Characterization of a multi-domain exo-ß-1,3-galactanase from Paenibacillus xylanexedens.

ß-1,3-Galactanases selectively degrade ß-1,3-galactan, thus it is an attractive enzyme technique to map high-galactan structure and prepare galactooligosaccharides. In this work, a gene encoding exo-ß-1,3-galactanase (PxGal43) was screened form Paenibacillus xylanexedens, consisting of a GH43 domain, a CBM32 domain and α-L-arabinofuranosidase B (AbfB) domain. Using ß-1,3-galactan (AG-II-P) as substrate, the recombined enzyme expressed in Escherichia coli BL21 (DE3) exhibited an optimal activity at pH 7.0 and 30 °C. The enzyme was thermostable, retaining >70 % activity after incubating at 50 °C for 2 h. In addition, it showed high tolerance to various metal ions, denaturants and detergents. Substrate specificity indicated that PxGal43 hydrolysis only ß-1,3-linked galactosyl oligosaccharides and polysaccharides, releasing galactose as an exo-acting manner. The function of the CBM32 and AbfB domain was revealed by their sequential deletion and suggested that their connection to the catalytic domain was crucial for the oligomerization, catalytic activity, substrate binding and thermal stability of PxGal43. The substrate docking and site-directed mutagenesis proposed that Glu191, Gln244, Asp138 and Glu81 served as the catalytic acid, catalytic base, pKa modulator, and substrate identifier in PxGal43, respectively. These results provide a better understanding and optimization of multi-domain bacterial GH43 ß-1,3-galactanase for the degradation of arabinogalactan.

Enzymatic characterization and thermostability improvement of an acidophilic endoxylanase PphXyn11 from Paenibacillus physcomitrellae XB.

GH11 enzyme is known to be specific and efficient for the hydrolysis of xylan. It has been isolated from many microorganisms, and its enzymatic characteristics and thermostability vary between species. In this study, a GH11 enzyme PphXyn11 from a novel xylan-degrading strain of Paenibacillus physcomitrellae XB was characterized, and five mutants were constructed to try to improve the enzyme's thermostability. The results showed that PphXyn11 was an acidophilic endo-ß-1,4-xylanase with the optimal reaction pH of 3.0-4.0, and it could deconstruct different kinds of xylan substrates efficiently, such as beechwood xylan, wheat arabinoxylan and xylo-oligosaccharides, to produce xylobiose and xylotriose as the main products at the optimal reaction temperature of 40 °C. Improvement of the thermal stability of PphXyn11 using site-directed mutagenesis revealed that three mutants, W33C/N47C, S127C/N174C and S49E, designed by adding the disulfide bonds at the N-terminal, C-terminal and increasing the charged residues on the surface of PphXyn11 respectively, could increase the enzymatic activity and thermal stablility significantly and make the optimal reaction temperature reach 50 °C. Molecular dynamics simulations as well as computed the numbers of salt bridges and hydrogen bonds indicated that the protein structures of these three mutants were more stable than the wild type, which provided theoretical support for their improved thermal stability. Certainly, further research is necessary to improve the enzymatic characteristics of PphXyn11 to achieve the bioconversion of hemicellulosic biomass on an applicable scale.

In Vitro Characterization of an O-Specific Glycosyltransferase Involved in Flagellin Glycosylation.

Glycosyltransferases play a fundamental role in the biosynthesis of glycoproteins and glycotherapeutics. In this study, we investigated protein glycosyltransferase FlgGT1, belonging to the GT2 family. The GT2 family includes cysteine S-glycosyltransferases involved in antimicrobial peptide biosyntheses, sharing conserved catalytic domains while exhibiting diverse C-terminal domains. Our in vitro studies revealed that FlgGT1 recognizes structural motifs rather than specific amino acid sequences when glycosylating the flagellin protein Hag. Notably, FlgGT1 is selective for serine or threonine O-glycosylation over cysteine S-glycosylation. Molecular dynamics simulations provided insights into the structural basis of FlgGT1's ability to accommodate various sugar nucleotides as donor substrates. Mutagenesis experiments on FlgGT1 demonstrated that truncating the relatively large C-terminal domain resulted in a loss of flagellin glycosylation activity. Our classification based on sequence similarity network analysis and AlphaFold2 structural predictions suggests that the acquisition of the C-terminal domain is a key evolutionary adaptation conferring distinct substrate specificities on glycosyltransferases within the GT2 family.

