Biochemical characterization of a thermophilic cellobiose 2-epimerase from a thermohalophilic bacterium, Rhodothermus marinus JCM9785

Cellobiose 2-epimerase (CE) reversibly converts glucose residue to mannose residue at the reducing end of β-1,4-linked oligosaccharides. It efficiently produces epilactose carrying prebiotic properties from lactose, but the utilization of known CEs is limited due to thermolability. We focused on thermoholophilic Rhodothermus marinus JCM9785 as a CE producer, since a CE-like gene was found in the genome of R. marinus DSM4252. CE activity was detected in the cell extract of R. marinus JCM9785. The deduced amino acid sequence of the CE gene from R. marinus JCM9785 (RmCE) was 94.2% identical to that from R. marinus DSM4252. The N-terminal amino acid sequence and tryptic peptide masses of the native enzyme matched those of RmCE. The recombinant RmCE was most active at 80 °C at pH 6.3, and stable in a range of pH 3.2-10.8 and below 80 °C. In contrast to other CEs, RmCE demonstrated higher preference for lactose over cellobiose.     

A highly thermostable endo-(1,4)-β-mannanase from the marine bacterium Rhodothermus marinus

Rhodothermus marinus ATCC 43812, a thermophilic bacterium isolated from marine hot springs, possesses hydrolytic activities for depolymerising substrates such as carob-galactomannan. Screening of expression libraries identified mannanase-positive clones. Subsequently, the corresponding DNA sequences were determined, eventually identifying a coding sequence specifying a 997 amino acid residue protein of 113 kDa. Analyses revealed an N-terminal domain of unknown function and a C-terminal mannanase domain of 550 amino acid residues with homology to known mannanases of glycosidase family 26. Action pattern analysis categorised the R. marinus mannanase as an endo-acting enzyme with a requirement for at least five sugar moieties for effective catalytic activity. When expressed in Escherichia coli, purified gene product with catalytic activity was mainly found as two protein fragments of 45 kDa and 50 kDa. The full-length protein of 113 kDa was only detected in crude extracts of R. marinus, while truncated protein-containing fractions of the original source resulted in a major active protein of 60 kDa. Biochemical analysis of the mannanase revealed a temperature and pH optimum of 85 °C and pH 5.4, respectively. Purified, E. coli-produced protein fragments showed high heat stability, retaining more than 70% and 25% of the initial activity after 1 h incubation at 70 °C and 90 °C, respectively. In contrast, R. marinus-derived protein retained 87% activity after 1 h at 90 °C. The enzyme hydrolysed carob-galactomannan (locust bean gum) effectively and to a smaller extent guar gum, but not yeast mannan. Received: 5 November 1999 / Received revision: 19 January 2000 / Accepted: 23 January 2000     

Enzyme Immobilization on Ion Exchangers by Forming an Enzyme Coating with Transglutaminase as a Crosslinker

Southern hybridization analysis using the genes encoding the α- and β-subunits of nitrile hydratase (NHase) from Rhodococcus sp. N-774 as probe suggested that two R. erythropolis strains, JCM6823 and JCM2892, among 31 strains mainly from Japan Culture of Microorganisms (JCM) have NHase genes. Restriction analysis of DNA fragments showing positive hybridization showed that each fragment carried a nucleotide sequence very similar to that of the NHase genes from Rhodococcus sp. N-774. Nucleotide sequence analysis of the DNA fragment cloned from R. erythropolis JCM6823 showed the presence of the genes encoding the α- and β-subunits of NHase, which show 94.7% and 96.2% identity in amino acid sequence to those of Rhodococcus sp. N-774, respectively, as well as a C-terminal portion of the amidase gene upstream from these genes. Despite the extremely high amino acid sequence similarity in both NHases and amidases from R. erythropolis JCM6823 and Rhodococcus sp. N-774, the NHases and amidases from R. erythropolis strains showed broader substrate specificity when compared to those from Rhodococcus sp. N-774. This suggests that a very limited number of amino acid residues are responsible for the difference in substrate specificity. Although the NHase of Rhodococcus sp. N-774 are constitutively produced, the NHases of both R. erythropolis strains were inducibly produced by addition of ε-caprolactam as an inducer.     

