Functional Characterization of Endo- and Exo-Hydrolase Genes in Arabinan Degradation Gene Cluster of Bifidobacterium longum subsp. suis.

Bifidobacteria are probiotic microorganisms commonly found in the gastrointestinal tract, some of which are known to utilize linear arabino-oligosaccharides (AOS) as prebiotic carbohydrates. In general, the synergistic actions of exo-type α-l-arabinofuranosidases (ABFs) and endo-α-1,5-l-arabinanases (ABNs) are required for efficient arabinan degradation. In this study, the putative gene cluster for arabinan degradation was discovered in the genome of Bifidobacterium longum subsp. suis. It consists of a variety of genes encoding exo- and endo-hydrolases, sugar-binding proteins, ABC-binding cassettes, and transcriptional regulators. Among them, two endo-ABNs GH43 (BflsABN43A and BflsABN43B), two exo-ABFs GH43 (BflsABF43A and BflsABF43B), and an exo-ABF GH51 (BflsABF51) were predicted to be the key hydrolases for arabinan degradation. These hydrolase genes were functionally expressed in Escherichia coli, and their enzymatic properties were characterized. Their synergism in arabinan degradation has been proposed from the detailed modes of action. Extracellular endo-BflsABN43A hydrolyzes sugar beet and debranched arabinans into the short-chain branched and linear AOS. Intracellularly, AOS can be further degraded into l-arabinose via the cooperative actions of endo-BflsABN43B, exo-BflsABF43A with debranching activity, α-1,5-linkage-specific exo-BflsABF43B, and exo-BflsABF51 with dual activities. The resulting l-arabinose is expected to be metabolized into energy through the pentose phosphate pathway by three enzymes expressed from the ara operon of bifidobacteria. It is anticipated that uncovering arabinan utilization gene clusters and their detailed functions in the genomes of diverse microorganisms will facilitate the development of customized synbiotics.

Development of a Gene-Based Soybean-Origin Discrimination Method Using Allele-Specific Polymerase Chain Reaction.

A low soybean self-sufficiency rate in South Korea has caused a high import dependence and considerable price variation between domestic and foreign soybeans, causing the false labeling of foreign soybeans as domestic. Conventional soybean origin discrimination methods prevent a single-grain analysis and rely on the presence or absence of several compounds or concentration differences. This limits the origin discrimination of mixed samples, demonstrating the need for a method that analyzes individual grains. Therefore, we developed a method for origin discrimination using genetic analysis. The whole-genome sequencing data of the Williams 82 reference cultivar and 15 soybean varieties cultivated in South Korea were analyzed to identify the dense variation blocks (dVBs) with a high single-nucleotide polymorphism density. The PCR primers were prepared and validated for the insertion-deletion (InDel) sequences of the dVBs to discriminate each soybean variety. Our method effectively discriminated domestic and foreign soybean varieties, eliminating their false labeling.

Design of hypoxia responsive CRISPR-Cas9 for target gene regulation.

The CRISPR-Cas9 system is a widely used gene-editing tool, offering unprecedented opportunities for treating various diseases. Controlling Cas9/dCas9 activity at specific location and time to avoid undesirable effects is very important. Here, we report a conditionally active CRISPR-Cas9 system that regulates target gene expression upon sensing cellular environmental change. We conjugated the oxygen-sensing transcription activation domain (TAD) of hypoxia-inducing factor (HIF-1α) with the Cas9/dCas9 protein. The Cas9-TAD conjugate significantly increased endogenous target gene cleavage under hypoxic conditions compared with that under normoxic conditions, whereas the dCas9-TAD conjugate upregulated endogenous gene transcription. Furthermore, the conjugate system effectively downregulated the expression of SNAIL, an essential gene in cancer metastasis, and upregulated the expression of the tumour-related genes HNF4 and NEUROD1 under hypoxic conditions. Since hypoxia is closely associated with cancer, the hypoxia-dependent Cas9/dCas9 system is a novel addition to the molecular tool kit that functions in response to cellular signals and has potential application for gene therapeutics.