The CBM91 module enhances the activity of ß-xylosidase/α-L-arabinofuranosidase PphXyl43B from Paenibacillus physcomitrellae XB by adopting a unique loop conformation at the top of the active pocket.

Carbohydrate-binding module (CBM) family 91 is a novel module primarily associated with glycoside hydrolase (GH) family 43 enzymes. However, our current understanding of its function remains limited. PphXyl43B is a ß-xylosidase/α-L-arabinofuranosidase bifunctional enzyme from physcomitrellae patens XB belonging to the GH43_11 subfamily and containing CBM91 at its C terminus. To fully elucidate the contributions of the CBM91 module, the truncated proteins consisting only the GH43_11 catalytic module (rPphXyl43B-dCBM91) and only the CBM91 module (rCBM91) of PphXyl43B were constructed, respectively. The result showed that rPphXyl43B-dCBM91 completely lost hydrolysis activity against both p-nitrophenyl-ß-D-xylopyranoside and p-nitrophenyl-α-L-arabinofuranoside; it also exhibited significantly reduced activity towards xylobiose, xylotriose, oat spelt xylan and corncob xylan compared to the control. Thus, the CBM91 module is crucial for the ß-xylosidase/α-L-arabinofuranosidase activities in PphXyl43B. However, rCBM91 did not exhibit any binding capability towards corncob xylan. Structural analysis indicated that CBM91 of PphXyl43B might adopt a loop conformation (residues 496-511: ILSDDYVVQSYGGFFT) to actively contribute to the catalytic pocket formation rather than substrate binding capability. This study provides important insights into understanding the function of CBM91 and can be used as a reference for analyzing the action mechanism of GH43_11 enzymes and their application in biomass energy conversion.

Expression and characterization of α-1,3-glucanase from Paenibacillus alginolyticus NBRC15375, which is classified into subgroup 2 (minor group) of GH family 87.

Bacterial α-1,3-glucanase, classified as glycoside hydrolase (GH) family 87, has been divided into 3 subgroups based on differences in gene sequences in the catalytic domain. The enzymatic properties of subgroups 1 and 3 of several bacteria have been previously investigated and reported; however, the chemical characterization of subgroup 2 enzymes has not been previously conducted. The α-1,3-glucanase gene from Paenibacillus alginolyticus NBRC15375 (PaAgl) belonging to subgroup 2 of GH family 87 was cloned and expressed in Escherichia coli. PgAgl-N1 (subgroup 3) and PgAgl-N2 (subgroup 1) from P. glycanilyticus NBRC16188 were expressed in E. coli, and their enzymatic characteristics were compared. The amino acid sequence of PaAgl demonstrated that the homology was significantly lower in other subgroups when only the catalytic domain was compared. The oligosaccharide products of the mutan-degrading reaction seemed to have different characteristics among subgroups 1, 2, and 3 in GH family 87.

Highly efficient bioconversion of icariin to icaritin by whole-cell catalysis.

BACKGROUND: Icaritin is an aglycone of flavonoid glycosides from Herba Epimedii. It has good performance in the treatment of hepatocellular carcinoma in clinical trials. However, the natural icaritin content of Herba Epimedii is very low. At present, the icaritin is mainly prepared from flavonoid glycosides by α-L-rhamnosidases and ß-glucosidases in two-step catalysis process. However, one-pot icaritin production required reported enzymes to be immobilized or bifunctional enzymes to hydrolyze substrate with long reaction time, which caused complicated operations and high costs. To improve the production efficiency and reduce costs, we explored α-L-rhamnosidase SPRHA2 and ß-glucosidase PBGL to directly hydrolyze icariin to icaritin in one-pot, and developed the whole-cell catalytic method for efficient icaritin production. RESULTS: The SPRHA2 and PBGL were expressed in Escherichia coli, respectively. One-pot production of icaritin was achieved by co-catalysis of SPRHA2 and PBGL. Moreover, whole-cell catalysis was developed for icariin hydrolysis. The mixture of SPRHA2 cells and PBGL cells transformed 200 g/L icariin into 103.69 g/L icaritin (yield 95.23%) in 4 h in whole-cell catalysis under the optimized reaction conditions. In order to further increase the production efficiency and simplify operations, we also constructed recombinant E. coli strains that co-expressed SPRHA2 and PBGL. Crude icariin extracts were also efficiently hydrolyzed by the whole-cell catalytic system. CONCLUSIONS: Compared to previous reports on icaritin production, in this study, whole-cell catalysis showed higher production efficiency of icaritin. This study provides promising approach for industrial production of icaritin in the future.

