Anticancer and anti-angiogenic activities of mannooligosaccharides extracted from coconut meal on colorectal carcinoma cells in vitro.

Colorectal carcinoma (CRC) is one of the most common malignancies, though there are no effective therapeutic regimens at present. This study aimed to investigate the inhibitory effects of mannooligosaccharides extracted from coconut meal (CMOSs) on the proliferation and migration of human colorectal cancer HCT116 cells in vitro. The results showed that CMOSs exhibited significant inhibitory activity against HCT116 cell proliferation in a concentration-dependent manner with less cytotoxic effects on the Vero normal cells. CMOSs displayed the ability to increase the activation of caspase-8, -9, and -3/7, as well as the generation of reactive oxygen species (ROS). Moreover, CMOSs suppressed HCT116 cell migration in vitro. Interestingly, treatment of human microvascular endothelial cells (HMVECs) with CMOSs resulted in the inhibition of cell proliferation, cell migration, and capillary-like tube formation, suggesting its anti-vascular angiogenesis. In summary, the results of this study indicate that CMOSs could be a valuable therapeutic candidate for CRC treatment.

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.

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.

Genomic analysis of Paenibacillus macerans strain I6, which can effectively saccharify oil palm empty fruit bunches under nutrient-free conditions.

The improper disposal of palm oil industrial waste has led to serious environmental pollution. In this study, we isolated Paenibacillus macerans strain I6, which can degrade oil palm empty fruit bunches generated by the palm oil industry in nutrient-free water, from bovine manure biocompost and sequenced its genome on PacBio RSII and Illumina NovaSeq 6000 platforms. We obtained 7.11 Mbp of genomic sequences with 52.9% GC content from strain I6. Strain I6 was phylogenetically closely related to P. macerans strains DSM24746 and DSM24 and was positioned close to the head of the branch containing strains I6, DSM24746, and DSM24 in the phylogenetic tree. We used the RAST (rapid annotation using subsystem technology) server to annotate the strain I6 genome and discovered genes related to biological saccharification; 496 genes were related to carbohydrate metabolism and 306 genes were related to amino acids and derivatives. Among them were carbohydrate-active enzymes (CAZymes), including 212 glycoside hydrolases. Up to 23.6% of the oil palm empty fruit bunches was degraded by strain I6 under anaerobic and nutrient-free conditions. Evaluation of the enzymatic activity of extracellular fractions of strain I6 showed that amylase and xylanase activity was highest when xylan was the carbon source. The high enzyme activity and the diversity in the associated genes may contribute to the efficient degradation of oil palm empty fruit bunches by strain I6. Our results indicate the potential utility of P. macerans strain I6 for lignocellulosic biomass degradation.

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.

Production of a Series of Long-Chain Isomaltooligosaccharides from Maltose by Bacillus subtilis AP-1 and Associated Prebiotic Properties.

Bacillus subtilis strain AP-1, which produces α-glucosidase with transglucosidase activity, was used to produce a series of long-chain isomaltooligosaccharides (IMOs) with degree of polymerization (DP) ranging from 2 to 14 by direct fermentation of maltose. A total IMOs yield of 36.33 g/L without contabacillusmination from glucose and maltose was achieved at 36 h of cultivation using 50 g/L of maltose, with a yield of 72.7%. IMOs were purified by size exclusion chromatography with a Superdex 30 Increase column. The molecular mass and DP of IMOs were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). Subsequently, linkages in produced oligosaccharides were verified by enzymatic hydrolysis with α-amylase and oligo-α-1,6-glucosidase. These IMOs showed prebiotic properties, namely tolerance to acidic conditions and digestive enzymes of the gastrointestinal tract, stimulation of probiotic bacteria growth to produce short-chain fatty acids and no stimulating effect on pathogenic bacteria growth. Moreover, these IMOs were not toxic to mammalian cells at up to 5 mg/mL, indicating their biocompatibility. Therefore, this research demonstrated a simple and economical method for producing IMOs with DP2-14 without additional operations; moreover, the excellent prebiotic properties of the IMOs offer great prospects for their application in functional foods.

