Membrane-bound amylopullulanase is essential for starch metabolism of Sulfolobus acidocaldarius DSM639.

Sulfolobus acidocaldarius DSM639 produced an acid-resistant membrane-bound amylopullulanase (Apu) during growth on starch as a sole carbon and energy source. The physiological role of Apu in starch metabolism was investigated by the growth and starch degradation pattern of apu disruption mutant as well as biochemical properties of recombinant Apu. The Δapu mutant lost the ability to grow in minimal medium in the presence of starch, and the amylolytic activity observed in the membrane fraction of the wild-type strain was not detected in the Δapu mutant when the cells were grown in YT medium. The purified membrane-bound Apu initially hydrolyzed starch, amylopectin, and pullulan into various sizes of maltooligosaccharides, and then produced glucose, maltose, and maltotriose in the end, indicating Apu is a typical endo-acting glycoside hydrolase family 57 (GH57) amylopullulanase. The maltose and maltotriose observed in the culture medium during the exponential and stationary phase growth indicates that Apu is the essential enzyme to initially hydrolyze the starch into small maltooligosaccharides to be transported into the cell.

Identification and characterization of MalA in the maltose/maltodextrin operon of Sulfolobus acidocaldarius DSM639.

A putative maltose/maltodextrin operon was found in the Sulfolobus acidocaldarius DSM639 genome. The gene cluster consisted of 7 genes (malA, trmB, amyA, malG, malF, malE, and malK). Here, we report the identification of MalA, which is responsible for the hydrolysis of maltose or maltodextrin to glucose in S. acidocaldarius. The transcription level of malA was increased 3-fold upon the addition of maltose or starch to the medium. Moreover, the α-glucosidase activity for maltose as a substrate in cell extracts of S. acidocaldarius DSM639 was also 11- and 10-fold higher during growth in YT medium (Brock's mineral salts, 0.1% [wt/vol] tryptone, and 0.005% [wt/vol] yeast extract) containing maltose or starch, respectively, than during growth on other sugars. The gene encoding MalA was cloned and expressed in S. acidocaldarius. The enzyme purified from the organism was a dodecamer in its active state and showed strong maltose-hydrolyzing activity at 100°C and pH 5.0. MalA was remarkably thermostable, with half-lives of 33.8 h, 10.6 h, and 1.8 h at 95°C, 100°C, and 105°C, respectively. Substrate specificity and kinetic studies of MalA with maltooligosaccharides indicated that MalA efficiently hydrolyzed maltose to maltopentaose, which is a typical characteristic of GH31-type α-glucosidases. However, glycogen or starch was not hydrolyzed. Reverse transcription-PCR, sugar uptake, and growth studies of the wild-type DSM639 and ΔmalEFG mutant on different sugars demonstrated that MalA located in the mal operon gene cluster is involved in maltose and starch metabolism in S. acidocaldarius.

Structure of a novel α-amylase AmyB from Thermotoga neapolitana that produces maltose from the nonreducing end of polysaccharides.

An intracellular α-amylase, AmyB, has been cloned from the hyperthermophilic bacterium Thermotoga neapolitana. AmyB belongs to glycoside hydrolase family 13 and liberates maltose from diverse substrates, including starch, amylose, amylopectin and glycogen. The final product of AmyB is similar to that of typical maltogenic amylases, but AmyB cleaves maltose units from the nonreducing end, which is a unique property of this α-amylase. In this study, the crystal structure of AmyB from T. neapolitana has been determined at 2.4 Šresolution, revealing that the monomeric AmyB comprises domains A, B and C like other α-amylases, but with structural variations. In the structure, a wider active site and a putative extra sugar-binding site at the top of the active site were found. Subsequent biochemical results suggest that the extra sugar-binding site is suitable for recognizing the nonreducing end of the substrates, explaining the unique activity of this enzyme. These findings provide a structural basis for the ability of an α-amylase that has the common α-amylase structure to show a diverse substrate specificity.

Identification of antigenic Edwardsiella tarda surface proteins and their role in pathogenesis.