Discovery of solabiose phosphorylase and its application for enzymatic synthesis of solabiose from sucrose and lactose.

Glycoside phosphorylases (GPs), which catalyze the reversible phosphorolysis of glycosides, are promising enzymes for the efficient production of glycosides. Various GPs with new catalytic activities are discovered from uncharacterized proteins phylogenetically distant from known enzymes in the past decade. In this study, we characterized Paenibacillus borealis PBOR_28850 protein, belonging to glycoside hydrolase family 94. Screening of acceptor substrates for reverse phosphorolysis, in which α-D-glucose 1-phosphate was used as the donor substrate, revealed that the recombinant PBOR_28850 produced in Escherichia coli specifically utilized D-galactose as an acceptor and produced solabiose (ß-D-Glcp-(1 → 3)-D-Gal). This indicates that PBOR_28850 is a new GP, solabiose phosphorylase. PBOR_28850 catalyzed the phosphorolysis and synthesis of solabiose through a sequential bi-bi mechanism involving the formation of a ternary complex. The production of solabiose from lactose and sucrose has been established. Lactose was hydrolyzed to D-galactose and D-glucose by ß-galactosidase. Phosphorolysis of sucrose and synthesis of solabiose were then coupled by adding sucrose, sucrose phosphorylase, and PBOR_28850 to the reaction mixture. Using 210 mmol lactose and 280 mmol sucrose, 207 mmol of solabiose was produced. Yeast treatment degraded the remaining monosaccharides and sucrose without reducing solabiose. Solabiose with a purity of 93.7% was obtained without any chromatographic procedures.

Heme-Edge Residues Modulate Signal Transduction within a Bifunctional Homo-Dimeric Sensor Protein.

Bifunctional enzymes, which contain two domains with opposing enzymatic activities, are widely distributed in bacteria, but the regulatory mechanism(s) that prevent futile cycling are still poorly understood. The recently described bifunctional enzyme, DcpG, exhibits unusual heme properties and is surprisingly able to differentially regulate its two cyclic dimeric guanosine monophosphate (c-di-GMP) metabolic domains in response to heme gaseous ligands. Mutagenesis of heme-edge residues was used to probe the heme pocket and resulted in decreased O2 dissociation kinetics, identifying roles for these residues in modulating DcpG gas sensing. In addition, the resonance Raman spectra of the DcpG wild type and heme-edge mutants revealed that the mutations alter the heme electrostatic environment, vinyl group conformations, and spin state population. Using small-angle X-ray scattering and negative stain electron microscopy, the heme-edge mutations were demonstrated to cause changes to the protein conformation, which resulted in altered signaling transduction and enzyme kinetics. These findings provide insights into molecular interactions that regulate DcpG gas sensing as well as mechanisms that have evolved to control multidomain bacterial signaling proteins.

Crystal structure of a novel putative sugar isomerase from the psychrophilic bacterium Paenibacillus sp. R4.

Sugar isomerases (SIs) catalyze the reversible conversion of aldoses to ketoses. A novel putative SI gene has been identified from the genome sequence information on the psychrophilic bacterium Paenibacillus sp. R4. Here, we report the crystal structure of the putative SI from Paenibacillus sp. R4 (PbSI) at 2.98 Å resolution. It was found that the overall structure of PbSI adopts the triose-phosphate isomerase (TIM) barrel fold. PbSI was also identified to have two heterogeneous metal ions as its cofactors at the active site in the TIM barrel, one of which was confirmed as a Zn ion through X-ray anomalous scattering and inductively coupled plasma mass spectrometry analysis. Structural comparison with homologous SI proteins from mesophiles, hyperthermophiles, and a psychrophile revealed that key residues in the active site are well conserved and that dimeric PbSI is devoid of the extended C-terminal region, which tetrameric SIs commonly have. Our results provide novel structural information on the cold-adaptable SI, including information on the metal composition in the active site.

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.

Differential ligand-selective control of opposing enzymatic activities within a bifunctional c-di-GMP enzyme.