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).

Utilization of Receptor-Binding Domain of SARS-CoV-2 Spike Protein Expressed in Escherichia coli for the Development of Neutralizing Antibody Assay.

The ongoing COVID-19 pandemic has resulted from widespread infection by the SARS-CoV-2 virus. As new variants of concern continue to emerge, understanding the correlation between the level of neutralizing antibodies (NAb) and clinical protection from SAR-CoV-2 infection could be critical in planning the next steps in COVID-19 vaccine programs. This study explored the potential usefulness of E. coli as an alternative expression system that can be used to produce a SARS-CoV-2 receptor-binding domain (RBD) for the development of an affordable and flexible NAb detection assay. We expressed the RBD of Beta, Delta, and Omicron variants in the E. coli BL21(DE3) strain and purified them from whole bacterial cells using His-tag-mediated affinity chromatography and urea-assisted refolding. Next, we conducted a head-to-head comparison of the binding activity of our E. coli-produced RBD (E-RBD) with commercial HEK293-produced RBD (H-RBD). The results of a direct binding assay revealed E-RBD and H-RBD binding with ACE2-hFc in similar signal strengths. Furthermore, in the NAb detection assay, % inhibition obtained from both E-RBD and H-RBD demonstrated comparable results in all the investigated assays, suggesting that non-glycosylated RBD produced from E. coli may offer a cost-effective alternative to the use of more expensive glycosylated RBD produced from human cells in the development of such an assay.

Genomics and cellulolytic, hemicellulolytic, and amylolytic potential of Iocasia fonsfrigidae strain SP3-1 for polysaccharide degradation.

Background: Cellulolytic, hemicellulolytic, and amylolytic (CHA) enzyme-producing halophiles are understudied. The recently defined taxon Iocasia fonsfrigidae consists of one well-described anaerobic bacterial strain: NS-1T. Prior to characterization of strain NS-1T, an isolate designated Halocella sp. SP3-1 was isolated and its genome was published. Based on physiological and genetic comparisons, it was suggested that Halocella sp. SP3-1 may be another isolate of I. fronsfrigidae. Despite being geographic variants of the same species, data indicate that strain SP3-1 exhibits genetic, genomic, and physiological characteristics that distinguish it from strain NS-1T. In this study, we examine the halophilic and alkaliphilic nature of strain SP3-1 and the genetic substrates underlying phenotypic differences between strains SP3-1 and NS-1T with focus on sugar metabolism and CHA enzyme expression. Methods: Standard methods in anaerobic cell culture were used to grow strains SP3-1 as well as other comparator species. Morphological characterization was done via electron microscopy and Schaeffer-Fulton staining. Data for sequence comparisons (e.g., 16S rRNA) were retrieved via BLAST and EzBioCloud. Alignments and phylogenetic trees were generated via CLUTAL_X and neighbor joining functions in MEGA (version 11). Genomes were assembled/annotated via the Prokka annotation pipeline. Clusters of Orthologous Groups (COGs) were defined by eegNOG 4.5. DNA-DNA hybridization calculations were performed by the ANI Calculator web service. Results: Cells of strain SP3-1 are rods. SP3-1 cells grow at NaCl concentrations of 5-30% (w/v). Optimal growth occurs at 37 °C, pH 8.0, and 20% NaCl (w/v). Although phylogenetic analysis based on 16S rRNA gene indicates that strain SP3-1 belongs to the genus Iocasia with 99.58% average nucleotide sequence identity to Iocasia fonsfrigida NS-1T, strain SP3-1 is uniquely an extreme haloalkaliphile. Moreover, strain SP3-1 ferments D-glucose to acetate, butyrate, carbon dioxide, hydrogen, ethanol, and butanol and will grow on L-arabinose, D-fructose, D-galactose, D-glucose, D-mannose, D-raffinose, D-xylose, cellobiose, lactose, maltose, sucrose, starch, xylan and phosphoric acid swollen cellulose (PASC). D-rhamnose, alginate, and lignin do not serve as suitable culture substrates for strain SP3-1. Thus, the carbon utilization profile of strain SP3-1 differs from that of I. fronsfrigidae strain NS-1T. Differences between these two strains are also noted in their lipid composition. Genomic data reveal key differences between the genetic profiles of strain SP3-1 and NS-1T that likely account for differences in morphology, sugar metabolism, and CHA-enzyme potential. Important to this study, I. fonsfrigidae SP3-1 produces and extracellularly secretes CHA enzymes at different levels and composition than type strain NS-1T. The high salt tolerance and pH range of SP3-1 makes it an ideal candidate for salt and pH tolerant enzyme discovery.

