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.

Secretion of the cytoplasmic and high molecular weight ß-galactosidase of Paenibacillus wynnii with Bacillus subtilis.

BACKGROUND: The gram-positive bacterium Bacillus subtilis is widely used for industrial enzyme production. Its ability to secrete a wide range of enzymes into the extracellular medium especially facilitates downstream processing since cell disruption is avoided. Although various heterologous enzymes have been successfully secreted with B. subtilis, the secretion of cytoplasmic enzymes with high molecular weight is challenging. Only a few studies report on the secretion of cytoplasmic enzymes with a molecular weight > 100 kDa. RESULTS: In this study, the cytoplasmic and 120 kDa ß-galactosidase of Paenibacillus wynnii (ß-gal-Pw) was expressed and secreted with B. subtilis SCK6. Different strategies were focused on to identify the best secretion conditions. Tailormade codon-optimization of the ß-gal-Pw gene led to an increase in extracellular ß-gal-Pw production. Consequently, the optimized gene was used to test four signal peptides and two promoters in different combinations. Differences in extracellular ß-gal-Pw activity between the recombinant B. subtilis strains were observed with the successful secretion being highly dependent on the specific combination of promoter and signal peptide used. Interestingly, signal peptides of both the general secretory- and the twin-arginine translocation pathway mediated secretion. The highest extracellular activity of 55.2 ± 6 µkat/Lculture was reached when secretion was mediated by the PhoD signal peptide and expression was controlled by the PAprE promoter. Production of extracellular ß-gal-Pw was further enhanced 1.4-fold in a bioreactor cultivation to 77.5 ± 10 µkat/Lculture with secretion efficiencies of more than 80%. CONCLUSION: For the first time, the ß-gal-Pw was efficiently secreted with B. subtilis SCK6, demonstrating the potential of this strain for secretory production of cytoplasmic, high molecular weight enzymes.

Recombinant production of Paenibacillus wynnii ß-galactosidase with Komagataella phaffii.

BACKGROUND: The ß-galactosidase from Paenibacillus wynnii (ß-gal-Pw) is a promising candidate for lactose hydrolysis in milk and dairy products, as it has a higher affinity for the substrate lactose (low KM value) compared to industrially used ß-galactosidases and is not inhibited by the hydrolysis-generated product D-galactose. However, ß-gal-Pw must firstly be produced cost-effectively for any potential industrial application. Accordingly, the yeast Komagataella phaffii was chosen to investigate its feasibility to recombinantly produce ß-gal-Pw since it is approved for the regulated production of food enzymes. The aim of this study was to find the most suitable way to produce the ß-gal-Pw in K. phaffii either extracellularly or intracellularly. RESULTS: Firstly, 11 different signal peptides were tested for extracellular production of ß-gal-Pw by K. phaffii under the control of the constitutive GAP promoter. None of the signal peptides resulted in a secretion of ß-gal-Pw, indicating problems within the secretory pathway of this enzyme. Therefore, intracellular ß-gal-Pw production was investigated using the GAP or methanol-inducible AOX1 promoter. A four-fold higher volumetric ß-galactosidase activity of 7537 ± 66 µkatoNPGal/Lculture was achieved by the K. phaffii clone 27 using the AOX1 promoter in fed-batch bioreactor cultivations, compared to the clone 5 using the GAP promoter. However, a two-fold higher specific productivity of 3.14 ± 0.05 µkatoNPGal/gDCW/h was achieved when using the GAP promoter for ß-gal-Pw production compared to the AOX1 promoter. After partial purification, a ß-gal-Pw enzyme preparation with a total ß-galactosidase activity of 3082 ± 98 µkatoNPGal was obtained from 1 L of recombinant K. phaffii culture (using AOX1 promoter). CONCLUSION: This study showed that the ß-gal-Pw was produced intracellularly by K. phaffii, but the secretion was not achieved with the signal peptides chosen. Nevertheless, a straightforward approach to improve the intracellular ß-gal-Pw production with K. phaffii by using either the GAP or AOX1 promoter in bioreactor cultivations was demonstrated, offering insights into alternative production methods for this enzyme.

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.

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.

A new ß-galactosidase from Paenibacillus wynnii with potential for industrial applications.

