Characterization of the trehalosyl dextrin-forming enzyme from the thermophilic archaeon <Emphasis Type="Italic">Sulfolobus solfataricus</Emphasis> ATCC 35092

The trehalosyl dextrin-forming enzyme (TDFE) mainly catalyzes an intramolecular transglycosyl reaction to form trehalosyl dextrins from dextrins by converting the -1,4-glucosidic linkage at the reducing end to an -1,1-glucosidic linkage. In this study, the treY gene encoding TDFE was PCR cloned from the genomic DNA of Sulfolobus solfataricus ATCC 35092 to an expression vector with a T7 lac promoter and then expressed in Escherichia coli. The recombinant TDFE was purified sequentially by using heat treatment, ultrafiltration, and gel filtration. The obtained recombinant TDFE showed an apparent optimal pH of 5 and an optimal temperature of 75°C. The enzyme was stable in a pH range of 4.5–11, and the activity remained unchanged after a 2-h incubation at 80°C. The transglycosylation activity of TDFE was higher when using maltoheptaose as substrate than maltooligosaccharides with a low degree of polymerization (DP). However, the hydrolysis activity of TDFE became stronger when low DP maltooligosaccharides, such as maltotriose, were used as substrate. The ratios of hydrolysis activity to transglycosylation activity were in the range of 0.2–14% and increased when the DP of substrate decreased. The recombinant TDFE was found to exhibit different substrate specificity, such as its preferred substrates for the transglycosylation reaction and the ratio of hydrolysis to transglycosylation of the enzyme reacting with maltotriose, when compared with other natural or recombinant TDFEs from Sulfolobus.     

Recombinant thermophilic enzymes for trehalose and trehalosyl dextrins production

Two thermophilic and thermostable enzymes, trehalosyl dextrins forming enzyme (TDFE) and trehalose forming enzyme (TFE), able to convert starch and dextrins to ,-trehalose were recently purified and characterized from Sulfolobales [I. Di Lernia, A. Morana, A. Ottombrino, S. Fusco, M. Rossi, M. De Rosa, Extremophiles, 2 (1998) 409; T. Nakada, S. Ikegami, H. Chaen, M. Kubota, S. Fukuda, T. Sugimoto, M. Kurimoto, Y. Tsujisaka, Biosci., Biotechnol., Biochem., 60 (1996) 267; T. Nakada, S. Ikegami, H. Chaen, M. Kubota, S. Fukuda, T. Sugimoto, M. Kurimoto, Y. Tsujisaka, Biosci., Biotechnol., Biochem., 60 (1996) 263; M. Kato, Y. Miura, M. Kettoku, K. Shindo, A. Iwamatsu, K. Kobayashi, Biosci., Biotechnol., Biochem., 60 (1996) 921; M. Kato, Y. Miura, M. Kettoku, K. Shindo, A. Iwamatsu, K. Kobayashi, Biosci., Biotechnol., Biochem., 60 (1996) 925]. The first enzyme transforms starch and dextrins to the corresponding trehalosyl derivatives, with an intramolecular transglycosylation process, which converts the glucosidic linkage at the reducing end from -1,4 to -1,1. The second, hydrolyzes the -1,4 linkage adjacent to the -1,1 bond of trehalosyl dextrins, forming trehalose and lower molecular weight dextrins. Herein, we report the cloning and high level expression of the two enzymes of Sulfolobus solfataricus strain MT4 in Escherichia coli using pTrc expression vector. The yield of TDFE and TFE obtained in this expression system was of 180 U/l and of 3630 U/l of medium, respectively.     

