Catalytic and molecular properties of alkaliphilic and thermotolerant ß-etherase from Altererythrobacter sp. B11.

Phenylpropanone monomers, including guaiacyl hydroxypropanone, are important precursors for the synthesis of various chemicals. The monomers are obtained in a three-step cascade reaction catalyzed by a group of enzymes in the ß-etherase system that cleaves the ß-O-4 bond, the major bond in lignin. In this study, one of the ß-etherase of the glutathione-S-transferase superfamily, AbLigF2, was discovered in genus Altererythrobacter, and the recombinant etherase was characterized. The enzyme showed maximal activity at 45 °C, maintained 30% of its activity after 2 h at 50 °C, and was the most thermostable among the previously reported enzymes. Moreover, N13, S14, and S115, located near the thiol group of glutathione, had a significant effect on the maximum reaction rate of enzyme activity. This study suggests that AbLigF2 has the potential to serve as a thermostable enzyme for lignin utilization and provides insights into its catalytic mechanism.

Degradation of ester linkages in rice straw components by Sphingobium species recovered from the sea bottom using a non-secretory tannase-family α/ß hydrolase.

Microbial decomposition of allochthonous plant components imported into the aquatic environment is one of the vital steps of the carbon cycle on earth. To expand the knowledge of the biodegradation of complex plant materials in aquatic environments, we recovered a sunken wood from the bottom of Otsuchi Bay, situated in northeastern Japan in 2012. We isolated Sphingobium with high ferulic acid esterase activity. The strain, designated as OW59, grew on various aromatic compounds and sugars, occurring naturally in terrestrial plants. A genomic study of the strain suggested its role in degrading hemicelluloses. We identified a gene encoding a non-secretory tannase-family α/ß hydrolase, which exhibited ferulic acid esterase activity. This enzyme shares the consensus catalytic triad (Ser-His-Asp) within the tannase family block X in the ESTHER database. The molecules, which had the same calculated elemental compositions, were produced consistently in both the enzymatic and microbial degradation of rice straw crude extracts. The non-secretory tannase-family α/ß hydrolase activity may confer an important phenotypic feature on the strain to accelerate plant biomass degradation. Our study provides insights into the underlying biodegradation process of terrestrial plant polymers in aquatic environments.

Effect of short-term treatment with sitagliptin or glibenclamide on daily glucose fluctuation in drug-naïve Japanese patients with type 2 diabetes mellitus.

AIMS: To compare the effect of a dipeptidyl peptidase-4 inhibitor (DPP4-i) and a sulfonylurea (SU) on daily glucose fluctuation in drug-naïve Japanese patients with type 2 diabetes mellitus (T2DM). MATERIALS AND METHODS: A total of 53 drug-naïve Japanese patients with T2DM (HbA1c, 7.0%-9.0%; fasting plasma glucose, 6.1 mmol/L or higher) were randomly assigned to either sitagliptin 50 mg qd or glibenclamide 2.5 mg per day (given in divided doses) in a 1:1 ratio. A continuous glucose monitoring (CGM) device was used to obtain 24-hour glucose profiles for each patient at baseline and at Week 2. The primary study endpoint was change from baseline in mean amplitude of glucose excursion (MAGE) during a 24-hour period. A key secondary endpoint was change from baseline in the standard deviation (SD) of 24-hour glucose levels. RESULTS: After 2 weeks of treatment, a numerically greater reduction in MAGE from baseline was observed in the sitagliptin group compared with the glibenclamide group, but the between-treatment difference was not statistically significant (LS mean difference [95% CI]: -0.48 mmol/L [-1.31, 0.34]; P = .245). However, a significantly greater reduction in the change from baseline in SD was observed in the sitagliptin group compared with the glibenclamide group (LS mean difference [95% CI]: -0.33 mmol/L [-0.62, -0.03]; P = .029). CONCLUSIONS: This study suggests that the DPP4 inhibitor sitagliptin has a greater ability to reduce daily glucose fluctuation than the SU glibenclamide in drug-naïve Japanese patients with T2DM. ClinicalTrials.gov: NCT02318693.

Involvement of flocculin in negative potential-applied ITO electrode adhesion of yeast cells.