The Distinctive Permutated Domain Structure of Periplasmic α-Amylase (MalS) from Glycoside Hydrolase Family 13 Subfamily 19.

Periplasmic α-amylase MalS (EC. 3.2.1.1), which belongs to glycoside hydrolase (GH) family 13 subfamily 19, is an integral component of the maltose utilization pathway in Escherichia coli K12 and used among Ecnterobacteriaceae for the effective utilization of maltodextrin. We present the crystal structure of MalS from E. coli and reveal that it has unique structural features of circularly permutated domains and a possible CBM69. The conventional C-domain of amylase consists of amino acids 120-180 (N-terminal) and 646-676 (C-terminal) in MalS, and the whole domain architecture shows the complete circular permutation of C-A-B-A-C in domain order. Regarding substrate interaction, the enzyme has a 6-glucosyl unit pocket binding it to the non-reducing end of the cleavage site. Our study found that residues D385 and F367 play important roles in the preference of MalS for maltohexaose as an initial product. At the active site of MalS, ß-CD binds more weakly than the linear substrate, possibly due to the positioning of A402. MalS has two Ca2+ binding sites that contribute significantly to the thermostability of the enzyme. Intriguingly, the study found that MalS exhibits a high binding affinity for polysaccharides such as glycogen and amylopectin. The N domain, of which the electron density map was not observed, was predicted to be CBM69 by AlphaFold2 and might have a binding site for the polysaccharides. Structural analysis of MalS provides new insight into the structure-evolution relationship in GH13 subfamily 19 enzymes and a molecular basis for understanding the details of catalytic function and substrate binding of MalS.

Biological characterization of D-lactate dehydrogenase responsible for high-yield production of D-phenyllactic acid in Sporolactobacillus inulinus.

PLA (3-D-phenyllactic acid) is an ideal antimicrobial and immune regulatory compound present in honey and fermented foods. Sporolactobacillus inulinus is regarded as a potent D-PLA producer that reduces phenylpyruvate (PPA) with D-lactate dehydrogenases. In this study, PLA was produced by whole-cell bioconversion of S. inulinus ATCC 15538. Three genes encoding D-lactate dehydrogenase (d-ldh1, d-ldh2, and d-ldh3) were cloned and expressed in Escherichia coli BL21 (DE3), and their biochemical and structural properties were characterized. Consequently, a high concentration of pure D-PLA (47 mM) was produced with a high conversion yield of 88%. Among the three enzymes, D-LDH1 was responsible for the efficient conversion of PPA to PLA with kinetic parameters of Km (0.36 mM), kcat (481.10 s-1 ), and kcat /Km (1336.39 mM-1  s-1 ). In silico structural analysis and site-directed mutagenesis revealed that the Ile307 in D-LDH1 is a key residue for excellent PPA reduction with low steric hindrance at the substrate entrance. This study highlights that S. inulinus ATCC 15538 is an excellent PLA producer, equipped with a highly specific and efficient D-LDH1 enzyme.

Hydrolysis of Arabinoxylo-oligosaccharides by α-L-Arabinofuranosidases and ß-D-Xylosidase from Bifidobacterium dentium.