A Novel Glycerol Kinase from Flavobacterium meningosepticum: Characterization,Gene Cloning and Primary Structure

A thermostable glycerol kinase (FGK) was purified 34-fold to homogeneity from Flavobacterium meningosepticum. The molecular masses of the enzyme were 200 kDa by gel filtration and 50 kDa by SDS-PAGE. The Km for glycerol and ATP were 0.088 and 0.030 mM, respectively. The enzyme was stable at 65°C for 10 min and at 37°C for two weeks. The enzyme gene was cloned into Escherichia coli and its complete DNA was sequenced. The FGK gene consists of an open reading frame of 1494-bp encoding a protein of 498 amino acids. The deduced amino acid sequence of the gene had 40-60% similarity to those of glycerol kinases from other origins and the amino acid sequence of the putative active site residue reported for E. coli GK is identical to the corresponding sequence of FGK except for one amino acid residue.     

Thermovorax subterraneus, gen. nov., sp. nov., a thermophilic hydrogen-producing bacterium isolated from geothermally active underground mine

A thermophilic, rod-shaped, motile, Gram-positive, spore-forming bacterium strain 70B^T was isolated from a geothermally active underground mine in Japan. The temperature and pH range for growth was 50–81°C (optimum 71°C) and 6.2–9.8 (optimum pH 7–7.5), respectively. Growth occurred in the presence 0–2% NaCl (optimum 1% NaCl). Strain 70B^T could utilize glucose, fructose, mannose, mannitol, pyruvate, cellobiose and tryptone as substrates. Thiosulfate was used as electron acceptor. Major whole-cell fatty acids were iso-C_15:0, C_16:0 DMA (dimethyl acetal), C_16:0 and anteiso-C_15:0. The G+C mol% of the DNA was 44.2%. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that the closest relatives of strain 70B^T were Thermosediminibacter oceani DSM 16646^T (94% similarity) and Thermosediminibacter litoriperuensis DSM 16647 (93% similarity). The phenotypic, chemotaxonomic and phylogenetic properties suggest that strain 70B^T represents a novel species in a new genus, for which the name Thermovorax subterraneus gen. nov., sp. nov. is proposed. The type strain of Thermovorax subterraneus is 70B^T (=DSM 21563 = JCM 15541).     

Cloning, Expression, and Characterization of a Methionyl Aminopeptidase from a Hyperthermophilic Archaeon Thermococcus sp. NA1

Genomic analysis of a hyperthermophilic archaeon Thermococcus sp. NA1 revealed the presence of an 885-bp open reading frame encoding a protein of 295 amino acids with a calculated molecular mass of 32,981 Da. Analysis of the deduced amino acid sequence showed that amino acid residues important for catalytic activity and the metal binding ligands conserved in all of methionyl aminopeptidases (MetAP) were also conserved and belonged to type IIa MetAP. The protein, designated TNA1_MetAP (Thermococcus sp. NA1 MetAP), was cloned and expressed in Escherichia coli. The recombinant enzyme was a Mn²⁺-, Ni²⁺-, Fe²⁺-, or Co²⁺-dependent metallopeptidase. Optimal MetAP activity against l-methionine p-nitroanilide (Met-pNA) (K _m = 0.68 mM) occurred at pH 7.0 and 80 to 90°C. The MetAP was very unstable compared to Pyrococcus furiosus MetAP, which was completely inactivated by heating at 80°C for 5 min. It seemed likely that the cysteine residue (Cys53) played a critical role in regulating the thermostability of TNA1_MetAP.     

High-level expression of the xylanase from <Emphasis Type="Italic">Thermomyces lanuginosus</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

According to the amino acid sequence, a codon-optimized xylanase gene (xynA1) from Thermomyces lanuginosus DSM 5826 was synthesized to construct the expression vector pHsh-xynA1. After optimization of the mRNA secondary structure in the translational initiation region of pHsh-xynA1, free energy of the 70 nt was changed from −6.56 to −4.96 cal/mol, and the spacing between AUG and the Shine-Dalgarno sequence was decreased from 15 to 8 nt. The expression level was increased from 1.3 to 13% of total cell protein. A maximum xylanase activity of 47.1 U/mL was obtained from cellular extract. The recombinant enzyme was purified 21.5-fold from the cellular extract of Escherichia coli by heat treatment, DEAE-Sepharose FF column and t-Butyl-HIC column. The optimal temperature and pH were 65 °C and pH 6.0, respectively. The purified enzyme was stable for 30 min over the pH range of 5.0–8.0 at 60 °C, and had a half-life of 3 h at 65 °C.     