Constitutive expression of a novel isoamylase from Bacillus lentus in Pichia pastoris for starch processing

Isoamylase is essential to saccharifying starch by cleavage of 1,6-glucoside linkages in starch molecules. In this study, a novel isoamylase gene from Bacillus lentus JNU3 was cloned. The open reading frame of the gene was 2412 base pairs long and encoded a polypeptide of 804 amino acids with a calculated molecular mass of 90 kDa. The deduced amino acid sequence shared less than 40% homology with that of microbial isoamylase ever reported, which indicated it was a novel isoamylase. A constitutive GAP promoter was used to express the recombinant isoamylase in the yeast Pichia pastoris by continuous high cell-density fermentation to avoid the use of methanol, which resulted in 318 U/mL extracellular isoamylase activity after 72 h in a 10 L fermenter. The recombinant enzyme was purified and characterized. It had an estimated molecular mass of 90 kDa, with its optimal activity at 70 °C, pH 6.5 and was quite stable between 30 °C and 70 °C. The recombinant isoamylase proves to be superior to pullulanase as an auxiliary enzyme in maltose production from starch. Therefore it will contribute significantly to the starch debranching process.     

Characterization of the maltooligosyl trehalose synthase from the thermophilic archaeon Sulfolobus acidocaldarius

We report the molecular characterization and the detailed study of the recombinant maltooligosyl trehalose synthase mechanism from the thermoacidophilic archaeon Sulfolobus acidocaldarius. The mts gene encoding a maltooligosyl trehalose synthase was overexpressed in Escherichia coli using the T7-expression system. The purified recombinant enzyme exhibited optimum activity at 75 degrees C and pH 5 with citrate-phosphate buffer and retained 60% of residual activity after 72 h of incubation at 80 degrees C. The recombinant enzyme was active on maltooligosaccharides such as maltotriose, maltotetraose, maltopentaose and maltoheptaose. Investigation of the enzyme action on maltooligosaccharides has brought much insight into the reaction mechanism. Results obtained from thin-layer chromatography suggested a possible mechanism of action for maltooligosyl trehalose synthase: the enzyme, after converting the alpha-1,4-glucosidic linkage to an alpha-1,1-glucosidic linkage at the reducing end of maltooligosaccharide glc(n) is able to release glucose and maltooligosaccharide glc(n-1) residues. And then, the intramolecular transglycosylation and the hydrolytic reaction continue, with the maltooligosaccharide glc(n-1) until the initial maltooligosaccharide is reduced to maltose. An hypothetical mechanism of maltooligosyl trehalose synthase acting on maltooligosaccharide is proposed.     

Expression and Characterization of the HMG-CoA Reductase of the Thermophilic Archaeon Sulfolobus solfataricus

The thermostable class I HMG-CoA reductase of Sulfolobus solfataricus offers potential for industrial applications and for the initiation of crystallization trials of a biosynthetic HMG-CoA reductase. However, of the 15 arginine codons of the hmgA gene that encodes S. solfataricus HMG-CoA reductase, 14 (93%) are AGA or AGG, the arginine codons used least frequently by Escherichia coli. The presence of these rare codons in tandem or in the first 20 codons of a gene can complicate expression of that gene in E. coli. Problems include premature chain termination and misincorporation of lysine for arginine. We therefore sought to improve the expression and subsequent yield of S. solfataricus HMG-CoA reductase by expanding the pool size of tRNA^AGA,AGG, the tRNA that recognizes these two rare codons. Coexpression of the S. solfataricus hmgA gene with the argU gene that encodes tRNA^AGA,AGG resulted in an over 10-fold increase in enzyme yield. This has provided significantly greater quantities of purified enzyme for potential industrial applications and for crystallographic characterization of a stable class I HMG-CoA reductase. It has, in addition, facilitated determination of kinetic parameters and of pH optima for all four catalyzed reactions, for determination of the K_i for inhibition by the statin drug mevinolin, and for comparison of the properties of the HMG-CoA reductase of this thermophilic archaeon to those of other class I HMG-CoA reductases.     