Two α-L-arabinofuranosidases (BfdABF1 and BfdABF3) and a ß-D-xylosidase (BfdXYL2) genes were cloned from Bifidobacterium dentium ATCC 27679, and functionally expressed in E. coli BL21(DE3). BfdABF1 showed the highest activity in 50 mM sodium acetate buffer at pH 5.0 and 25°C. This exo-enzyme could hydrolyze p-nitrophenyl arabinofuranoside, arabino-oligosaccharides (AOS), arabinoxylo-oligosaccharides (AXOS) such as 32-α-L-arabinofuranosyl-xylobiose (A3X), and 23-α-Larabinofuranosyl-xylotriose (A2XX), whereas hardly hydrolyzed polymeric substrates such as debranched arabinan and arabinoxylans. BfdABF1 is a typical exo-ABF with the higher specific activity on the oligomeric substrates than the polymers. It prefers to α-(1,2)-L-arabinofuranosidic linkages compared to α-(1,3)-linkages. Especially, BfdABF1 could slowly hydrolyze 23,33-di-α-L-arabinofuranosyl-xylotriose (A2+3XX). Meanwhile, BfdABF3 showed the highest activity in sodium acetate at pH 6.0 and 50°C, and it has the exclusively high activities on AXOS such as A3X and A2XX. BfdABF3 mainly catalyzes the removal of L-arabinose side chains from various AXOS. BfdXYL2 exhibited the highest activity in sodium citrate at pH 5.0 and 55°C, and it specifically hydrolyzed p-nitrophenyl xylopyranoside and xylo-oligosaccharides (XOS). Also, BfdXYL2 could slowly hydrolyze AOS and AXOS such as A3X. Based on the detailed hydrolytic modes of action of three exo-hydrolases (BfdABF1, BfdABF3, and BfdXYL2) from Bf. dentium, their probable roles in the hemiceulloseutilization system of Bf. dentium are proposed in the present study. These intracellular exo-hydrolases can synergistically produce L-arabinose and D-xylose from various AOS, XOS, and AXOS.

Arabinoxylo- and Arabino-Oligosaccharides-Specific α-L-Arabinofuranosidase GH51 Isozymes from the Amylolytic Yeast Saccharomycopsis fibuligera.

Two genes encoding probable α-L-arabinofuranosidase (E.C. 3.2.1.55) isozymes (ABFs) with 92.3% amino acid sequence identity, ABF51A and ABF51B, were found from chromosomes 3 and 5 of Saccharomycopsis fibuligera KJJ81, an amylolytic yeast isolated from Korean wheat-based nuruk, respectively. Each open reading frame consists of 1,551 nucleotides and encodes a protein of 517 amino acids with the molecular mass of approximately 59 kDa. These isozymes share approximately 49% amino acid sequence identity with eukaryotic ABFs from filamentous fungi. The corresponding genes were cloned, functionally expressed, and purified from Escherichia coli. SfABF51A and SfABF51B showed the highest activities on p-nitrophenyl arabinofuranoside at 40~45°C and pH 7.0 in sodium phosphate buffer and at 50°C and pH 6.0 in sodium acetate buffer, respectively. These exo-acting enzymes belonging to the glycoside hydrolase (GH) family 51 could hydrolyze arabinoxylo-oligosaccharides (AXOS) and arabino-oligosaccharides (AOS) to produce only L-arabinose, whereas they could hardly degrade any polymeric substrates including arabinans and arabinoxylans. The detailed product analyses revealed that both SfABF51 isozymes can catalyze the versatile hydrolysis of α-(1,2)-and α-(1,3)-L-arabinofuranosidic linkages of AXOS, and α-(1,2)-, α-(1,3)-, and α-(1,5)-linkages of linear and branched AOS. On the contrary, they have much lower activity against the α-(1,2)-and α-(1,3)-double-substituted substrates than the single-substituted ones. These hydrolases could potentially play important roles in the degradation and utilization of hemicellulosic biomass by S. fibuligera.

Functional Expression and Characterization of Acetyl Xylan Esterases CE Family 7 from Lactobacillus antri and Bacillus halodurans.

Acetyl xylan esterase (AXE; E.C. 3.1.1.72) is one of the accessory enzymes for xylan degradation, which can remove the terminal acetate residues from xylan polymers. In this study, two genes encoding putative AXEs (LaAXE and BhAXE) were cloned from Lactobacillus antri DSM 16041 and Bacillus halodurans C-125, and constitutively expressed in Escherichia coli. They possess considerable activities towards various substrates such as p-nitrophenyl acetate, 4-methylumbelliferyl acetate, glucose pentaacetate, and 7-amino cephalosporanic acid. LaAXE and BhAXE showed the highest activities at pH 7.0 and 8.0 at 50°C, respectively. These enzymes are AXE members of carbohydrate esterase (CE) family 7 with the cephalosporine-C deacetylase activity for the production of antibiotics precursors. The simultaneous treatment of LaAXE with Thermotoga neapolitana ß-xylanase showed 1.44-fold higher synergistic degradation of beechwood xylan than the single treatment of xylanase, whereas BhAXE showed no significant synergism. It was suggested that LaAXE can deacetylate beechwood xylan and enhance the successive accessibility of xylanase towards the resulting substrates. The novel LaAXE originated from a lactic acid bacterium will be utilized for the enzymatic production of D-xylose and xylooligosaccharides.