Edwardsiella tarda causes an infectious fish disease called edwardsiellosis. Several outer membrane proteins (OMPs) are associated with virulence factors and are attractive as vaccine candidates. In this study, 4 immuno-reactive OMPs of E. tarda were detected using anti-sera from flounder infected with E. tarda. Using matrix-assisted laser desorption/ionization mass spectrometry analyses, 2 of the 4 OMPs were identified as OmpA and murein lipoprotein (Lpp), which are highly conserved surface proteins in gram-negative bacteria. For further characterization of these surface proteins, we generated ompA- and lpp-inactivated mutants by insertion of a kanamycin cassette in the corresponding genes, and named these mutants E. tarda CK99 and CK164, respectively. As expected, immuno-reactive OmpA and Lpp proteins were absent in E. tarda CK99 and CK164, respectively, confirming that OmpA and Lpp are antigenic surface proteins. Interestingly, the LD(50) value of E. tarda CK164 in fish (2.0 × 10(8) colony-forming unit [CFU]/fish) was greater than that of the parental strain (3.0 × 10(7) CFU/fish). The LD(50) of E. tarda CK99 did not differ from that of its parental strain. After administering attenuated E. tarda CK164 to fish, we monitored the E. tarda-specific immune response profile. We observed that the E. tarda-specific serum IgM titer increased in a time-dependent manner, and was much higher than the value observed after the administration of a heat-killed E. tarda control. Moreover, fish vaccinated with E. tarda CK164 were 100% protected when challenged by CK41, a pathogenic strain. Our results suggest that E. tarda CK164 can potentially be used for developing an effective live attenuated vaccine for edwardsiellosis that can be applied in the aquaculture industry.

Treatment of asymptomatic pulmonary cryptococcosis in immunocompetent hosts with oral fluconazole.

BACKGROUND: Pulmonary cryptococcosis is occasionally detected on routine imaging studies in healthy hosts with no or mild symptoms. Isolated pulmonary cryptococcosis may be observed without specific therapy in asymptomatic immunocompetent hosts. However, considering that dissemination from a pulmonary infection can occur in patients with no immunologic defects, treatment of asymptomatic pulmonary cryptococcosis in immunocompetent hosts remains controversial. The aim of this study was to determine the role of fluconazole therapy in the management of isolated pulmonary cryptococcosis in asymptomatic healthy hosts. METHODS: We retrospectively analyzed the medical records and radiographic findings of 10 healthy subjects with isolated pulmonary cryptococcosis diagnosed incidentally and treated with oral fluconazole. RESULTS: All patients had no respiratory or constitutional symptoms. The most common radiological findings were pulmonary nodules, and the number of nodules in each patient was from 1 to 9. After histological confirmation, all patients were treated with oral fluconazole at a dosage of 400 mg per day for a median period of 6.4 months. No patient developed an adverse reaction to fluconazole. The mean interval between the initiation of antifungal therapy and final radiological response was 8.3 months. Seven of the 10 patients showed complete resolution, and the other 3 patients were assessed as having partial resolution. During the average follow-up period of 11.9 months, all patients showed a favourable outcome with no relapse. The overall cure rate was 70%. CONCLUSION: These results suggest that fluconazole may be an attractive therapeutic option for asymptomatic pulmonary cryptococcosis in immunocompetent hosts.

Identification of the Genes Related to the Glycogen Metabolism in Hyperthermophilic Archaeon, Sulfolobus acidocaldarius.

Glycogen is a polysaccharide that comprises α-1,4-linked glucose backbone and α-1,6-linked glucose polymers at the branching points. It is widely found in organisms ranging from bacteria to eukaryotes. The physiological role of glycogen is not confined to being an energy reservoir and carbon source but varies depending on organisms. Sulfolobus acidocaldarius, a thermoacidophilic archaeon, was observed to accumulate granular glycogen in the cell. However, the role of glycogen and genes that are responsible for glycogen metabolism in S. acidocaldarius has not been identified clearly. The objective of this study is to identify the gene cluster, which is composed of enzymes that are predicted to be involved in the glycogen metabolism, and confirm the role of each of these genes by constructing deletion mutants. This study also compares the glycogen content of mutant and wild type and elucidates the role of glycogen in this archaeon. The glycogen content of S. acidocaldarius MR31, which is used as a parent strain for constructing the deletion mutant in this study, was increased in the early and middle exponential growth phases and decreased during the late exponential and stationary growth phases. The pattern of the accumulated glycogen was independent to the type of supplemented sugar. In the comparison of the glycogen content between the gene deletion mutant and MR31, glycogen synthase (GlgA) and α-amylase (AmyA) were shown to be responsible for the synthesis of glycogen, whereas glycogen debranching enzyme (GlgX) and glucoamylase (Gaa) appeared to affect the degradation of glycogen. The expressions of glgC-gaa-glgX and amyA-glgA were detected by the promoter assay. This result suggests that the gradual decrease of glycogen content in the late exponential and stationary phases occurs due to the increase in the gene expression of glgC-gaa-glgX. When the death rate in nutrient limited condition was compared among the wild type strain, the glycogen deficient strain and the strain with increased glycogen content, the death rate of the glycogen deficient strain was found to be higher than any other strain, thereby suggesting that the glycogen in S. acidocaldarius supports cell maintenance in harsh conditions.

Characterization of an exo-acting intracellular alpha-amylase from the hyperthermophilic bacterium Thermotoga neapolitana.