Characterization of a GH Family 43 ß-Xylosidase Having a Novel Carbohydrate-binding Module from Paenibacillus xylaniclasticus Strain TW1.

Paenibacillus xylaniclasticus strain TW1, a gram-positive facultative anaerobic bacterium, was isolated as a xylanolytic microorganism from the wastes of a pineapple processing factory. A gene encoding one of its xylanolytic enzymes, a ß-xylosidase, was cloned and sequenced. Sequence analysis revealed that this ß-xylosidase, named PxXyl43A, was composed of a glycoside hydrolase (GH) family 43 subfamily 12 catalytic module and an unknown function module (UM). The full-length PxXyl43A (PxXyl43A) was heterologously expressed in Escherichia coli and purified. Recombinant PxXyl43A exhibited hydrolysis activity against both p-nitrophenyl-ß-D-xylopyranoside (pNPX) and p-nitrophenyl-α-L-arabinofuranoside at specific activities of 250 and 310 mU/mg, respectively. The optimal reaction pH and temperature for pNPX hydrolysis were 7.1 and 54 ˚C, respectively. At pH 7.0 and 54 ˚C, the K m and k cat for pNPX were 1.2 mM and 2.8 ± 0.15 s-1, respectively. It was also discovered that the recombinant unknown function module of PxXyl43A (PxXyl43A-UM) could bind to insoluble xylans like birchwood xylan and oat spelt xylan, whereas it did not bind to cellulosic substrates such as ball-milled cellulose, carboxymethyl cellulose or lichenan. The PxXyl43A-UM's binding constant value K a for oat spelt xylan was 2.0 × 10-5 M-1. These results suggest that PxXyl43A possesses a novel carbohydrate-binding module, named as CBM91, specific for xylan-containing polysaccharides.

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.

Digestibility of Bacillus firmus K-1 pretreated rice straw by different commercial cellulase cocktails.

Removal of xylan in plant biomass is believed to increase cellulose hydrolysis by uncovering cellulose surfaces for cellulase adsorption and, in turn, catalysis reaction. Herein, we describe an eco-friendly method by culturing a xylanolytic Bacillus firmus K-1 on rice straw to remove xylan. The bacterium was grown on 2.5% (w/v) rice straw with different biomass particle sizes for two days at 37 °C. We found that the particle sizes ranged from <1 to 5 mm gave a similar xylan removal degree (about 21%). Besides, the porosity and disintegration of the rice straw fibers were observed at the molecular level. The digestibility of pretreated rice straw was tested with different commercial cellulase cocktails. We found that the pretreated rice straw was more susceptible to enzymatic hydrolysis, giving 30-70% glucan conversion than the untreated one. The degree of cellulose hydrolysis depended strongly on the kinds of enzyme and their formulations. HighlightCulturing B. firmus K-1 on rice straw yielded about 21% removal of xylan.Particle sizes (of 1-5 mm) had negligible effects on xylan removal efficiency.The degree of glucan conversion in pretreated biomass relied on enzyme formulation.

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.

Characterization of a thermophilic facultatively anaerobic bacterium Paenibacillus sp. strain DA-C8 that exhibits xylan degradation under anaerobic conditions.