Commercial ß-galactosidases exhibit undesirable kinetic properties regarding substrate affinity (Michaelis-Menten constant [KM] for lactose) and product inhibition (inhibitor constant [Ki] for galactose). An in silico screening of gene sequences was done and identified a putative ß-galactosidase (Paenibacillus wynnii ß-galactosidase, BgaPw) from the psychrophilic bacterium Paenibacillus wynnii. The cultivation of the wild-type P. wynnii strain resulted in very low ß-galactosidase activities of a maximum of 150 nkat per liter of medium with o-nitrophenyl-ß-d-galactopyranoside (oNPGal) as substrate. The recombinant production of BgaPw in Escherichia coli BL21(DE3) increased the yield ∼9,000-fold. Here, a volumetric activity of 1,350.18 ± 11.82 µkatoNPGal/Lculture was achieved in a bioreactor cultivation. The partly purified BgaPw showed a pH optimum at 7.0, a temperature maximum at 40°C, and an excellent stability at 8°C with a half-life of 77 d. Kinetic studies with BgaPw were done in milk or in milk-imitating synthetic buffer (Novo buffer), respectively. Remarkably, the KM value of BgaPw with lactose was as low as 0.63 ± 0.045 mM in milk. It was found that the resulting products of lactose hydrolysis, namely galactose and glucose, did not inhibit the ß-galactosidase activity of BgaPw, but instead showed a striking activating effect in both cases (up to 144%). In a comparison study in milk, lactose was completely hydrolyzed by BgaPw in 72 h at 8°C, whereas 2 other known ß-galactosidases were less powerful and converted only about 90% of lactose in the same time. Finally, the formation of galactooligosaccharides (GOS) was demonstrated with the new BgaPw, starting with pharma-lactose (400 g/L). A GOS production of about 144 g/L was achieved after 24 h (36.0% yield).

Display of Bacterial Exochitanase on Bacillus subtilis Spores Improved Enzyme Stability and Recyclability.

Chitin is the second most prevalent polysaccharide found in nature, following cellulose. Amino-oligosaccharides, the byproducts of chitin degradation, exhibit favorable biological properties and potential for various uses. Chitinases play a crucial function in the breakdown of chitin, and their exceptionally effective production has garnered significant interest. Here, in this study, the exochitinase PbChiA, obtained from Paenibacillus barengoltzii, was recombinantly produced and immobilized using the CotG surface protein of Bacillus subtilis WB800N. The resulting strain Bacillus subtilis WB800N pHS-CotG-Chi exhibited exceptional heat stability and efficacy across various pH levels. The chitinolytic activity of the enzyme, which had been isolated and immobilized on the spore surface, was measured to be approximately 16.06 U/mL. Including Ni2+, Zn+2, and K+, and EDTA at various concentration levels in the reaction system, has significantly enhanced the activity of the immobilized enzyme. The immobilized exochitinase demonstrated a notable rate of recycling, as the recombinant spores sustained a relative enzyme activity of more than 70% after three cycles and 62.7% after four cycles. These findings established a basis for additional investigation into the role and practical use of the immobilized bacterial exochitinase in industry.

Production of a bacterial secretome highly efficient for the deconstruction of xylans.

Bacteria within the Paenibacillus genus are known to secrete a diverse array of enzymes capable of breaking down plant cell wall polysaccharides. We studied the extracellular xylanolytic activity of Paenibacillus xylanivorans and examined the complete range of secreted proteins when grown on carbohydrate-based carbon sources of increasing complexity, including wheat bran, sugar cane straw, beechwood xylan and sucrose, as control. Our data showed that the relative abundances of secreted proteins varied depending on the carbon source used. Extracellular enzymatic extracts from wheat bran (WB) or sugar cane straw (SCR) cultures had the highest xylanolytic activity, coincidently with the largest representation of carbohydrate active enzymes (CAZymes). Scaling-up to a benchtop bioreactor using WB resulted in a significant enhancement in productivity and in the overall volumetric extracellular xylanase activity, that was further concentrated by freeze-drying. The enzymatic extract was efficient in the deconstruction of xylans from different sources as well as sugar cane straw pretreated by alkali extrusion (SCRe), resulting in xylobiose and xylose, as primary products. The overall yield of xylose released from SCRe was improved by supplementing the enzymatic extract with a recombinant GH43 ß-xylosidase (EcXyl43) and a GH62 α-L-arabinofuranosidase (CsAbf62A), two activities that were under-represented. Overall, we showed that the extracellular enzymatic extract from P. xylanivorans, supplemented with specific enzymatic activities, is an effective approach for targeting xylan within lignocellulosic biomass.