Construction, Expression, and Characterization of a Thermostable Xylanase

A hybrid gene, btx, encoding a thermostable xylanase, Btx, was constructed by substituting the 31 N-terminal amino acid residues of the Thermomonospora fusca xylanase A (TfxA) for the corresponding region of 22 amino acid residues of the Bacillus subtilis xylanase A (BsxA). The btx gene was expressed in Escherichia coli BL21. The halo size produced by xylanase Btx on a Remanzol brilliant blue R (RBB) xylan plate at 60°C and pH 6.0 was larger than those of BsxA and TfxA. The molecular weight of Btx was 22 kDa. Temperature and pH optima for Btx were at 50–60°C and 6.0, respectively. Btx showed activity over 80% over a pH range of 5.0–9.0, which was wider than that of BsxA, and was also more acid-resistant than TfxA. Btx exhibited significant thermostability compared with BsxA. The results show the importance of the N-terminal sequence of TfxA in thermostability.     

Molecular cloning of preprocathepsin E cDNA from the stomach of bullfrog Rana catesbeiana

A cDNA library was constructed from a poly(A)⁺ RNA fraction of the gastric mucosa of bullfrog Rana catesbeiana. We cloned a cDNA encoding preprocathepsin E (Pre-Pro-CE) from the library. The present study is the first demonstration of the Pre-Pro-CE cDNA of lower vertebrate such as amphibian. Amino acid sequence deduced from the cDNA was compared with partial amino acid sequence determined by Edman degradation, suggesting that the cDNA comprises an open reading frame encoding a signal peptide (16 amino acids), a pro-sequence (33 amino acids) and a mature protein region (348 amino acids). Two consensus tri-peptide sequences (FDT and VDT) as active site and positions of seven cysteine residues were conserved in this amphibian CE. Although the bullfrog CE was deduced to contain one potential N-linked glycosylation site, its position (Asn¹³⁹-Leu¹⁴⁰-Thr¹⁴¹) was different from that of mammalian CEs. Molecular phylogenetic analysis showed that the bullfrog Pro-CE belongs to the typical Pro-CE group among various aspartic proteinases.     

Characterization of the single psbA gene of Prochlorococcus marinus CCMP 1375 (Prochlorophyta)

DNA sequence, copy number, expression and phylogenetic relevance of the psbA gene from the abundant marine prokaryote P. marinus CCMP 1375 was analyzed. The 7 amino acids near the C-terminus missing in higher plant and in Prochlorothrix hollandica D1 proteins are present in the derived amino acid sequence. P. marinus contains only a single psbA gene. Thus, this organism lacks the ability to adapt its photosystem II by replacement of one type of D1 by another, as several cyanobacteria do. Phylogenetic trees suggested the D1-1 iso-form from Synechococcus PCC 7942 as the next related D1 protein and place P. Marinus separately from Prochlorothrix hollandica among the cyanobacteria.     

Cloning,expression, and characterization of an (R)-specific alcohol dehydrogenase from Lactobacillus kefir

Lactobacillus kefir DSM 20587 produces an (R)-specific NADP-dependent alcohol dehydrogenase (ADH) with a broad substrate specificity. The gene of this ADH was isolated and the complete nucleotide sequence determined. The adh gene comprises 759?bp and encodes a protein of 252 amino acids with a calculated molecular weight of 26 781?Da. The deduced amino acid sequence indicated a high degree of similarity to short-chain dehydrogenases. After cloning and expression in Escherichia coli the enzyme was purified and characterized. For the reduction of acetophenone the specific activity of the homogeneous recombinant ADH was 558?U?mg^?1. The enzyme shows its maximum activity at 50°C while the pH optimum was at pH?7.0. In order to demonstrate its preparative application, purified ADH was used for the stereoselective reduction of several aliphatic and aromatic ketones as well as β-keto esters. Glucose dehydrogenase was added for the regeneration of NADPH. All prochiral ketones were stereoselectively reduced to the corresponding alcohols with >99% ee and in the case of diketones >99% de.     

A new xylanase from thermoalkaline <Emphasis Type="Italic">Anoxybacillus</Emphasis> sp. E2 with high activity and stability over a broad pH range

A xylanase gene, xynE2, was cloned from thermoalkaline Anoxybacillus sp. E2 and was expressed in Escherichia coli BL21 (DE3). The gene consisted of 987 bp and encoded a 328-residue xylanase with a calculated molecular weight of 38.8 kDa. On the basis of amino acid sequence similarities, this enzyme was assigned as a member of glycoside hydrolase family 10. Purified recombinant XynE2 showed maximal activity at pH 7.8 and 65°C, and was thermostable at 60°C. The enzyme was highly active and stable over a broad pH range, showing more than 90% of maximal activity at pH 6.6–pH 8.6 and retaining more than 80% of activity at pH 4.6–pH 12.0, 37°C for 1 h, respectively. These favorable properties make XynE2 a good candidate in the pulp and paper industries. This is the first report on gene cloning, expression and characterization of a xylanase from the genus Anoxybacillus.     