A carboxylesterase from the thermoacidophilic archaeon Sulfolobus solfataricus P1; purification,characterization, and expression

The carboxylesterase, a 34 kDa monomeric enzyme, was purified from the thermoacidophilic archaeon Sulfolobus solfataricus P1. The optimum temperature and pH were 85 °C and 8.0, respectively. The enzyme showed remarkable thermostability: 41% of its activity remained after 5 days of incubation at 80 °C. In addition, the purified enzyme exhibited stability against denaturing agents, including various detergents, urea, and organic solvents. The enzyme has broad substrate specificity towards various PNP esters and short acyl chain triacylglycerols such as tributyrin (C_4:0). Among the PNP esters tested, the best substrate was PNP-caprylate (C₈) with K_m and k_cat values of 71 μM and 14,700 s⁻¹, respectively. The carboxylesterase gene consisted of 915 bp corresponding to 305 amino acid residues. We demonstrated that active recombinant S. solfataricus carboxylesterase could be expressed in Escherichia coli. The enzyme was identified as a serine esterase belonging to mammalian hormone-sensitive lipases (HSL) family and contained a catalytic triad composed of serine, histidine, and aspartic acid in the active site.     

Purification, characterization, and cDNA structure of isoamylase from developing endosperm of rice

Isoamylase (EC 3.2.1.68) in rice (Oryza sativa L.) was efficiently purified within a day to homogeneity, as confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), from developing endosperm by sequential use of Q Sepharose HP anion- exchange chromatography, ammonium sulfate fractionation, and TSKgel G4000SW_XL and G3000SW_XL gel filtration chromatography. Although the protein exhibited a molecular size of ca. 83 kDa on SDS-PAGE, the apparent size of the native enzyme was approximately 340 and 490 kDa on TSKgel G3000SW_XL and G4000SW_XL gel filtration chromatograms, respectively, suggesting that rice isoamylase exists in a homo-tetramer to homo-hexamer form in developing endosperm. The purified rice isoamylase was able to debranch glycogen, phytoglycogen and amylopectin but could not attack pullulan. The optimum pH and temperature for isoamylase activity were found to be pH 6.5 to 7.0 and 30 °C, respectively. The enzyme activity was completely inhibited by HgCl₂ and p-chloromercuribenzoate at 1 mM. These results indicate that rice isoamylase possesses properties which are distinct from those reported for bacterial isoamylase. Complementary-DNA clones for rice endosperm isoamylase were isolated with a polymerase-chain-reaction product as probe which was generated by primers designed from nucleotides conserved in cDNA for maize Sugary-1 isoamylase (M.G. James et al., 1995, Plant Cell 7: 417–429) and a Pseudomonas amyloderamosa gene encoding isoamylase (A. Amemura et al., 1988, J Biol Chem 263: 9271–9275). The nucleotide sequence and deduced amino acid sequence of the longest clone showed a high similarity to those of maize Surgary-1 isoamylase, but a lesser similarity to those of Pseudomonas amyloderamosa isoamylase. Southern blot analysis and gene mapping analysis indicated that the isoamylase gene exists as a single copy in the rice genome and is located on chromosome 8 of cv. Nipponbare which belongs to the Japonica rice group. Phylogenetic analysis indicated that isoamylases from maize and rice are more closely related to a number of glgX gene products of the blue green alga Synechocystis and various bacteria than to isoamylases from Pseudomonas and Flavobacterium. Hence, it is proposed that glgX proteins are classified as isoamylase-type debranching enzymes. Our tree also showed that all starch- and glycogen-debranching enzymes from plants and bacteria tested can be classified into two distinct types, an isoamylase-type and a pullulanase-type. Received: 29 October 1998 / Accepted: 10 December 1998     

Cloning,expression and evolution of the gene encoding the elongation factor 1alpha from a low thermophilic Sulfolobus solfataricus strain

The gene encoding the elongation factor 1alpha (EF-1alpha) from the archaeon Sulfolobus solfataricus strain MT3 (optimum growth temperature 75 degrees C) was cloned, sequenced and expressed in Escherichia coli. The structural and biochemical properties of the purified enzyme were compared to those of EF-1alpha isolated from S. solfataricus strain MT4 (optimum growth temperature 87 degrees C). Only one amino acid change (Val15-->Ile) was found. Interestingly, the difference was in the first guanine nucleotide binding consensus sequence G(13)HIDHGK and was responsible for a reduced efficiency in protein synthesis, which was accompanied by an increased affinity for both guanosine diphosphate (GDP) and guanosine triphosphate (GTP), and an increased efficiency in the intrinsic GTPase activity. Despite the different thermophilicities of the two microorganisms, only very marginal effects on the thermal properties of the enzyme were observed. Molecular evolution among EF-1alpha genes from Sulfolobus species showed that the average rate of nucleotide substitution per site per year (0.0312x10(-9)) is lower than that reported for other functional genes.     