The purpose of this study was to develop novel methods for attachment and cultivation of specifically positioned single yeast cells on a microelectrode surface with the application of a weak electrical potential. Saccharomyces cerevisiae diploid strains attached to an indium tin oxide/glass (ITO) electrode to which a negative potential between -0.2 and -0.4 V vs. Ag/AgCl was applied, while they did not adhere to a gallium-doped zinc oxide/glass electrode surface. The yeast cells attached to the negative potential-applied ITO electrodes showed normal cell proliferation. We found that the flocculin FLO10 gene-disrupted diploid BY4743 mutant strain (flo10Δ /flo10Δ) almost completely lost the ability to adhere to the negative potential-applied ITO electrode. Our results indicate that the mechanisms of diploid BY4743 S. cerevisiae adhesion involve interaction between the negative potential-applied ITO electrode and the Flo10 protein on the cell wall surface. A combination of micropatterning techniques of living single yeast cell on the ITO electrode and omics technologies holds potential of novel, highly parallelized, microchip-based single-cell analysis that will contribute to new screening concepts and applications.

Crystal structure of the catalytic domain of a GH16 ß-agarase from a deep-sea bacterium, Microbulbifer thermotolerans JAMB-A94.

A deep-sea bacterium, Microbulbifer thermotolerans JAMB-A94, has a ß-agarase (MtAgaA) belonging to the glycoside hydrolase family (GH) 16. The optimal temperature of this bacterium for growth is 43-49 °C, and MtAgaA is stable at 60 °C, which is one of the most thermostable enzymes among GH16 ß-agarases. Here, we determined the catalytic domain structure of MtAgaA. MtAgaA consists of a ß-jelly roll fold, as observed in other GH16 enzymes. The structure of MtAgaA was most similar to two ß-agarases from Zobellia galactanivorans, ZgAgaA, and ZgAgaB. Although the catalytic cleft structure of MtAgaA was similar to ZgAgaA and ZgAgaB, residues at subsite -4 of MtAgaA were not conserved between them. Also, an α-helix, designated as α4', was uniquely located near the catalytic cleft of MtAgaA. A comparison of the structures of the three enzymes suggested that multiple factors, including increased numbers of arginine and proline residues, could contribute to the thermostability of MtAgaA.

Thalassospira alkalitolerans sp. nov. and Thalassospira mesophila sp. nov., isolated from a decaying bamboo sunken in the marine environment, and emended description of the genus Thalassospira.

Two marine bacteria, designated strains MBE#61(T) and MBE#74(T), were isolated from a piece of sunken bamboo in the marine environment in Japan. Both of these strains were Gram-stain-negative, but had different cell shapes: MBE#61(T) was spiral, whereas MBE#74(T) was rod-shaped. The temperature, pH and salt concentration ranges for growth of strain MBE#61(T) were 4-38 °C (optimal at 32 °C), pH 4.5-11.0 (optimal at pH 7.0-8.0) and 1-11 % (optimal at 2 %) NaCl, whereas those of strain MBE#74(T) were 4-36 °C (optimal at 30 °C), pH 4.0-10.5 (optimal at pH 7.0-8.0) and 1-12 % (optimal at 4 %) NaCl. Phylogenetic analysis based on partial 16S rRNA gene sequences revealed that both strains belong to the genus Thalassospira within the class Alphaproteobacteria. Similarity between the 16S rRNA gene sequence of strain MBE#61(T) and those of the type strains of species of the genus Thalassospira was 97.5-99.0 %, and that of strain MBE#74(T) was 96.9-98.6 %; these two isolates were most closely related to Thalassospira lucentensis QMT2(T). However, the DNA-DNA hybridization values between T. lucentensis QMT2(T) and strain MBE#61(T) or MBE#74(T) were only 16.0 % and 7.1 %, respectively. The DNA G+C content of strain MBE#61(T) was 54.4 mol%, and that of strain MBE#74(T) was 55.9 mol%. The predominant isoprenoid quinone of the two strains was Q-10 (MBE#61(T), 97.3 %; MBE#74(T), 93.5 %). The major cellular fatty acids of strain MBE#61(T) were C18 : 1ω7c (31.1 %), summed feature 3 comprising C16 : 0ω7c/iso-C15 : 0 2-OH (26.1 %) and C16 : 0 (20.9 %); those of strain MBE#74(T) were C16 : 0 (26.2 %), C17 : 0 cyclo (19.9 %) and C18 : 1ω7c (12.1 %). On the basis of these results, strain MBE#61(T) and strain MBE#74(T) are considered to represent novel species of the genus Thalassospira, for which names Thalassospira alkalitolerans sp. nov. and Thalassospira mesophila sp. nov. are proposed. The type strains are MBE#61(T) ( = JCM 18968(T) = CECT 8273(T)) and MBE#74(T) ( = JCM 18969(T) = CECT 8274(T)), respectively. An emended description of the genus Thalassospira is also proposed.