A new xylanase from Streptomyces megasporus DSM 41476 with high yield of xylobiose

A new xylanase gene, xynBM4, was cloned from Streptomyces megasporus DSM 41476 and expressed in Pichia pastoris. The full-length gene consists of 1,443 bp and encodes 480 amino acids including a putative 49-residue signal peptide. The deduced amino acid sequence of xynBM4 shows the highest identity of 66.3% to the xylanase Xys1L from Streptomyces halstedii JM8. The purified recombinant XYNBM4 had a high specific activity of 350.7 U mg^-1 towards soluble wheat arabinoxylan, exhibited optimal activity at pH 6.0 and 57°C, showed broad pH adaptability (>75% of the maximum activity at pH 2.5–9.0), was resistant to neutral proteases and most chemicals, and produced simple products. The hydrolysis products of birchwood xylan and corncob xylan were predominantly xylobiose (76.9 and 90.8%, respectively) and no xylose. These characteristics suggest that XYNBM4 has potential in various applications, especially in the food industry.     

<Emphasis Type="Italic">Tindallia texcoconensis</Emphasis> sp. nov., a new haloalkaliphilic bacterium isolated from lake Texcoco,Mexico

A new alkaliphilic and moderately halophilic, strictly anaerobic, fermentative bacterium (strain IMP-300^T) was isolated from a groundwater sample in the zone of the former soda lake Texcoco in Mexico. Strain IMP-300^T was Gram-positive, non-sporulated, motile and rod-shaped. It grew within a pH range from 7.5 to 10.5, and an optimum at 9.5. The organism was obligately dependent on the presence of sodium salts. Growth showed an optimum at 35°C with absence of growth above 45°C. It fermented peptone and a few amino acids, preferentially arginine and ornithine, with production of acetate, propionate, and ammonium. Its fatty acid pattern was mainly composed of straight chain saturated, unsaturated, and cyclopropane fatty acids. The G + C content of genomic DNA was 40.0 mol%. Analysis of the 16S rRNA gene sequence indicated that the new isolate belongs to the genus Tindallia, in the low G + C Gram-positive phylum. Phylogenetically, strain IMP-300^T has Tindallia californiensis, as closest relative with a 97.5% similarity level between their 16S rDNA gene sequences, but the DNA–DNA re-association value between the two DNAs was only 42.2%. On the basis of differences in genotypic, phenotypic, and phylogenetic characteristics, strain IMP-300^T is proposed as a new species of the genus Tindallia, T. texcoconensis sp. nov. (type strain IMP-300^T = DSM 18041^T = JCM 13990^T).     

Molecular modeling of CPT-11 metabolism by carboxylesterases (CEs): use of pnb CE as a model

CPT-11 is a prodrug that is converted in vivo to the topoisomerase I poison SN-38 by carboxylesterases (CEs). Among the CEs studied thus far, a rabbit liver CE (rCE) converts CPT-11 to SN-38 most efficiently. Despite extensive sequence homology, however, the human homologues of this protein, hCE1 and hiCE, metabolize CPT-11 with significantly lower efficiencies. To understand these differences in drug metabolism, we wanted to generate mutations at individual amino acid residues to assess the effects of these mutations on CPT-11 conversion. We identified a Bacillus subtilis protein (pnb CE) that could be used as a model for the mammalian CEs. We demonstrated that pnb CE, when expressed in Escherichia coli, metabolizes both the small esterase substrate o-NPA and the bulky prodrug CPT-11. Furthermore, we found that the pnb CE and rCE crystal structures show an only 2.4 A rmsd variation over 400 residues of the alpha-carbon trace. Using the pnb CE model, we demonstrated that the "side-door" residues, S218 and L362, and the corresponding residues in rCE, L252 and L424, were important in CPT-11 metabolism. Furthermore, we found that at position 218 or 252 the size of the residue, and at position 362 or 424 the hydrophobicity and charge of the residue, were the predominant factors in influencing drug activation. The most significant change in CPT-11 metabolism was observed with the L424R variant rCE that converted 10-fold less CPT-11 than the wild-type protein. As a result, COS-7 cells expressing this mutant were 3-fold less sensitive to CPT-11 than COS-7 cells expressing the wild-type protein.     