Purification, characterization and crystallization of pyrroline-5-carboxylate reductase from the hyperthermophilic archeon Sulfolobus Solfataricus

The gene SSO0495 (proC), which encodes pyrroline-5-carboxylate reductase (P5CR) from the thermoacidophilic archeon Sulfolobus solfataricus P2 (Ss-P5CR), was cloned and expressed. The purified recombinant enzyme catalyzes the thioproline dehydrogenase with concomitant oxidation of NAD(P)H to NAD(P)⁺. This archeal enzyme has an optimal alkaline pH in this reversible reaction and is thermostable with a half-life of approximately 30 min at 80 °C. At pH 9.0, the reverse activation rate is nearly 3-fold higher than at pH 7.0. The homopolymer was characterized by cross-linking and size exclusion gel filtration chromatography. Ss-P5CR was crystallized by the hanging-drop vapor-diffusion method at 37 °C. Diffraction data were obtained to a resolution of 3.5 Å and were suitable for X-ray structure determination.     

Methanogenesis from acetate: Physiology of a thermophilic, acetate-utilizing methanogenic bacterium

Abstract The biosynthesis of K88, K99 and F41 fibrillae by enterotoxigenic Escherichia coli strains was shown to be dependent on the growth phase of the cultures. An increase in adhesin production was observed, during exponential growth reaching its maximum at the end of this phase; thereafter adhesin production was arrested.
A simple and rapid purification procedure was developed for adhesins isolated from exponentially growing cells.     

Oligomeric and functional properties of a debranching enzyme (TreX) from the archaeon Sulfolobus solfataricus P2

A gene, treX, encoding a debranching enzyme previously cloned from the trehalose biosynthesis gene cluster of Sulfolobus solfataricus P2 was expressed in Escherichia coli as a His-tagged protein and the biochemical properties were studied. The specific activity of the S. solfataricus debranching enzyme (TreX) was highest at 75°C and pH 5.5. The enzyme exhibited hydrolysing activity toward α-1,6-glycosidic linkages of amylopectin, glycogen, pullulan, and other branched substrates, and glycogen was the preferred substrate. TreX has a high specificity for hydrolysis of maltohexaosyl α-1,6-β-cyclodextrin, indicating the high preference for side chains consisting of 6 glucose residues or more. The enzyme also exhibited 4-α-sulfoxide-glucan transferase activity, catalysing transfer of α-1,4-glucan oligosaccharides from one chain to another. Dimethyl sulfoxide (10%, v/v) increased the hydrolytic activity of TreX. Gel permeation chromatography and sedimentation equilibrium analytical ultracentrifugation revealed that the enzyme exists mostly as a dimer at pH 7.0, and as a mixture of dimers and tetramers at pH 5.5. Interestingly, TreX existed as a tetramer in the presence of DMSO at pH 5.5–6.5. The tetramer showed a 4-fold higher catalytic efficiency than the dimer. The enzyme catalysed not only intermolecular trans-glycosylation of malto-oligosaccharides (disproportionation) to produce linear α-1,4-glucans, but also intramolecular trans-glycosylation of glycogen. The results presented in this study indicated that TreX may be associated with glycogen metabolism by selective cleavage of the outer side chain.     