Enhancing thermostability and the structural characterization of Microbacterium saccharophilum K-1 ß-fructofuranosidase.

A ß-fructofuranosidase from Microbacterium saccharophilum K-1 (formerly known as Arthrobacter sp. K-1) is useful for producing the sweetener lactosucrose (4(G)-ß-D-galactosylsucrose). Thermostability of the ß-fructofuranosidase was enhanced by random mutagenesis and saturation mutagenesis. Clones with enhanced thermostability included mutations at residues Thr47, Ser200, Phe447, Phe470, and Pro500. In the highest stability mutant, T47S/S200T/F447P/F470Y/P500S, the half-life at 60 °C was 182 min, 16.5-fold longer than the wild-type enzyme. A comparison of the crystal structures of the full-length wild-type enzyme and three mutants showed that various mechanisms appear to be involved in thermostability enhancement. In particular, the replacement of Phe447 with Val or Pro induced a conformational change in an adjacent residue His477, which results in the formation of a new hydrogen bond in the enzyme. Although the thermostabilization mechanisms of the five residue mutations were explicable on the basis of the crystal structures, it appears to be difficult to predict which amino acid residues should be modified to obtain thermostabilized enzymes.

The etherase system of Novosphingobium sp. MBES04 functions as a sensor of lignin fragments through phenylpropanone production to induce specific transcriptional responses.

The MBES04 strain of Novosphingobium accumulates phenylpropanone monomers as end-products of the etherase system, which specifically and reductively cleaves the ß-O-4 ether bond (a major bond in lignin molecules). However, it does not utilise phenylpropanone monomers as an energy source. Here, we studied the response to the lignin-related perturbation to clarify the physiological significance of its etherase system. Transcriptome analysis revealed two gene clusters, each consisting of four tandemly linked genes, specifically induced by a lignin preparation extracted from hardwood (Eucalyptus globulus) and a ß-O-4-type lignin model biaryl compound, but not by vanillin. The most strongly induced gene was a 2,4'-dihydroxyacetophenone dioxygenase-like protein, which leads to energy production through oxidative degradation. The other cluster was related to multidrug resistance. The former cluster was transcriptionally regulated by a common promoter, where a phenylpropanone monomer acted as one of the effectors responsible for gene induction. These results indicate that the physiological significance of the etherase system of the strain lies in its function as a sensor for lignin fragments. This may be a survival strategy to detect nutrients and gain tolerance to recalcitrant toxic compounds, while the strain preferentially utilises easily degradable aromatic compounds with lower energy demands for catabolism.

Microbacterium saccharophilum sp. nov., isolated from a sucrose-refining factory.

A Gram-stain-positive, non-motile, non-spore-forming, rod-shaped bacterium, designated strain K-1(T), was isolated from soil at a sucrose refinery in Japan. The strain grew at 9-37 °C (optimum, 30 °C) and at pH 6-11 (optimum, pH 7.0). Phylogenetic analysis based on the full-length 16S rRNA gene sequence of strain K-1(T) revealed that it was a member of the genus Microbacterium. High 16S rRNA gene sequence similarities were found between strains K-1(T) and both Microbacterium pumilum NBRC 101279(T) (99.7 %) and Microbacterium deminutum NRRL B-24453(T) (99.5 %). However, the DNA-DNA hybridization values between strain K-1(T) and M. pumilum NBRC 101279(T) and M. deminutum NRRL B-24453(T) were only 12 % and 10 %, respectively. The DNA G+C content of strain K-1(T) was 73 mol%. The major fatty acids of strain K-1(T) were anteiso-C15 : 0 and anteiso-C17 : 0, and the major menaquinones were MK-12 and MK-13. The diamino acid in the cell-wall peptidoglycan was lysine. On the basis of these results, strain K-1(T) is considered to represent a novel species of the genus Microbacterium, for which the name Microbacterium saccharophilum sp. nov. is proposed. The type strain is K-1(T) (= NBRC 108778(T) = NCIMB 14782(T)).