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.

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.

Characterization of the ß-glucuronidase Pn3Pase as the founding member of glycoside hydrolase family GH169.

Paenibacillus sp. 32352 is a soil-dwelling bacterium capable of producing an enzyme, Pn3Pase that degrades the capsular polysaccharide of Streptococcus pneumoniae serotype 3 (Pn3P). Recent reports on Pn3Pase have demonstrated its initial characterization and potential for protection against highly virulent S. pneumoniae serotype 3 infections. Initial experiments revealed this enzyme functions as an exo-ß1,4-glucuronidase cleaving the ß(1,4) linkage between glucuronic acid and glucose. However, the catalytic mechanism of this enzyme is still unknown. Here, we report the detailed biochemical analysis of Pn3Pase. Pn3Pase shows no significant sequence similarity to known glycoside hydrolase (GH) families, thus this novel enzyme establishes a new carbohydrate-active enzyme (CAZy) GH family. Site-directed mutagenesis studies revealed two catalytic residues along with truncation mutants defining essential domains for function. Pn3Pase and its mutants were screened for activity, substrate binding and kinetics. Additionally, nuclear magnetic resonance spectroscopy analysis revealed that Pn3Pase acts through a retaining mechanism. This study exhibits Pn3Pase activity at the structural and mechanistic level to establish the new CAZy GH family GH169 belonging to the large GH-A clan. This study will also serve toward generating Pn3Pase derivatives with optimal activity and pharmacokinetics aiding in the use of Pn3Pase as a novel therapeutic approach against type 3 S. pneumoniae infections.

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.

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.

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.

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).

Characterization of an acidic pectin methylesterase from Paenibacillus xylanexedens and its application in fruit processing.

A pectinase-producing bacterial isolate, identified as Paenibacillus xylanexedens SZ 29, was screened by using the soil dilution plate with citrus pectin and congo red. A pectin methylesterase gene (Pxpme) was cloned and expressed in Escherichia coli. The gene coded for a protein with 334 amino acids and a calculated molecular mass of 36.76 kDa. PxPME showed the highest identity of 32.4% with the characterized carbohydrate esterase family 8 pectin methylesterase from Daucus carota. The recombined PxPME showed a specific activity with 39.38 U/mg against citrus pectin with >65% methylesterification. The optimal pH and temperature for PxPME activity were 5.0 and 45 °C. Its Km and Vmax value were determined to be 1.43 mg/mL and 71.5 µmol/mg·min, respectively. Moreover, PxPME could increase the firmness of pineapple cubes by 114% when combined with CaCl2. The acidic and mesophilic properties make PxPME a potential candidate for application in the fruit processing.

Purification and characterization of chitinase from Paenibacillus sp. .

The chitinase-producing bacteria Paenibacillus sp. was isolated from soil samples. The chitinase was purified successively by ammonia sulfate fractional precipitation followed by chromatography on DEAE 52-cellulose column and then on Sephadex G-75 column. The chitinase has a molecular weight of ca. 30 kDa as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis. Its optimum pH is 4.5, and its optimum temperature is 50 °C with colloidal chitin as a substrate. The enzyme is stable below 45 °C and in pH ranges between 4.5 and 5.5. It is activated by glucosamine, glucose, N-acetylglucosamine, and metal ions including Ca2+ , Fe2+ , Fe3+ , and Ni2+ . It is inhibited by SDS, H2 O2 , ascorbic acid, Cu2+ , Mg2+ , Ba2+ , Sn2+ , Cr3+ , and K+ . With colloidal chitin as substrate, the Km and the Vmax of the chitinase are 4.28 mg/mL and 14.29 µg/(Min·mL), respectively, whereas the end products of the enzymatic hydrolysis are 14.33% monomer and 85.67% dimer of N-acetylglucosamine. The viscosity of carboxymethyl chitin decreased rapidly at the initial stages when subjected to chitinase hydrolysis, which indicates that the chitinase acts in an endosplitting pattern.

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.

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.

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.