Characterization, sequence and mode of replication of plasmid pNB2 from the thermophilic bacterium Clostridium thermosaccharolyticum

 The 1882-bp nucleotide sequence of the cryptic plasmid pNB2 isolated from the thermophilic bacterium Clostridium thermosaccharolyticum was determined. pNB2 DNA has very low GC content (27%) and may serve as a model for studying the modes of maintenance and replication of AT-rich DNA under conditions of thermophilic growth. The plasmid sequence revealed three open reading frames (ORFs) which would encode polypeptides of 289, 68 and 59 amino acids, respectively, and these proteins were synthesized in E. coli extracts primed with the plasmid. We found that the product of ORF289 may be initiated at the non-ATG start codon, TTG, and has similarities with the conserved motifs of Rep proteins encoded by rolling circle (RC) plasmids of the pC194/pUB110 family. Southern hybridization analysis of lysates of C. thermosaccharolyticum cells harboring pNB2 revealed single-stranded intermediates, suggesting that this plasmid is able to replicate in clostridial cells via the RC mechanism. The most significant similarities are found between pNB2 Rep protein and the Rep proteins of three RC plasmids of the pC194 family (pTB913, pBC1 and pST1) isolated from thermophilic bacteria. Comparative analysis of these Rep proteins showed that despite the significant level of divergence, these Rep proteins share a high degree of similarity in the regions of five well-known conserved domains of RC Rep proteins and fall into two groups in accordance with the similarities found in their active sites. Received: 25 April 1996 / Accepted: 20 June 1996     

Immobilization of thermostable β-glucosidase from Sulfolobus shibatae by cross-linking with transglutaminase

Thermostable β-glucosidase from Sulfolobus shibatae was immobilized on silica gel modified or not modified with 3-aminopropyl-triethoxysilane using transglutaminase as a cross-linking factor. Obtained preparations had specific activity of 3883 U/g of the support, when measured at 70 °C using o-nitrophenyl β-d-galactopyranoside (GalβoNp) as substrate. The highest immobilization yield of the enzyme was achieved at pH 5.0 in reaction media. The most active preparations of immobilized β-glucosidase were obtained at a transglutaminase concentration of 40 mg/ml at 50 °C. The immobilization was almost completely terminated after 100 min of the reaction and prolonged time of this process did not cause considerable changes of the activity of the preparations. The immobilization did not influence considerably on optimum pH and temperature of GalβoNp hydrolysis catalyzed by the investigated enzyme (98 °C, pH 5.5). The broad substrate specifity and properties of the thermostable β-glucosidase from S. shibatae immobilized on silica-gel indicate its suitability for hydrolysis of lactose during whey processing.     

Enzymes from Sulfolobus shibatae for the production of trehalose and glucose from starch

Enzymes that convert starch and dextrins to α,α-trehalose and glucose were found in cell homogenates of the hyperthermophilic acidophilic archaeon Sulfolobus shibatae DMS 5389. Three enzymes were purified and characterized. The first, the S. shibatae trehalosyl dextrin-forming enzyme (SsTDFE), transformed starch and dextrins to the corresponding trehalosyl derivatives with an intramolecular transglycosylation process that converted the glucosidic linkage at the reducing end from α-1,4 to α-1,1. The second, the S. shibatae trehalose-forming enzyme (SsTFE), hydrolyzed the α-1,4 linkage adjacent to the α-1,1 bond of trehalosyl dextrins, forming trehalose and lower molecular weight dextrins. These two enzymes had molecular masses of 80 kDa and 65 kDa, respectively, and showed the highest activities at pH 4.5. The apparent optimal temperature for activity was 70°C for SsTDFE and 85°C for SsTFE. The third enzyme identified was an α-glycosidase (SsαGly), which catalyzed the hydrolysis of the α-1,4 glucosidic linkages in starch and dextrins, releasing glucose in a stepwise manner from the nonreducing end of the polysaccharide chain. The enzyme had a molecular mass of 313 kDa and showed the highest activity at pH 5.5 and at 85°C. Received: October 29, 1997 / Accepted: April 29, 1998     

Biochemical and structural studies of a l-haloacid dehalogenase from the thermophilic archaeon Sulfolobus tokodaii