Crystal structure of the Csm5 subunit of the type III-A CRISPR-Cas system.

The Csm complex eliminates foreign RNA and DNA in the microbial defense CRISPR-Cas system. Csm5, one of the five subunits in the complex, facilitates crRNA maturation and target RNA binding in the type III system. However, the exact functional mechanism of Csm5 has remained elusive. Here, we report the crystal structure of the apo form of the Csm5 subunit at a resolution of 2.6 Å. Structural comparison of amino acids in the complex bound to RNA exhibits notable conformational changes in the crRNA and the target RNA binding sites. Shifts in the ß-hairpin motif (ß5-ß6), α13 helix (resides 352-383), and G-rich loop (residues 335-337) in the C-terminal domain indicate an induced movement by crRNA binding. The positively charged residues (Lys 92, Arg 95 and Lys 96) located in the ß-α4 loop of the target RNA interface show high conformational flexibility, while three-helix bundles (α1-α3) of the N-domain involved in Csm2 binding exhibit a rotational shift. The altered architecture of the Csm5 subunit demonstrates remarkable versatility of the ferredoxin-like fold in the RNA binding protein and provides a structural basis for the mechanism for crRNA and target RNA binding in the type III-A Crispr-Cas system.

Enzymatic modification of daidzin using heterologously expressed amylosucrase in Bacillus subtilis.

Amylosucrases (ASase, EC 2.4.1.4) from Deinococcus geothermalis (DGAS) and Neisseria polysaccharea (NPAS) were heterologously expressed in Bacillus subtilis. While DGAS was successfully expressed, NPAS was not. Instead, NPAS was expressed in Escherichia coli. Recombinant DGAS and NPAS were purified using nickel-charged affinity chromatography and employed to modify daidzin to enhance its water solubility and bioavailability. Analyses by LC/MS revealed that the major products of transglycosylation using DGAS were daidzein diglucoside and daidzein triglucoside, whereas that obtained by NPAS was only daidzein diglucoside. The optimal bioconversion conditions for daidzein triglucoside, which was predicted to have the highest water-solubility among the daidzin derivatives, was determined to be 4% (w/v) sucrose and 250 mg/L daidzin in sodium phosphate pH 7.0, with a reaction time of 12 h. Taken together, we suggest that the yield and product specificity of isoflavone daidzin transglycosylation may be modulated by the source of ASase and reaction conditions.

Detailed Mode of Action of Arabinan-Debranching α-L-Arabinofuranosidase GH51 from Bacillus velezensis.

The gene encoding an α-L-arabinofuranosidase (BvAF) GH51 from Bacillus velezensis FZB42 was cloned and expressed in Escherichia coli. The corresponding open reading frame consists of 1,491 nucleotides which encode 496 amino acids with the molecular mass of 56.9 kDa. BvAF showed the highest activity against sugar beet (branched) arabinan in 50 mM sodium acetate buffer (pH 6.0) at 45°C. However, it could hardly hydrolyze debranched arabinan and arabinoxylans. The time-course hydrolyses of branched arabinan and arabinooligosaccharides (AOS) revealed that BvAF is a unique exo-hydrolase producing exclusively L-arabinose. BvAF could cleave α-(1,2)- and/or α-(1,3)-L-arabinofuranosidic linkages of the branched substrates to produce the debranched forms of arabinan and AOS. Although the excessive amount of BvAF could liberate L-arabinose from linear AOS, it was extremely lower than that on branched AOS. In conclusion, BvAF is the arabinan-specific exo-acting α-L-arabinofuranosidase possessing high debranching activity towards α-(1,2)- and/or α-(1,3)-linked branches of arabinan, which can facilitate the successive degradation of arabinan by endo-α-(1,5)-L-arabinanase.

