic/xylanolytic/cellulolytic multienzyme complex from Clostridium manihotivorum that hydrolyzes polysaccharides in cassava pulp.

Some anaerobic bacteria, particularly Clostridium species, produce extracellular cellulolytic and xylanolytic enzymes as multienzyme complexes (MECs). However, an amylolytic/xylanolytic/cellulolytic multienzyme complex (AXC-MEC) from anaerobic bacteria is rarely found. In this work, the glycoprotein AXC-MEC, composed of subunits of amylolytic, xylanolytic, and cellulolytic enzymes, was isolated from crude extracellular enzyme of the mesophilic anaerobic bacterium Clostridium manihotivorum CT4, grown on cassava pulp, using a milled cassava pulp column and Sephacryl S-500 gel filtration chromatography. The isolated AXC-MEC showed a single band upon native-polyacrylamide gel electrophoresis (native-PAGE). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed at least eight protein bands of the multienzyme complex which predominantly exhibited amylolytic enzyme activity, followed by xylanolytic and cellulolytic enzyme activities. The AXC-MEC is highly capable of degrading starch and non-starch polysaccharides present in cassava pulp into glucose and oligosaccharides, without conventional pretreatment. Base on the genomic analysis of C. manihotivorum CT4, we found no evidence of the known structural components of the well-known multienzyme complexes from Clostridium species, cellulosomes such as scaffoldin, cohesin, and dockerin, indicating that AXC-MEC from strain CT4 exhibit a different manner of assembly from the cellulosomes. These results suggest that AXC-MEC from C. manihotivorum CT4 is a new MEC capable of hydrolyzing cassava pulp into value-added products, which will benefit the starch industry. KEY POINTS: • Glycoprotein AXC-MEC was first reported in Clostridium manihotivorum. • Unlike cellulosomes, AXC-MEC consists of amylase, xylanase, and cellulase. • Glucose and oligosaccharides were hydrolysis products from cassava pulp by AXC-MEC.

Effect of decomposing oil palm trunk fibers on plant growth and soil microbial community composition.

Oil palm trunks (OPT) are logged for replantation and the fiber residues are disposed of into the palm plantation area. The fiber residues are expected to increase soil fertility through recycling of carbon and minerals via fiber decomposition. This study investigated the effects of OPT fiber disposal and other lignocellulosic biomass on plant growth and microbial diversity in the soil environment. Four treatment plots were tested: (A) soil+OPT fiber (1:20), (B) soil+sugarcane bagasse (1:20), (C) soil+cellulose powder (1:20), and (D) unamended soil as a negative control. Low plant height, decreased chlorophyll content, and low biomass was observed in corn grown on soil mixed with OPT fiber, cellulose, and sugarcane bagasse, when compared with those of the control. The plants grown with OPT fiber were deficient in total nitrogen and magnesium when compared with those without fiber amendment, which suggested that nitrogen and minerals in soil might be taken up by changing microflora because of the OPT fibers presence. To confirm differences in the soil microflora, metagenomics analysis was performed on untreated soil and soil from each lignocellulose treatment. The microflora of soils mixed with OPT fiber, cellulose and sugarcane bagasse revealed substantial increases in bacteria such as families Cytophagaceae and Oscillospiraceae, and two major fungal genera, Trichoderma and Trichocladium, that are involved in lignocellulose degradation. OPT fiber resulted in a drastic increase in the ratios and amounts of Trichocladium in the soil when compared with those of cellulose and sugarcane bagasse. These results indicate that unregulated disposal of OPT fiber into plantation areas could result in nutrient loss from soil by increasing the abundance of microorganisms involved in lignocellulose decomposition.

A novel multifunctional GH9 enzyme from Paenibacillus curdlanolyticus B-6 exhibiting endo/exo functions of cellulase, mannanase and xylanase activities.

PcMulGH9, a novel glycoside hydrolase family 9 (GH9) from Paenibacillus curdlanolyticus B-6, was successfully expressed in Escherichia coli. It is composed of a catalytic domain of GH9, two domains of carbohydrate-binding module family 3 (CBM3) and two domains of fibronectin type 3 (Fn3). The PcMulGH9 enzyme showed broad activity towards the ß-1,4 glycosidic linkages of cellulose, mannan and xylan, including cellulose and xylan contained in lignocellulosic biomass, which is rarely found in GH9. The enzyme hydrolysed substrates with bifunctional endo-/exotypes cellulase, mannanase and xylanase activities, but predominantly exhibited exo-activities. This enzyme released cellobiose as a major product from cellohexaose, while mannotriose and xylotriose were major hydrolysis products from mannohexaose and xylohexaose, respectively. Moreover, PcMulGH9 could hydrolyse untreated corn hull and rice straw into xylo- and cello-oligosaccharides. Enzyme kinetics, site-directed mutagenesis and molecular docking revealed that Met394, located at the binding subsite + 2, was involved in broad substrate specificity of PcMulGH9 enzyme. This study offers new knowledge of the multifunctional cellulase/mannanase/xylanase in GH9. The PcMulGH9 enzyme showed a novel function of GH9, which increases its potential for saccharification of lignocellulosic biomass into value-added products, especially oligosaccharides.