Salinisphaera japonica sp. nov., a moderately halophilic bacterium isolated from the surface of a deep-sea fish, Malacocottus gibber, and emended description of the genus Salinisphaera.

A moderately halophilic, slightly acidophilic, aerobic bacterium, designated strain YTM-1(T), was isolated from the body surface of Malacocottus gibber. Cells were Gram-stain-negative, short rods or cocci, approximately 0.9-1.1 µm long and 1.0-1.8 µm wide. Strain YTM-1(T) was able to grow with 1-30% NaCl (optimum, 7.5-10%, w/v), at 4-30 °C (optimum, 20-25 °C) and at pH 3.8-9.5 (optimum, pH 5.0-5.5). Phylogenetic analysis based on 16S rRNA gene sequence similarities showed that strain YTM-1(T) belonged to the genus Salinisphaera with low similarity values to the type strains of recognized species of this genus (<94.8-94.4%). The polar lipids of strain YTM-1(T) consisted of diphosphatidylglycerol, phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine, three unknown phospholipids and one unknown lipid. The predominant isoprenoid quinone was Q-8. The major fatty acids were C19:0ω8c cyclo, C18:1ω7c, C16:1ω5c and C16:0. The DNA G+C content of strain YTM-1(T) was 67.3 mol%. These phylogenetic, physiological and chemotaxonomic data indicated that strain YTM-1(T) represents a novel species of the genus Salinisphaera, for which the name Salinisphaera japonica sp. nov. is proposed. The type strain is YTM-1(T) (=JCM 18087(T)=CECT 8012(T)). An emended description of the genus Salinisphaera is also proposed.

Thermophilic and halophilic ß-agarase from a halophilic archaeon Halococcus sp. 197A.

An agar-degrading archaeon Halococcus sp. 197A was isolated from a solar salt sample. The agarase was purified by hydrophobic column chromatography using a column of TOYOPEARL Phenyl-650 M. The molecular mass of the purified enzyme, designated as Aga-HC, was ~55 kDa on both SDS-PAGE and gel-filtration chromatography. Aga-HC released degradation products in the order of neoagarohexose, neoagarotetraose and small quantity of neoagarobiose, indicating that Aga-HC was a ß-type agarase. Aga-HC showed a salt requirement for both stability and activity, being active from 0.3 M NaCl, with maximal activity at 3.5 M NaCl. KCl supported similar activities as NaCl up to 3.5 M, and LiCl up to 2.5 M. These monovalent salts could not be substituted by 3.5 M divalent cations, CaCl2 or MgCl2. The optimal pH was 6.0. Aga-HC was thermophilic, with optimum temperature of 70 °C. Aga-HC retained approximately 90 % of the initial activity after incubation for 1 hour at 65-80 °C, and retained 50 % activity after 1 hour at 95 °C. In the presence of additional 10 mM CaCl2, approximately 17 % remaining activity was detected after 30 min at 100 °C. This is the first report on agarase purified from Archaea.

Unique substrate specificity of a thermostable glycosyl hydrolase from an uncultured Anaerolinea, derived from bacterial mat on a subsurface geothermal water stream.

To investigate novel extremozymes encoded by sequenced metagenes from a microbial community in an extreme environment, we have characterized a recombinant glycosyl hydrolase (rGH) from an uncultured bacterium within the order Chloroflexi. rGH formed insoluble bodies in an Escherichia coli protein expression system. The protein was partially dissolved by a surfactant and was enzymatically characterized. The MW of the monomeric peptide was ~62 kDa, and it formed a homodimers in buffer. It was optimally active at 65 °C and from pH 4 to 8. rGH showed hydrolytic activity for α-1,1, α-1,2 and α-1,6 linkages, including isomaltose, but not α-1,4 and ß-linkages.

Salarchaeum japonicum gen. nov., sp. nov., an aerobic, extremely halophilic member of the Archaea isolated from commercial salt.