We cloned and expressed the gene for an intracellular alpha-amylase, designated AmyB, from the hyperthermophilic bacterium Thermotoga neapolitana in Escherichia coli. The putative intracellular amylolytic enzyme contained four regions that are highly conserved among glycoside hydrolase family (GH) 13 alpha-amylases. AmyB exhibited maximum activity at pH 6.5 and 75 degrees C, and its thermostability was slightly enhanced by Ca2+. However, Ca2+ was not required for the activity of AmyB as EDTA had no effect on enzyme activity. AmyB hydrolyzed the typical substrates for alpha-amylase, including soluble starch, amylose, amylopectin, and glycogen, to liberate maltose and minor amount of glucose. The hydrolytic pattern of AmyB is most similar to those of maltogenic amylases (EC 3.2.1.133) among GH 13 alpha-amylases; however, it can be distinguished by its inability to hydrolyze pullulan and beta-cyclodextrin. AmyB enzymatic activity was negligible when acarbose, a maltotetraose analog in which a maltose residue at the nonreducing end was replaced by acarviosine, was present, indicating that AmyB cleaves maltose units from the nonreducing end of maltooligosaccharides. These results indicate that AmyB is a new type exo-acting intracellular alpha-amylase possessing distinct characteristics that distinguish it from typical alpha-amylase and cyclodextrin-/pullulan-hydrolyzing enzymes.

Hydrophobization of Cellulose Sheets by Gas Grafting of Palmitoyl Chloride by Using Hot Press.

The purpose of this study was to investigate the improvement in the hydrophobicity of cellulose through gas grafting treatment with long chain fatty acid chloride using high pressure during pressing at high temperature. To do this, the gas grafting treatment was performed on the cellulose sheet using a hot pressing method, and then the hydrophobization effect was analyzed. It was found that the gas grafting treatment by hot pressing using high pressure during pressing at high temperature produced cellulose sheets of high hydrophobicity. Especially, it was notable that the hydrophobization efficiency enhanced with an increase of the pressing pressure. In addition, the gas grafting efficiency was improved when polyvinyl alcohol (PVA) was coated to obtain high resistance to air permeability. These results indicate that protecting the loss of fatty acid gas by coating of polyvinyl alcohol (PVA) on the cellulose sheet surface contributed to the improvement of gas grafting efficiency.

Necrotizing fasciitis in a patient treated with etanercept for dermatomyositis.

We first report a case of patient with refractory dermatomyositis (DM) who successfully treated with tumor necrosis factor-alpha blocker, etanercept and developed necrotizing fasciitis (NF) after the use of it. NF was diagnosed early by MRI and managed with antimicrobial therapy and timely debridement of necrotic tissue with favorable outcome. We suggest that etanercept can be a useful therapeutic agent in patients with DM refractory to steroid and immunosuppressive agents and NF may occur in DM in association with etanercept therapy. We also suggest that management strategies for early diagnosis and treatment are needed for this complication.

Probing the critical residues for intramolecular fructosyl transfer reaction of a levan fructotransferase.

Levan fructotransferase (LFTase) preferentially catalyzes the transfructosylation reaction in addition to levan hydrolysis, whereas other levan-degrading enzymes hydrolyze levan into a levan-oligosaccharide and fructose. Based on sequence comparisons and enzymatic properties, the fructosyl transfer activity of LFTase is proposed to have evolved from levanase. In order to probe the residues that are critical to the intramolecular fructosyl transfer reaction of the Microbacterium sp. AL-210 LFTase, an error-prone PCR mutagenesis process was carried out, and the mutants that led to a shift in activity from transfructosylation towards hydrolysis of levan were screened by the DNS method. After two rounds of mutagenesis, TLC and HPLC analyses of the reaction products by the selected mutants revealed two major products; one is a di-D-fructose- 2,6':6,2'-dianhydride (DFAIV) and the other is a levanbiose. The newly detected levanbiose corresponds to the reaction product from LFTase lacking transferring activity. Two mutants (2-F8 and 2-G9) showed a high yield of levanbiose (38-40%) compared with the wild-type enzyme, and thus behaved as levanases. Sequence analysis of the individual mutants responsible for the enhanced hydrolytic activity indicated that Asn-85 was highly involved in the transfructosylation activity of LFTase.

Saci_1816: A Trehalase that Catalyzes Trehalose Degradation in the Thermoacidophilic Crenarchaeon Sulfolobus acidocaldarius.