Cyclic dimeric guanosine monophosphate (c-di-GMP) serves as a second messenger that modulates bacterial cellular processes, including biofilm formation. While proteins containing both c-di-GMP synthesizing (GGDEF) and c-di-GMP hydrolyzing (EAL) domains are widely predicted in bacterial genomes, it is poorly understood how domains with opposing enzymatic activity are regulated within a single polypeptide. Herein, we report the characterization of a globin-coupled sensor protein (GCS) from Paenibacillus dendritiformis (DcpG) with bifunctional c-di-GMP enzymatic activity. DcpG contains a regulatory sensor globin domain linked to diguanylate cyclase (GGDEF) and phosphodiesterase (EAL) domains that are differentially regulated by gas binding to the heme; GGDEF domain activity is activated by the Fe(II)-NO state of the globin domain, while EAL domain activity is activated by the Fe(II)-O2 state. The in vitro activity of DcpG is mimicked in vivo by the biofilm formation of P. dendritiformis in response to gaseous environment, with nitric oxide conditions leading to the greatest amount of biofilm formation. The ability of DcpG to differentially control GGDEF and EAL domain activity in response to ligand binding is likely due to the unusual properties of the globin domain, including rapid ligand dissociation rates and high midpoint potentials. Using structural information from small-angle X-ray scattering and negative stain electron microscopy studies, we developed a structural model of DcpG, providing information about the regulatory mechanism. These studies provide information about full-length GCS protein architecture and insight into the mechanism by which a single regulatory domain can selectively control output domains with opposing enzymatic activities.

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.

Simple stain-free screening method for pectinolytic microorganisms under alkalophilic conditions.

OBJECTIVES: To develop a simple pectin-degrading microorganism screening method. RESULTS: We developed a method utilizing the phenomenon whereby cooling an alkaline agar medium containing pectin causes the agar to become cloudy. This highly simplified method involves culturing the microorganisms on pectin-containing agar medium until colony formation is observed, and subsequent overnight cooling of the agar medium to 4 °C. Using this simple procedure, we successfully identified pectin-degrading microorganisms by observing colonies with halos on the clouded agar medium. We used alkaline pectinase and Bacillus halodurans, which is known to secrete alkaline pectinase, to establish the screening method. We demonstrated the screening of pectin-degrading microorganisms using the developed method and successfully isolated pectin-degrading microorganisms (Paenibacillus sp., Bacillus clausii, and Bacillus halodurans) from a soil sample. CONCLUSIONS: The developed method is useful for identifying pectin-degrading microorganisms.

Structural determination of the sheath-forming polysaccharide of Sphaerotilus montanus using thiopeptidoglycan lyase which recognizes the 1,4 linkage between α-d-GalN and ß-d-GlcA.

Sphaerotilus natans is a filamentous sheath-forming bacterium commonly found in activated sludge. Its sheath is assembled from a thiolic glycoconjugate called thiopeptidoglycan. S. montanus ATCC-BAA-2725 is a sheath-forming member of stream biofilms, and its sheath is morphologically similar to that of S. natans. However, it exhibits heat susceptibility, which distinguishes it from the S. natans sheath. In this study, chemical composition and solid-state NMR analyses suggest that the S. montanus sheath is free of cysteine, indicating that disulfide linkage is not mandatory for sheath formation. The S. montanus sheath was successfully solubilized by N-acetylation, allowing solution-state NMR analysis to determine the sugar sequence. The sheath was susceptible to thiopeptidoglycan lyase prepared from the thiopeptidoglycan-assimilating bacterium, Paenibacillus koleovorans. The reducing ends of the enzymatic digests were labeled with 4-aminobenzoic acid ethyl ester, followed by HPLC. Two derivatives were detected, and their structures were determined. We found that the sheath has no peptides and is assembled as follows: [→4)-ß-d-GlcA-(1→4)-ß-d-Glc-(1→3)-ß-d-GalNAc-(1→4)-α-d-GalNAc-(1→4)-α-d-GalN-(1→]n (ß-d-Glc and α-d-GalNAc are stoichiometrically and substoichiometrically 3-O-acetylated, respectively). Thiopeptidoglycan lyase was thus confirmed to cleave the 1,4 linkage between α-d-GalN and ß-d-GlcA, regardless of the peptide moiety. Furthermore, vital fluorescent staining of the sheath demonstrated that elongation takes place at the tips, as with the S. natans sheath.

Biochemical characterization of a novel acidic chitinase with antifungal activity from Paenibacillus xylanexedens Z2-4.