Strain YSM-79(T) was isolated from commercial salt made from seawater in Yonaguni island, Okinawa, Japan. The strain is an aerobic, Gram-negative, chemo-organotrophic and extremely halophilic archaeon. Cells are short rods that lyse in distilled water. Growth occurs at 1.5-5.3 M NaCl (optimum 2.5-3.0 M), pH 5.0-8.8 (optimum pH 5.2-6.3) and 20-55 °C (optimum 40 °C). Mg²âº is required for growth, with maximum growth at 200-300 mM Mg²âº. Polar lipid analysis revealed the presence of phosphatidylglycerol, phosphatidylglycerophosphate methyl ester, sulfated diglycosyl diether-1 and five unidentified glycolipids. The G+C content of the DNA was 64 mol%. On the basis of 16S rRNA gene sequence analysis, strain YSM-79(T) was determined to be a member of the family Halobacteriaceae, with the closest related genus being Halobacterium (94 % sequence identity). In addition, the rpoB' gene sequence of strain YSM-79(T) had <88 % sequence similarity to those of other members of the family Halobacteriaceae. The results of phenotypic, chemotaxonomic and phylogenetic analysis suggested that strain YSM-79(T) should be placed in a new genus, Salarchaeum gen. nov., as a representative of Salarchaeum japonicum sp. nov. The type strain is YSM-79(T) ( = JCM 16327(T)  = CECT 7563(T)).

Biochemical synthesis of novel, self-assembling glycolipids from ricinoleic acid by a recombinant α-glucosidase from Geobacillus sp.

PURPOSE OF WORK: To explore a novel glycolipid, we performed biochemical reactions using a recombinant α-glucosidase from Geobacillus sp. which shows excellent transglycosylation reaction to hydroxyl groups in a variety of compounds. Two different glycolipids (GL-1 and GL-2) were prepared from ricinoleic acid using a recombinant α-glucosidase from Geobacillus sp. The molecular structure of GL-1 was confirmed as 12-O-α-D-glucopyranosyl-9-hexadecenoic acid by 1D and 2D NMR analyses. According to MALDI-TOF/MS, GL-1 and GL-2 showed single major peaks at m/z 483.82 and 645.97, respectively. The peaks corresponded to the [M + Na](+) ions of the glycolipids. GL-2 was estimated as 12-O-α-D-glucopyranosyl-(4'-O-α-glucopyranosyl)-9-hexadecenoic acid. Light polarization microscopy revealed that GL-2 easily formed self-assembled vesicles in aqueous solution.

Natronoarchaeum mannanilyticum gen. nov., sp. nov., an aerobic, extremely halophilic archaeon isolated from commercial salt.

Strain YSM-123(T) was isolated from commercial salt made from Japanese seawater in Niigata prefecture. Optimal NaCl and Mg(2+) concentrations for growth were 4.0-4.5 M and 5 mM, respectively. The isolate was a mesophilic and slightly alkaliphilic haloarchaeon, whose optimal growth temperature and pH were 37 °C and pH 8.0-9.0. Phylogenetic analysis based on 16S rRNA gene sequence analysis suggested that strain YSM-123(T) is a member of the phylogenetic group defined by the family Halobacteriaceae, but there were low similarities to type strains of other genera of this family (≤90 %); for example, Halococcus (similarity <89 %), Halostagnicola (<89 %), Natronolimnobius (<89 %), Halobiforma (<90 %), Haloterrigena (<90 %), Halovivax (<90 %), Natrialba (<90 %), Natronobacterium (<90 %) and Natronococcus (<90 %). The G+C content of the DNA was 63 mol%. Polar lipid analysis revealed the presence of phosphatidylglycerol, phosphatidylglycerophosphate methyl ester, disulfated diglycosyl diether and an unknown glycolipid. On the basis of the data presented, we propose that strain YSM-123(T) should be placed in a new genus and species, Natronoarchaeum mannanilyticum gen. nov., sp. nov. The type strain of Natronoarchaeum mannanilyticum is strain YSM-123(T) (=JCM 16328(T) =CECT 7565(T)).

Isolation and evaluation of the radical-scavenging activity of the antioxidants in the leaves of an edible plant, Mallotus japonicus.