Previously, a cytosolic trehalase (TreH) from the hyperthermophilic archaeon Sulfolobus acidocaldarius was reported; however, the gene responsible for the trehalase activity was not identified. Two genes, saci_1816 and saci_1250, that encode the glycoside hydrolase family 15 type glucoamylase-like proteins in S. acidocaldarius were targeted and expressed in Escherichia coli, and their abilities to hydrolyze trehalose were examined. Recombinant Saci_1816 hydrolyzed trehalose exclusively without any help from a cofactor. The mass spectrometric analysis of partially purified native TreH also confirmed that Saci_1816 was involved in proteins exhibiting trehalase activity. Optimal trehalose hydrolysis activity of the recombinant Saci_1816 was observed at pH 4.0 and 60°C. The pH dependence of the recombinant enzyme was similar to that of the native enzyme, but its optimal temperature was 20-25°C lower, and its thermostability was also slightly reduced. From the biochemical and structural results, Saci_1816 was identified as a trehalase responsible for trehalose degradation in S. acidocaldarius. Identification of the treH gene confirms that the degradation of trehalose in Sulfolobus species occurs via the TreH pathway.

Purification and characterization of a new fibrinolytic enzyme of Bacillus licheniformis KJ-31, isolated from Korean traditional Jeot-gal.

Jeot-gal is a traditional Korean fermented seafood and has long been used for seasoning. We isolated 188 strains from shrimp, anchovy, and yellow corvina Jeot-gal, and screened sixteen strains that showed strong fibrinolytic activities on a fibrin plate. Among those strains, the strain that had the largest halo zone was chosen and identified as Bacillus licheniformis by using 16S rDNA sequencing and an API CHB kit. The fibrinolytic activity of Bacillus licheniformis was characterized and designated as bpKJ-31. The active component of bpKJ-31 was identified as a 37 kDa protein, designated bacillopeptidase F, by internal peptide mapping and N-terminal sequencing. The optimum activity of bpKJ-31 was shown at pH 9 and 40 degrees C, with a chromogenic substrate for plasmin. It had high degrading activity for the Bbeta-chain and Aalpha-chain of fibrin(ogen), and also acted on thrombin, but not skim milk and casein. The amidolytic activity of bpKJ-31 was inhibited by 1 mM phenylmethanesulfonyl fluoride, but 1 mM EDTA did not affect the enzyme activity, indicating that bpKJ-31 is an alkaline serine protease, like a plasmin. The bpKJ-31 showed approximately 14.3% higher fibrinolytic activity than the plasmin. These features of bpKJ-31 make it attractive as a health-promoting biomaterial.

Characterization of a thermostable glycoside hydrolase family 36 α-galactosidase from Caldicellulosiruptor bescii.

The putative gene cluster involved in the degradation of the raffinose family oligosaccharides (RFO) was identified in Caldicellulosiruptor bescii. Within the cluster, the gene encoding a putative α-galactosidase (CbAga36) was cloned and expressed in Escherichia coli. Size exclusion chromatography of the purified rCbAga36 indicated that the native form was a tetramer. Its primary sequence was similar to the family of glycoside hydrolase 36. The purified recombinant CbAga36 (rCbAga36) was optimally active at pH 5.0 and 70°C and had a half-life of 15 h and 10 h at 70°C and 80°C, respectively. rCbAga36 showed high activity with the artificial substrate (p-nitrophenyl α-d-galactopyranoside, pNPαGal) exhibiting lower Km and higher kcat than natural substrates such as melibiose and raffinose. Although rCbAga36 demonstrated preferential activity toward the hydrolysis of RFO such as raffinose and stachyose, it did not degrade the polymeric galactomannans. Our results imply that CbAga36 may play a role in the degradation of RFO, transported into the cytoplasm via a transporter into galactose, which is further utilized as an energy source in C. bescii. Furthermore, its ability to synthesize novel oligosaccharides by transglycosylation renders this enzyme potentially useful for the production of dietary oligosaccharides with novel function.

Characterization of a trehalose-degrading enzyme from the hyperthermophilic archaeon Sulfolobus acidocaldarius.

We purified a cytosolic trehalase (TreH) from a thermoacidophilic archaeon Sulfolobus acidocaldarius. Enzyme activity in cell-free extracts indicated that trehalose degradation in the cell occurred via the hydrolytic activity of TreH, and not via TreP (phosphorolytic activity) or TreT (transfer activity). TreH was purified to near-homogeneity by DEAE anion-exchange chromatography, followed by size exclusion and HiTrap Q anion-exchange chromatography, and its molecular mass was estimated as 40 kDa. Maximum activity was observed at 85°C and pH 4.5. The half-life of TreH was 53 and 41 min at 90°C and 95°C, respectively. TreH was highly specific for trehalose and was inhibited by glucose with a Ki of 0.05 mM. Compared with TreH from other trehalases, TreH from S. acidocaldarius is the most thermostable trehalase reported so far. Furthermore, this is the first trehalase characterized in the Archaea domain.