Functional expression and enzymatic characterization of Lactobacillus plantarum cyclomaltodextrinase catalyzing novel acarbose hydrolysis.

Cyclomaltodextrinases (CDases) belong to Glycoside Hydrolases (GH) family 13, which show versatile hydrolyzing and/or transglycosylation activity against cyclodextrin (CD), starch, and pullulan. Especially, some CDases have been reported to hydrolyze acarbose, a potent α-glucosidase inhibitor, and transfer the resulting acarviosine-glucose to various acceptors. In this study, a novel CDase (LPCD) gene was cloned from Lactobacillus plantarum WCFS1, which encodes 574 amino acids (64.6 kDa) and shares less than 44% of identities with the known CDase-family enzymes. Recombinant LPCD with C-terminal six-histidines was produced and purified from Escherichia coli. It showed the highest activity on ß-CD at 45°C and pH 5.0, respectively. Gel permeation chromatography analysis revealed that LPCD exists as a dodecameric form (~826 kDa). Its hydrolyzing activity on ß- CD is almost same as that on starch, whereas it can hardly attack pullulan. Most interestingly, LPCD catalyzed the unique modes of action in acarbose hydrolysis to produce maltose and acarviosine, as well as to glucose and acarviosineglucose.

One-pot synthesis of GDP-l-fucose by a four-enzyme cascade expressed in Lactococcus lactis.

GDP-l-fucose is an l-fucose donor to synthesize fucosylated compounds such as human milk oligosaccharides or Lewis antigen. In this study, we used Lactococcus lactis subsp. cremoris NZ9000 to express 4 enzymes, ManB, ManC, Gmd, and WcaG and produced GDP-l-fucose by using one-pot synthesis method with mannose-6-phosphate as substrate and the enzymes as biocatalyst. For preparation of enzyme mixture, 4 genes (manB, manC, gmd, and wcaG) cloned from Escherichia coli were transformed into L. lactis strains using pNZ8008 and the recombinant cell lysates were obtained after cultivation. When mannose-6-phosphate was used as the substrate, the consecutive reactions with ManB, ManC, Gmd, and WcaG resulted in the successful production of GDP-l-fucose (0.13mM). When GDP-d-mannose was used as the substrate, it was entirely converted to GDP-l-fucose (0.2mM; 0.12g/L) via 2 enzymatic reactions mediated by Gmd and WcaG. This is the first report of GDP-l-fucose production by using multiple enzymes expressed in lactic acid bacteria.

Elimination of the cryptic plasmid in Leuconostoc citreum by CRISPR/Cas9 system.

Leuconostoc spp. are important lactic acid bacteria for the fermentation of foods. In particular, L. citreum strains isolated from various foods have been used as host strains for genetic and metabolic engineering studies. In order to develop a food-grade genetic engineering system, L. citreum CB2567 was isolated from Kimchi. However, the isolated bacterium contained a cryptic plasmid which was difficult to eliminate. As the existence of the plasmid might hinder strain engineering, we eliminated the plasmid using an RNA-guided DNA endonuclease CRISPR/Cas9 system. We demonstrated that a plasmid-free L. citreum CB2567 host strain could be efficiently constructed through a two-step procedure: 1) transformation of the "killer" plasmid expressing Cas9 endonuclease and a guide RNA (gRNA) targeting for a specific sequence in the cryptic plasmid, and 2) serial subculture without antibiotics for curing the killer plasmid. When the crude extract of L. citreum expressing Cas9 and the guide RNA was incubated with a PCR fragment containing the specific sequence recognized by the guide RNA, the PCR fragment was cleaved. Also, the cryptic plasmid pCB42 was successfully eliminated from the host strain after transforming the plasmid harboring Cas9 and the guide RNA. The Cas9 and gRNA expression plasmid used in this study can be applied for genome engineering purposes by additionally introducing an editing DNA template to repair the double strand DNA breakage caused by Cas9 in the genome of L. citreum. This study demonstrates the feasibility of developing CRISPR/Cas9-based genetic engineering tools to develop a safe host strain and construct food-grade lactic acid bacteria without residual antibiotic markers.