Haloacid dehalogenases have potential applications in the pharmaceutical and fine chemical industry as well as in the remediation of contaminated land. The l-2-haloacid dehalogenase from the thermophilic archaeon Sulfolobus tokodaii has been cloned and over-expressed in Escherichia coli and successfully purified to homogeneity. Here we report the structure of the recombinant dehalogenase solved by molecular replacement in two different crystal forms. The enzyme is a homodimer with each monomer being composed of a core-domain of a β-sheet bundle surrounded by α-helices and an α-helical sub-domain. This fold is similar to previously solved mesophilic l-haloacid dehalogenase structures. The monoclinic crystal form contains a putative inhibitor l-lactate in the active site. The enzyme displays haloacid dehalogenase activity towards carboxylic acids with the halide attached at the C2 position with the highest activity towards chloropropionic acid. The enzyme is thermostable with maximum activity at 60°C and a half-life of over 1 h at 70°C. The enzyme is relatively stable to solvents with 25% activity lost when incubated for 1 h in 20% v/v DMSO.     

Discovery of a putative acetoin dehydrogenase complex in the hyperthermophilic archaeon Sulfolobus solfataricus

Like many other aerobic archaea, the hyperthermophile Sulfolobus solfataricus possesses a gene cluster encoding components of a putative 2-oxoacid dehydrogenase complex. In the current paper, we have cloned and expressed the first two genes of this cluster and demonstrate that the protein products form an α₂β₂ hetero-tetramer possessing the catalytic activity characteristic of the first component enzyme of an acetoin dehydrogenase multienzyme complex. This represents the first report of an acetoin multienzyme complex in archaea, and contrasts with the branched-chain 2-oxoacid dehydrogenase complex activities characterised in two other archaea, Thermoplasma acidophilum and Haloferax volcanii.     

Characterization of an inducible nitrilase from a thermophilic bacillus

Nitrilase activity was induced in the thermophilic bacterium Bacillus pallidus strain Dac521 by growth on benzonitrile-supplemented minimal medium. The enzyme had a subunit relative molecular mass of 41 kDa but was purified as a complex with a putative GroEL protein (total M _r, 600 kDa). The enzyme catalyzed the hydrolysis of aliphatic, aromatic, and heterocyclic nitriles with widely varying k _cat/K _M values, primarily the result of differences in substrate affinity. Of the nitriles tested, 4-cyanopyridine was hydrolyzed at the fastest rate. Substitution of benzonitrile at the meta or para position either had no effect on catalytic rate or enhanced k _cat, while ortho-substitution was strongly inhibitory, probably because of steric hindrance. The effect of catalytic inhibitors was consistent with the presence of active site thiol residues although activity was little affected by putative thiol reagents such as iodoacetate, iodoacetamide, and N-methylmaleimide. Enzymatic activity was constant between pH 6 and 9 with an optimum at pH 7.6. The optimal temperature for activity was 65°C with rapid activity loss at higher temperatures. The purified nitrilase-GroEL complex had the following half-lives of activity: 8.4 h at 50°C, 2.5 h at 60°C, 13 min at 70°C, and less than 3 min at 80°C. Received: March 1, 1999 / Accepted: August 3, 1999     

Purification and properties of thermostable lipase from a thermophilic Bacillus stearothermophilus MC 7

Extracellular thermostable lipase produced by the thermophilic Bacillus stearothermophilus MC 7 was purified to 19.25-fold with 10.2% recovery. The molecular weight of the purified enzyme determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was shown to be 62 500 Da. The purified enzyme expressed maximum activity at 75–80 °C and its half life was 30 min at 70 °C. The K_m and V_max were calculated to be, respectively, 0.33 mM and 188 μM min⁻¹ mg⁻¹ with p-nitrophenyl palmitate (pNPP) as a substrate. Enzyme activity was inhibited by divalent ions of heavy metals, thiol and serine inhibitors, whereas calcium ion stimulated its activity. The most advantageous method for immobilization was found to be ionic binding to DEAE Cellulose. The enzyme was able to hydrolyze both soluble and insoluble emulsified substrates and was classified as a lipase, expressing some esterase activity as well.