A novel AA10 from Paenibacillus curdlanolyticus and its synergistic action on crystalline and complex polysaccharides.

Lytic polysaccharide monooxygenases (LPMOs) play an important role in the degradation of complex polysaccharides in lignocellulosic biomass. In the present study, we characterized a modular LPMO (PcAA10A), consisting of a family 10 auxiliary activity of LPMO (AA10) catalytic domain, and non-catalytic domains including a family 5 carbohydrate-binding module, two fibronectin type-3 domains, and a family 3 carbohydrate-binding module from Paenibacillus curdlanolyticus B-6, which was expressed in a recombinant Escherichia coli. Comparison of activities between full-length PcAA10A and the catalytic domain polypeptide (PcAA10A_CD) indicates that the non-catalytic domains are important for the deconstruction of crystalline cellulose and complex polysaccharides contained in untreated lignocellulosic biomass. Interestingly, PcAA10A_CD acted not only on cellulose and chitin, but also on xylan, mannan, and xylan and cellulose contained in lignocellulosic biomass, which has not been reported for the AA10 family. Mutation of the key residues, Trp51 located at subsite - 2 and Phe171 located at subsite +2, in the substrate-binding site of PcAA10A_CD revealed that these residues are substantially involved in broad substrate specificity toward cellulose, xylan, and mannan, albeit with a low effect toward chitin. Furthermore, PcAA10A had a boosting effect on untreated corn hull degradation by P. curdlanolyticus B-6 endo-xylanase Xyn10D and Clostridium thermocellum endo-glucanase Cel9A. These results suggest that PcAA10A is a unique LPMO capable of cleaving and enhancing lignocellulosic biomass degradation, making it a good candidate for biotechnological applications. KEY POINTS: • PcAA10A is a novel modular LPMO family 10 from Paenibacillus curdlanolyticus. • PcAA10A showed broad substrate specificity on ß-1,4 glycosidic linkage substrates. • Non-catalytic domains are important for degrading complex polysaccharides. • PcAA10A is a unique LPMO capable of enhancing lignocellulosic biomass degradation.

Capillibacterium thermochitinicola gen. nov., sp. nov., a novel anaerobic thermophilic chitinolytic bacterium from compost.

A novel Gram-negative, spore forming, obligately anaerobic, thermophilic, chitin-degrading bacterium, designated UUS1-1T, was isolated from compost on Ishigaki Island, Japan by enrichment culturing using chitin powder as the carbon source. The strain has unique, long, hair-like rod morphological features and exhibits strong degradation activity toward crystalline chitin under thermophilic conditions. Growth of the novel strain was observed at 45-65 °C (optimum, 55 °C) and pH 6.5-7.5 (optimum, pH 7.0). In addition to chitin, the strain utilized several other carbon sources, including N-acetylglucosamine, glucose, galactose, mannose, maltose, cellobiose, fructose and sucrose. The end products of chitin degradation were acetate, lactate, H2 and CO2. Phylogenetic tree analysis based on 16S rRNA gene sequences revealed a clear affiliation of the proposed bacterium to the phylum Firmicutes; the most closely related species were Hydrogenispora ethanolica LX-BT and Desulfotomaculum thermobenzoicum DSM6193T with similarities of 90.4 and 87.8 %, respectively. The G+C content of the genomic DNA was 52.1 mol%. The average nucleotide identity and digital DNA-DNA hybridization values between the genomes of UUS1-1T and H. ethanolica LX-BT were 65.5 and 21.0 %, respectively. The cellular fatty acid composition of the strain was C16 : 0, anteiso-C15 : 0, C14 : 0, C12 : 0 3-OH and dimethyl acetal-C13 : 0. Based on phenotypic, chemotaxonomic and genotypic analysis, strain UUS1-1T represents a novel genus and species, for which the name Capillibacterium thermochitinicola gen. nov., sp. nov. is proposed. The type strain is UUS1-1T (=JCM 33882T=DSM 111537T).

Chemical Pretreatment-Independent Saccharifications of Xylan and Cellulose of Rice Straw by Bacterial Weak Lignin-Binding Xylanolytic and Cellulolytic Enzymes.