A chitinase gene (PxChi52) from Paenibacillus xylanexedens Z2-4 was cloned and heterologously expressed in Escherichia coli BL21 (DE3). PxChi52 shared the highest identity of 91% with a glycoside hydrolase family 18 chitinase (ChiD) from Bacillus circulans. The recombinant enzyme (PxChi52) was purified and biochemically characterized. PxChi52 had a molecular mass of 52.8 kDa. It was most active at pH 4.5 and 65 °C, respectively, and stable in a wide pH range of 4.0-13.0 and up to 50 °C. The enzyme exhibited the highest specific activity of 16.0 U/mg towards colloidal chitin, followed by ethylene glycol chitin (5.4 U/mg) and ball milled chitin (0.4 U/mg). The Km and Vmax values of PxChi52 towards colloidal chitin were determined to be 3.06 mg/mL and 71.38 U/mg, respectively, PxChi52 hydrolyzed colloidal chitin and chitooligosaccharides with degree of polymerization 2-5 to release mainly N-acetyl chitobiose. In addition, PxChi52 displayed inhibition effects on the growth of some phytopathogenic fungi, including Alternaria alstroemeriae, Botrytis cinerea, Rhizoctonia solani, Sclerotinia sclerotiorum and Valsa mali. The unique properties of PxChi52 may enable it potential application in agriculture field as a biocontrol agent.

Rational design and structure-based engineering of alkaline pectate lyase from Paenibacillus sp. 0602 to improve thermostability.

BACKGROUND: Ramie degumming is often carried out at high temperatures; therefore, thermostable alkaline pectate lyase (PL) is beneficial for ramie degumming for industrial applications. Thermostable PLs are usually obtained by exploring new enzymes or reconstructing existing enzyme by rational design. Here, we improved the thermostability of an alkaline pectate lyase (PelN) from Paenibacillus sp. 0602 with rational design and structure-based engineering. RESULTS: From 26 mutants, two mutants of G241A and G241V showed a higher thermostability compared with the wild-type PL. The mutant K93I showed increasing specific activity at 45 °C. Subsequently, we obtained combinational mutations (K93I/G241A) and found that their thermostability and specific activity improved simultaneously. The K93I/G241A mutant showed a half-life time of 15.9 min longer at 60 °C and a melting temperature of 1.6 °C higher than those of the wild PL. The optimum temperature decreased remarkably from 67.5 °C to 60 °C, accompanied by a 57% decrease in Km compared with the Km value of the wild-type strain. Finally, we found that the intramolecular interaction in PelN was the source in the improvements of molecular properties by comparing the model structures. Rational design of PelN was performed by stabilizing the α-helices with high conservation and increasing the stability of the overall structure of the protein. Two engineering strategies were applied by decreasing the mutation energy calculated by Discovery Studio and predicting the free energy in the process of protein folding by the PoPMuSiC algorithm. CONCLUSIONS: The results demonstrated that the K93I/G241A mutant was more suitable for industrial production than the wild-type enzyme. Furthermore, the two forementioned strategies could be extended to reveal engineering of other kinds of industrial enzymes.

Structures and functions of carbohydrate-active enzymes of chitinolytic bacteria Paenibacillus sp. str. FPU-7.

Chitin and its derivatives have valuable potential applications in various fields that include medicine, agriculture, and food industries. Paenibacillus sp. str. FPU-7 is one of the most potent chitin-degrading bacteria identified. This review introduces the chitin degradation system of P. str. FPU-7. In addition to extracellular chitinases, P. str. FPU-7 uses a unique multimodular chitinase (ChiW) to hydrolyze chitin to oligosaccharides on the cell surface. Chitin oligosaccharides are converted to N-acetyl-d-glucosamine by ß-N-acetylhexosaminidase (PsNagA) in the cytosol. The functions and structures of ChiW and PsNagA are also summarized. The genome sequence of P. str. FPU-7 provides opportunities to acquire novel enzymes. Genome mining has identified a novel alginate lyase, PsAly. The functions and structure of PsAly are reviewed. These findings will inform further improvement of the sustainable conversion of polysaccharides to functional materials.

Biochemical characterization of a novel bifunctional chitosanase from Paenibacillus barengoltzii for chitooligosaccharide production.