The screening, identification, and study of the functional properties of cellulolytic xylanolytic bacteria are crucial for the construction of applicable bioprocesses. The thermophilic facultatively anaerobic, xylanolytic bacterial strain DA-C8 (=JCM34211=DSM111723) exhibiting efficient xylan degradation was newly isolated from compost. Strain DA-C8 completely degraded 1% beechwood xylan within 4 days under anaerobic conditions. By 16S rRNA gene sequence homology and phylogenetic tree analysis, strain DA-C8 was closely related to Paenibacillus cisolokensis and Xylanibacillus composti; however, the average nucleotide identity and digital DNA-DNA hybridization values based on genome information and the carbon source utilization properties indicated that strain DA-C8 belongs to Paenibacillus rather than Xylanibacillus. The gene numbers of xylanase and endoglucanase of strain DA-C8 and X. composti were not different; however, strain DA-C8 had higher abundance of α-L-arabinofuranosidase, ß-xylosidase, and ß-glucosidase than X. composti. Strain DA-C8 showed decreased xylan and corn hull degradation abilities and growth on xylan medium under aerobic conditions. Quantitative PCR showed high expression of xylan and cellulose degradation genes under anaerobic conditions, but the genes were repressed under aerobic conditions, indicating that strain DA-C8 controls polysaccharide degradation depending on the aeration conditions. Strain DA-C8 is a new species of Paenibacillus with a unique polysaccharide degradation system.

Draft genome sequence data of Paenibacillus cisolokensis strain LC2-13A and Xylanibacillus composti strain K-13.

To discover more efficient degradation processes of lignocellulosic biomass, it is still important to analyze genomic and enzymatic data from bacteria that have strong xylanolytic ability. Here, we present the draft genome sequences of the xylanolytic bacteria Paenibacillus cisolokensis strain LC2-13A and Xylanibacillus composti strain K-13 that are closest to Paenibacillus sp. strain DA-C8, which has strong xylan degradation ability under anaerobic growth conditions. Whole-genome sequencing on the Ion GeneStudio S5 System yielded 277 contigs with total size 5,305,208 bp and G+C content 52.3 mol% for strain LC2-13A and 115 contigs with total size 4,652,266 bp and G+C content of 56.2 mol% for strain K-13. The LC2-13A genome had 5,744 protein-coding sequences (CDSs), 57 tRNAs, and 4 clustered regularly interspaced short palindromic repeats (CRISPRs), and the K-13 genome had 4,388 CDSs, 1 rRNA gene, 45 tRNAs, and 5 CRISPRs. The CDSs of LC2-13A and K-13 encoded the following carbohydrate-active enzymes: 98 and 67 glycoside hydrolases, 31 and 29 glycosyl transferases, 23 and 17 carbohydrate esterases, and 13 and 37 carbohydrate-binding modules, respectively. The whole-genome sequences of LC2-13A and K-13 have been deposited in DDBJ/ENA/GenBank under accession numbers BOVK00000000 and BOVJ00000000. The versions described in this paper are version 1.

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.

Bioconversion of Untreated Corn Hull into L-Malic Acid by Trifunctional Xylanolytic Enzyme from Paenibacillus curdlanolyticus B-6 and Acetobacter tropicalis H-1.

L-Malic acid (L-MA) is widely used in food and non-food products. However, few microorganisms have been able to efficiently produce L-MA from xylose derived from lignocellulosic biomass (LB). The objective of this work is to convert LB into L-MA with the concept of a bioeconomy and environmentally friendly process. The unique trifunctional xylanolytic enzyme, PcAxy43A from Paenibacillus curdlanolyticus B-6, effectively hydrolyzed xylan in untreated LB, especially corn hull to xylose, in one step. Furthermore, the newly isolated, Acetobacter tropicalis strain H1 was able to convert high concentrations of xylose derived from corn hull into L-MA as the main product, which can be easily purified. The strain H1 successfully produced a high L-MA titer of 77.09 g/l, with a yield of 0.77 g/g and a productivity of 0.64 g/l/h from the xylose derived from corn hull. The process presented in this research is an efficient, low-cost and environmentally friendly biological process for the green production of L-MA from LB.

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.