The antioxidative properties of a hot water extract of the leaves of Mallotus japonicus were evaluated. The extract had a high phenolic content and strong antioxidative activity, compared with green tea, rooibos tea, and red wine. Six phenolic compounds were isolated as antioxidative components by HPLC. They were identified as mallotinic acid, mallotusinic acid, corilagin, geraniin, rutin, and ellagic acid. These antioxidative compounds were subjected to DPPH radical-scavenging, superoxide radical-scavenging, and hydroxyl radical-scavenging assays, and compared with other antioxidative compounds. Four of the compounds, mallotinic acid, mallotusinic acid, corilagin and geraniin, exhibited much stronger antioxidative activity than gallic acid, rutin, ellagic acid, quercetin, and chlorogenic acid, and were as active as epigallocatechin gallate (EGCG), a strong antioxidant in green tea. Mallotus japonicus leaves are an excellent source of strong natural antioxidative materials.

Complete Genome Sequence of Sphingobium sp. Strain YG1, a Lignin Model Dimer-Metabolizing Bacterium Isolated from Sediment in Kagoshima Bay, Japan.

Sphingobium sp. strain YG1 is a lignin model dimer-metabolizing bacterium newly isolated from sediment in Kagoshima, Japan, at a depth of 102 m. Here, we report the complete genome nucleotide sequence of strain YG1.

Complete Genome Sequence of Altererythrobacter sp. Strain B11, an Aromatic Monomer-Degrading Bacterium, Isolated from Deep-Sea Sediment under the Seabed off Kashima, Japan.

Altererythrobacter sp. strain B11 is an aromatic monomer-degrading bacterium newly isolated from sediment under the seabed off Kashima, Japan, at a depth of 2,100 m. Here, we report the complete nucleotide sequence of the genome of strain B11.

Enzymatic Specific Production and Chemical Functionalization of Phenylpropanone Platform Monomers from Lignin.

Enzymatic catalysis is an ecofriendly strategy for the production of high-value low-molecular-weight aromatic compounds from lignin. Although well-definable aromatic monomers have been obtained from synthetic lignin-model dimers, enzymatic-selective synthesis of platform monomers from natural lignin has not been accomplished. In this study, we successfully achieved highly specific synthesis of aromatic monomers with a phenylpropane structure directly from natural lignin using a cascade reaction of ß-O-4-cleaving bacterial enzymes in one pot. Guaiacylhydroxylpropanone (GHP) and the GHP/syringylhydroxylpropanone (SHP) mixture are exclusive monomers from lignin isolated from softwood (Cryptomeria japonica) and hardwood (Eucalyptus globulus). The intermediate products in the enzymatic reactions show the capacity to accommodate highly heterologous substrates at the substrate-binding sites of the enzymes. To demonstrate the applicability of GHP as a platform chemical for bio-based industries, we chemically generate value-added GHP derivatives for bio-based polymers. Together with these chemical conversions for the valorization of lignin-derived phenylpropanone monomers, the specific and enzymatic production of the monomers directly from natural lignin is expected to provide a new stream in "white biotechnology" for sustainable biorefineries.

A novel enzyme, lambda-carrageenase, isolated from a deep-sea bacterium.

A lambda-carrageenan-degrading Pseudoalteromonas bacterium, strain CL19, was isolated from a deep-sea sediment sample. A lambda-carrageenase from the isolate was purified to homogeneity from cultures containing lambda-carrageenan as a carbon source. This is the first report of the isolation of lambda-carrageenase together with the gene sequence for the enzyme. The molecular mass of the purified enzyme was approximately 100 kDa on both SDS-PAGE and gel-filtration chromatography, suggesting that the enzyme is a monomer. The optimal pH and temperature for activity were about 7 and 35 degrees C, respectively. The enzyme had specific activity of 253 U/mg protein. The enzyme required monovalent salts for the activity. Carbohydrates, such as sorbitol, sucrose, trehalose, improved the enzyme stability. The pattern of lambda-carrageenan hydrolysis showed that the enzyme is an endo-type lambda-carrageenase, and the final main product was a tetrasaccharide of the lambda-carrageenan ideal structure with galactose 2,6-disulfate at the reducing end, indicating the enzyme cleaves the beta-1,4 linkages of its backbone structure. Furthermore, the gene (cglA) encoding the enzyme was sequenced. It encoded a mature protein of 103 kDa (917 amino acids). Remarkably, the deduced amino acid sequence showed no similarity to any reported proteins.