Heterologous expression and enzymatic characterization of γ-glutamyltranspeptidase from Bacillus amyloliquefaciens.

γ-Glutamyltranspeptidase (GGT) catalyzes the cleavage of γ-glutamyl compounds and the transfer of γ-glutamyl moiety to water or to amino acid/peptide acceptors. GGT can be utilized for the generation of γ-glutamyl peptides or glutamic acid, which are used as food taste enhancers. In the present study, Bacillus amyloliquefaciens SMB469 with high GGT activity was isolated from Doenjang, a traditional fermented soy food of Korea. The gene encoding GGT from B. amyloliquefaciens SMB469 (BaGGT469) was cloned from the isolate, and heterologously expressed in E. coli and B. subtilis. For comparison, three additional GGT genes were cloned from B. subtilis 168, B. licheniformis DSM 13, and B. amyloliquefaciens FZB42. The BaGGT469 protein was composed of 591 amino acids. The final protein comprises two separate polypeptide chains of 45.7 and 19.7 kDa, generated via autocatalytic cleavage. The specific activity of BaGGT469 was determined to be 17.8 U/mg with γ-L-glutamyl-p-nitroanilide as the substrate and diglycine as the acceptor. GGTs from B. amyloliquefaciens showed 1.4- and 1.7-fold higher transpeptidase activities than those from B. subtilis and B. licheniformis, respectively. Especially, recombinant B. subtilis expressing BaGGT469 demonstrated 11- and 23-fold higher GGT activity than recombinant E. coli and the native B. amyloliquefaciens, respectively, did. These results suggest that BaGGT469 can be utilized for the enzymatic production of various γ-glutamyl compounds.

Physicochemical and in vitro binding properties of barley ß-glucan treated with hydrogen peroxide.

This study investigated the changes in content, purity, physical properties, and in vitro binding properties of barley ß-glucan by oxidation treatment. Barleys (Hordeum vulgare) were oxidized, using different concentrations of hydrogen peroxide (0.2-1.0% H2O2). The total and soluble ß-glucan contents ranged from 8.41% and 4.81% in the control to 9.48% and 6.45% in the 0.6% H2O2 treatment. With increasing H2O2 concentration, the purity of ß-glucan increased from 35% to 70%, whereas molecular weight (MW), viscosity, and water-binding capacities decreased to 2.0 × 10(4)Da, 3.9 cP, and 4.45 g water/g ß-glucan, respectively. Oil binding capacities ranged from 8.29 g of oil/g in non-oxidized ß-glucan to 9.42 g of oil/g in ß-glucan oxidized with 0.6% H2O2. The MW, viscosity, and binding capacities of waxy barley ß-glucan were higher than those of non-waxy barley ß-glucan. Oxidation by hydrogen peroxide improved the physical properties and in vitro binding capacity of barley ß-glucan.

In vitro digestion and fermentation properties of linear sugar-beet arabinan and its oligosaccharides.

This study was conducted to investigate the prebiotic effects of linear arabino-oligosaccharides (LAOS) and debranched (linear) sugar beet arabinan (LAR) for the development of new prebiotics. LAOS were prepared from LAR by enzymatic hydrolysis with endo-arabinanase from Bacillus licheniformis, followed by removal of the arabinose fraction by incubation with resting cells of Leuconostoc mesenteroides. The resulting LAOS contained DP2 (28.7%), DP3 (49.9%), DP4 (20.1%), and DP5 (1.16%). A standardized digestibility test showed that LAOS and LAR were not digestible. Individual cultures of 24 strains of gastrointestinal bacteria showed that LAOS and LAR stimulated growth of Lactobacillus brevis, Bifidobacterium longum, and Bacteroides fragilis. In vitro batch fermentation using human fecal samples showed that LAOS had higher bifidogenic properties than LAR; LAOS increased the population of bifidobacteria which produced short-chain fatty acids (SCFAs). LAOS was fermented slowly compared to fructo-oligosaccharides and this may permit SCFA production in the distal colon. This study demonstrates that LAOS prepared from LAR are promising dietary substrates for improvement of human intestinal health.