Purification and biochemical characterization of a native invertase from the hydrogen-producing Thermotoga neapolitana (DSM 4359)

This is the first report describing the purification and enzymatic properties of a native invertase (β-D-fructosidase) in Thermotogales. The invertase of the hydrogen-producing thermophilic bacterium Thermotoga neapolitana DSM 4359 (hereby named Tni) was a monomer of about 47 kDa having an amino acid sequence quite different from other invertases studied up to now. Its properties and substrates specificity let us classify this protein as a solute-binding protein with invertase activity. Tni was specific for the fructose moiety and the enzyme released fructose from sucrose and raffinose and the fructose polymer inulin was hydrolyzed in an endo-type fashion. Tni had an optimum temperature of 85°C at pH 6.0. At temperatures of 80–85°C, the enzyme retained at least 50% of its initial activity during a 6 h preincubation period. Tni had a K _m and k _cat /K _m values (at 85°C and pH 6.0) of about 14 mM and 5.2 × 10⁸ M⁻¹ s⁻¹, respectively. Dedicated to the memory of Prof. R. A. Nicolaus, founder of the Institute (1968).     

Characterization of a thermostable xylanase from an alkaliphilic Bacillus sp.

A xylanase gene (xyn10) from alkaliphilic Bacillus sp. N16-5 was cloned and expressed in Pichia pastoris. The deduced amino acid sequence has 85% identity with xylanase xyn10A from B. halodurans and contains two potential N-glycosylation sites. The glycosylated Xyn10 with MW 48 kDa can hydrolyze birchwood and oatspelt xylan. The enzyme had optimum activity at pH 7 and 70°C, with the specific activity of 92.5U/mg. The Xyn10 retained over 90% residual activity at 60°C for 30 min but lost all activity at 80°C over 15 min. Most tested ions showed no or slight inhibition effects on enzyme activity.     

Oceanobacillus kimchii sp. nov. Isolated from a traditional Korean fermented food

A moderate halophile, strain X50^T, was isolated from mustard kimchi, a traditional Korean fermented food. The organism grew under conditions ranging from 0–15.0% (w/v) NaCl (optimum: 3.0%), pH 7.0–10.0 (optimum: pH 9.0) and 15–45°C (optimum: 37°C). The morphological, physiological, and biochemical features and the 16S rRNA gene sequences of strain X50^T were characterized. Colonies of the isolate were creamcolored and the cells were rod-shaped. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain X50^T belongs to the genus Oceanobacillus and is closely related phylogenetically to the type strain O. iheyensis HTE831^T (98.9%) and O. oncorhynchi subsp. oncorhynchi R-2^T (97.0%). The cellular fatty acid profiles predominately included anteiso-C_15:0 and iso-C_15:0. The G+C content of the genomic DNA of the isolate was 37.9 mol% and the major isoprenoid quinone was MK-7. Analysis of the 16S rRNA gene sequences, DNA-DNA relatedness and physiological and biochemical tests indicated genotypic and phenotypic differences among strain X50^T and reference species in the genus Oceanobacillus. Therefore, strain X50^T was proposed as a novel species and named Oceanobacillus kimchii. The type strain of the new species is X50^T (=JCM 16803^T =KACC 14914^T =DSM 23341^T).     

Synthesis of Some Isocoumarin Derivatives

An alkaline κ-carrageenase, Cgk-K142, was found in the culture broth of a deep-sea bacterium, Pseudoalteromonas tetraodonis JAM-K142. A gene for the enzyme was cloned and expressed. Purified recombinant Cgk-K142 (rCgk-K142) showed an optimal pH of about 8.8 in glycine-NaOH buffer at 30 °C and of about 8.0 in MOPS buffer at 50 °C. The optimal temperature for the enzyme was 55 °C at pH 8.0. rCgk-K142 was unstable, but λ- and ι-carrageenans, non-degradative substrate homologs, extensively enhanced its stability. The nucleotide sequence of the gene for Cgk-K142 comprised 1,194 bp, and the deduced amino acid sequence (397 amino acids) showed a high level of similarity to the κ-carrageenase of P. carrageenovora, with 94% identity. Another gene for a κ-carrageenase-like protein was found downstream of the gene for Cgk-K142. The nucleotide sequence of that gene consisted of 966 bp (321 amino acids), and it showed the highest similarity, at 64% identity, to protein CgkB of P. carrageenovora, which has been reported as an incomplete 57-amino acid sequence.