Complete utilization of carbohydrate fractions is one of the prerequisites for obtaining economically favorable lignocellulosic biomass conversion. This study shows that xylan in untreated rice straw was saccharified to xylose in one step without chemical pretreatment, yielding 58.2% of the theoretically maximum value by Paenibacillus curdlanolyticus B-6 PcAxy43A, a weak lignin-binding trifunctional xylanolytic enzyme, endoxylanase/ß-xylosidase/arabinoxylan arabinofuranohydrolase. Moreover, xylose yield from untreated rice straw was enhanced to 78.9% by adding endoxylanases PcXyn10C and PcXyn11A from the same bacterium, resulting in improvement of cellulose accessibility to cellulolytic enzyme. After autoclaving the xylanolytic enzyme-treated rice straw, it was subjected to subsequent saccharification by a combination of the Clostridium thermocellum endoglucanase CtCel9R and Thermoanaerobacter brockii ß-glucosidase TbCglT, yielding 88.5% of the maximum glucose yield, which was higher than the glucose yield obtained from ammonia-treated rice straw saccharification (59.6%). Moreover, this work presents a new environment-friendly xylanolytic enzyme pretreatment for beneficial hydrolysis of xylan in various agricultural residues, such as rice straw and corn hull. It not only could improve cellulose saccharification but also produced xylose, leading to an improvement of the overall fermentable sugar yields without chemical pretreatment.IMPORTANCE Ongoing research is focused on improving "green" pretreatment technologies in order to reduce energy demands and environmental impact and to develop an economically feasible biorefinery. The present study showed that PcAxy43A, a weak lignin-binding trifunctional xylanolytic enzyme, endoxylanase/ß-xylosidase/arabinoxylan arabinofuranohydrolase from P. curdlanolyticus B-6, was capable of conversion of xylan in lignocellulosic biomass such as untreated rice straw to xylose in one step without chemical pretreatment. It demonstrates efficient synergism with endoxylanases PcXyn10C and PcXyn11A to depolymerize xylan in untreated rice straw and enhanced the xylose production and improved cellulose hydrolysis. Therefore, it can be considered an enzymatic pretreatment. Furthermore, the studies here show that glucose yield released from steam- and xylanolytic enzyme-treated rice straw by the combination of CtCel9R and TbCglT was higher than the glucose yield obtained from ammonia-treated rice straw saccharification. This work presents a novel environment-friendly xylanolytic enzyme pretreatment not only as a green pretreatment but also as an economically feasible biorefinery method.

A novel GH6 cellobiohydrolase from Paenibacillus curdlanolyticus B-6 and its synergistic action on cellulose degradation.

We recently discovered a novel glycoside hydrolase family 6 (GH6) cellobiohydrolase from Paenibacillus curdlanolyticus B-6 (PcCel6A), which is rarely found in bacteria. This enzyme is a true exo-type cellobiohydrolase which exhibits high substrate specificity on amorphous cellulose and low substrate specificity on crystalline cellulose, while this showed no activity on substitution substrates, carboxymethyl cellulose and xylan, distinct from all other known GH6 cellobiohydrolases. Product profiles, HPLC analysis of the hydrolysis products and a schematic drawing of the substrate-binding subsites catalysing cellooligosaccharides can explain the new mode of action of this enzyme which prefers to hydrolyse cellopentaose. PcCel6A was not inhibited by glucose or cellobiose at concentrations up to 300 and 100 mM, respectively. A good synergistic effect for glucose production was found when PcCel6A acted together with processive endoglucanase Cel9R from Clostridium thermocellum and ß-glucosidase CglT from Thermoanaerobacter brockii. These properties of PcCel6A make it a suitable candidate for industrial application in the cellulose degradation process.

The C-terminal region of xylanase domain in Xyn11A from Paenibacillus curdlanolyticus B-6 plays an important role in structural stability.

Paenibacillus curdlanolyticus B-6 produces an extracellular multienzyme complex containing a major xylanase subunit, designated Xyn11A, which includes two functional domains belonging to glycosyl hydrolase family-11 (GH11) and carbohydrate binding module family-36 (CBM36) and possesses a glycine and asparagine-rich linker (linker). To clarify the roles of each functional domain, recombinant proteins XynXL and XynX (CBM36 deleted and CBM36 and linker deleted, respectively) were constructed. Their xylanase activities were similar toward soluble xylan, whereas XynXL showed decreased hydrolysis activity toward insoluble xylan while XynX had no xylanase activity. To determine the significance of the linker and its neighbor region, XynX was subjected to secondary structural alignments using circular dichroism (CD) spectroscopy and three-dimensional (3D) structural analysis. A seven amino acid (NTITIGG) neighbor linker sequence was highly conserved among GH11 xylanases of Paenibacillus species. Although XynX exhibited a typical GH11 xylanase structure, conformational gaps were observed in the ß6- and ß12-sheets and in CD spectra. Flipping of the Arg163 side chains in the subsite was also observed upon analysis of superimposed models. Docking analysis using xylohexaose indicated that flipping of the Arg163 side chains markedly affected substrate binding in the subsite. To identify the amino acids related to stabilizing the substrate binding site, XynX with an extended C-terminal region was designed. At least seven amino acids were necessary to recover substrate binding and xylanase activity. These results indicated that the seven amino acid neighbor Xyn11A linker plays an important role in the activity and conformational stability of the xylanase domain.