Physicochemical Properties of ß-Glucan from Acid Hydrolyzed Barley.

This study was performed to investigate changes in the content and purity, as well as physical characteristics of ß-glucan extracted from acid hydrolyzed whole grain barleys. Waxy and non-waxy barleys (Hordeum vulgare) were hydrolyzed with different concentrations of HCl (0.1~0.5 N) for 1 h. As the HCl concentration increased, the contents of total and soluble ß-glucan from acid hydrolyzed barley decreased. However the ratio of soluble/total ß-glucan content and purities of ß-glucan significantly increased. The ratio of ß-(1→4)/ß-(1→3) linkages, molecular weight, and viscosity of soluble ß-glucan of raw barleys were 2.28~2.52, 6.0~7.0×10(5) g/mol, and 12.8~32.8 centipoise (cP). Those of isolated soluble ß-glucan were significantly decreased to 2.05~2.15, 6.6~7.8×10(3) g/mol, and 3.6~4.2 cP, respectively, with increasing acid concentration. The re-solubility of raw barley ß-glucan was about 50%, but increased to 97% with increasing acid concentration. Acid hydrolysis was shown to be an effective method to produce ß-glucan with high ratio of soluble ß-glucan content, purity, water solubility, and low viscosity.

Isolation of dextran-hydrolyzing intestinal bacteria and characterization of their dextranolytic activities.

The aim of this study was to isolate dextran-hydrolyzing bacteria from the human intestines and to identify their dextranolytic enzymes. For this, dextranase-producing microorganisms were screened from fecal samples by using blue dextran-containing media. Colonies producing a decolorized zone were isolated and they were grouped using RAPD-PCR. 16S rRNA gene sequencing analysis revealed the isolates were Bacteroides (B.) thetaiotaomicron, B. ovatus, B. vulgatus, B. dorei, B. xylanisolvens, B. uniformis, and Veillonella (V.) rogosae. Thin layer chromatography analysis showed that the dextranases exhibit mainly endo-type activity and produce various oligosaccharides including isomaltose and isomaltotriose. Zymogram analysis demonstrated that enzymes localized mainly in the cell membrane fraction and the molecular weight was 50-70 kDa. When cultured in a dextran-containing medium, all strains isolated in this study produced short-chain fatty acids, with butyric acid as the major compound. This is the first study to report that human intestinal B. xylanisolvens, B. dorei, and V. rogosae metabolize dextran utilizing dextranolytic enzymes.

Synergistic action modes of arabinan degradation by exo- and endo-arabinosyl hydrolases.

Two recombinant arabinosyl hydrolases, α-L-arabinofuranosidase from Geobacillus sp. KCTC 3012 (GAFase) and endo-(1,5)-α-L-arabinanase from Bacillus licheniformis DSM13 (BlABNase), were overexpressed in Escherichia coli, and their synergistic modes of action against sugar beet (branched) arabinan were investigated. Whereas GAFase hydrolyzed 35.9% of L-arabinose residues from sugar beet (branched) arabinan, endo-action of BlABNase released only 0.5% of L-arabinose owing to its extremely low accessibility towards branched arabinan. Interestingly, the simultaneous treatment of GAFase and BlABNase could liberate approximately 91.2% of L-arabinose from arabinan, which was significantly higher than any single exo-enzyme treatment (35.9%) or even stepwise exo- after endo-enzyme treatment (75.5%). Based on their unique modes of action, both exo- and endo-arabinosyl hydrolases can work in concert to catalyze the hydrolysis of arabinan to L-arabinose. At the early stage in arabinan degradation, exo-acting GAFase could remove the terminal arabinose branches to generate debranched arabinan, which could be successively hydrolyzed into arabinooligosaccharides via the endoaction of BlABNase. At the final stage, the simultaneous actions of exo- and endo-hydrolases could synergistically accelerate the L-arabinose production with high conversion yield.