A novel chitosanase gene, designated as PbCsn8, was cloned from Paenibacillus barengoltzii. It shared the highest identity of 73% with the glycoside hydrolase (GH) family 8 chitosanase from Bacillus thuringiensis JAM-GG01. The gene was heterologously expressed in Bacillus subtilis as an extracellular protein, and the highest chitosanase yield of 1, 108 U/mL was obtained by high-cell density fermentation in a 5-L fermentor. The recombinant chitosanase (PbCsn8) was purified to homogeneity and biochemically characterized. PbCsn8 was most active at pH 5.5 and 70 °C, respectively. It was stable in a wide pH range of 5.0-11.0 and up to 55 °C. PbCsn8 was a bifunctional enzyme, exhibiting both chitosanase and glucanase activities, with the highest specificity towards chitosan (360 U/mg), followed by barley ß-glucan (72 U/mg) and lichenan (13 U/mg). It hydrolyzed chitosan to release mainly chitooligosaccharides (COSs) with degree of polymerization (DP) 2-3, while hydrolyzed barley ß-glucan to yield mainly glucooligosaccharides with DP > 5. PbCsn8 was further applied in COS production, and the highest COS yield of 79.3% (w/w) was obtained. This is the first report on a GH family 8 chitosanase from P. barengoltzii. The high yield and remarkable hydrolysis properties may make PbCsn8 a good candidate in industrial application.

The Buzz about ADP-Ribosylation Toxins from Paenibacillus larvae, the Causative Agent of American Foulbrood in Honey Bees.

The Gram-positive, spore-forming bacterium Paenibacillus larvae is the etiological agent of American Foulbrood, a highly contagious and often fatal honey bee brood disease. The species P. larvae comprises five so-called ERIC-genotypes which differ in virulence and pathogenesis strategies. In the past two decades, the identification and characterization of several P. larvae virulence factors have led to considerable progress in understanding the molecular basis of pathogen-host-interactions during P. larvae infections. Among these virulence factors are three ADP-ribosylating AB-toxins, Plx1, Plx2, and C3larvin. Plx1 is a phage-born toxin highly homologous to the pierisin-like AB-toxins expressed by the whites-and-yellows family Pieridae (Lepidoptera, Insecta) and to scabin expressed by the plant pathogen Streptomyces scabiei. These toxins ADP-ribosylate DNA and thus induce apoptosis. While the presumed cellular target of Plx1 still awaits final experimental proof, the classification of the A subunits of the binary AB-toxins Plx2 and C3larvin as typical C3-like toxins, which ADP-ribosylate Rho-proteins, has been confirmed experimentally. Normally, C3-exoenzymes do not occur together with a B subunit partner, but as single domain toxins. Interestingly, the B subunits of the two P. larvae C3-like toxins are homologous to the B-subunits of C2-like toxins with striking structural similarity to the PA-63 protomer of Bacillus anthracis.

Synthesis of novel oligomeric anionic alkyl glycosides using laccase/TEMPO oxidation and cyclodextrin glucanotransferase (CGTase)-catalyzed transglycosylation.

Modification of alkyl glycosides, to alter their properties and widen the scope of potential applications, is of considerable interest. Here, we report the synthesis of new anionic alkyl glycosides with long carbohydrate chains, using two different approaches: laccase/2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) oxidation of a long-carbohydrate-chain alkyl glycoside and cyclodextrin glucanotransferase (CGTase)-catalyzed elongation of anionic alkyl glycosides. The laccase/TEMPO oxidation of dodecyl ß- d-maltooctaoside proceeded efficiently with the formation of aldehyde and acid products. However, depolymerization occurred to a large extent, limiting the product yield and purity. On the other hand, CGTase-catalyzed coupling/disproportionation reactions with α-cyclodextrin and dodecyl ß- d-maltoside diuronic acid (DDM-2COOH) or octyl ß- d-glucuronic acid (OG-COOH) as substrates gave high conversions, especially when the CGTase Toruzyme was used. It was found that pH had a strong influence on both the enzyme activity and the acceptor specificity. With non-ionic substrates (dodecyl ß- d-maltoside and octyl ß- d-glucoside), Toruzyme exhibited high catalytic activity at pH 5-6, but for the acidic substrates (DDM-2COOH and OG-COOH) the activity was highest at pH 4. This is most likely due to the enzyme favoring the protonated forms of DDM-2COOH and OG-COOH, which exist at lower pH (pKa about 3).