Discovery of a Novel Cellobiose Dehydrogenase from Cellulomonas palmilytica EW123 and Its Sugar Acids Production.

Cellobiose dehydrogenases (CDHs) are a group of enzymes belonging to the hemoflavoenzyme group, which are mostly found in fungi. They play an important role in the production of acid sugar. In this research, CDH annotated from the actinobacterium Cellulomonas palmilytica EW123 (CpCDH) was cloned and characterized. The CpCDH exhibited a domain architecture resembling class-I CDH found in Basidiomycota. The cytochrome c and flavin-containing dehydrogenase domains in CpCDH showed an extra-long evolutionary distance compared to fungal CDH. The amino acid sequence of CpCDH revealed conservative catalytic amino acids and a distinct flavin adenine dinucleotide region specific to CDH, setting it apart from closely related sequences. The physicochemical properties of CpCDH displayed optimal pH conditions similar to those of CDHs but differed in terms of optimal temperature. The CpCDH displayed excellent enzymatic activity at low temperatures (below 30°C), unlike other CDHs. Moreover, CpCDH showed the highest substrate specificity for disaccharides such as cellobiose and lactose, which contain a glucose molecule at the non-reducing end. The catalytic efficiency of CpCDH for cellobiose and lactose were 2.05 x 105 and 9.06 x 104 (M-1 s-1), respectively. The result from the Fourier-transform infrared spectroscopy (FT-IR) spectra confirmed the presence of cellobionic and lactobionic acids as the oxidative products of CpCDH. This study establishes CpCDH as a novel and attractive bacterial CDH, representing the first report of its kind in the Cellulomonas genus.

Sustainable oil palm trunk fibre based activated carbon for the adsorption of methylene blue.

Activated carbon (AC) is becoming the limelight due to its widespread application as an adsorbent for wastewater treatment, gases, and catalysis. However, its high consumption and price have drawn more attention to the sustainable use of natural resources as precursor for AC production. This study focuses on synthesising AC from two types of oil palm trunk (OPT) fibres, a significant agricultural waste products produced by Malaysia's thriving palm oil industries. The BET surface area of about 2057.9 m2 g-1 was achieved by chemical activation with phosphoric acid (H3PO4). The efficiency of the synthesised AC was critically analysed based on the adsorption experiments with methylene blue (MB) by varying several parameters (dosage of adsorbent, pH, initial dye concentration, and temperature of the solution) to elucidate the adsorption mechanism(s). A maximum adsorption capacity of 320.4 mg g-1 at 50 °C was achieved, and the Temkin (r2 = 0.98, 0.95, 0.95) and Langmuir (r2 = 0.94, 0.93, 0.95) isotherm models fitted the adsorption process better than the Freundlich (r2 = 0.95, 0.90, 0.86) model. Besides, the pseudo-second-order model (r2 > 0.90) best described the adsorption process, favouring chemisorption over physisorption. Thermodynamics showed MB adsorption on AC was spontaneous except at the highest dye concentration. It was exothermic at lower dye concentrations (50 and 100 mg L-1) and endothermic at higher ones (300, 500, and 700 mg L-1). In a nutshell, this study reveals that OPT fibre is a promising precursor for synthesising highly porous AC for the adsorption of MB dye.

Transcriptomic responses of oil palm (Elaeis guineensis) stem to waterlogging at plantation in relation to precipitation seasonality.

Global warming-induced climate change causes significant agricultural problems by increasing the incidence of drought and flooding events. Waterlogging is an inevitable consequence of these changes but its effects on oil palms have received little attention and are poorly understood. Recent waterlogging studies have focused on oil palm seedlings, with particular emphasis on phenology. However, the transcriptomic waterlogging response of mature oil palms remains elusive in real environments. We therefore investigated transcriptomic changes over time in adult oil palms at plantations over a two-year period with pronounced seasonal variation in precipitation. A significant transcriptional waterlogging response was observed in the oil palm stem core but not in leaf samples when gene expression was correlated with cumulative precipitation over two-day periods. Pathways and processes upregulated or enriched in the stem core response included hypoxia, ethylene signaling, and carbon metabolism. Post-waterlogging recovery in oil palms was found to be associated with responses to heat stress and carotenoid biosynthesis. Nineteen transcription factors (TFs) potentially involved in the waterlogging response of mature oil palms were also identified. These data provide new insights into the transcriptomic responses of planted oil palms to waterlogging and offer valuable guidance on the sensitivity of oil palm plantations to future climate changes.