A thermostable #x003B2;-ketothiolase of polyhydroxyalkanoates (PHAs) in <Emphasis Type="Italic">Thermus thermophilus</Emphasis>: Purification and biochemical properties

Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyalkanoates (HAs) synthesised by numerous bacteria as intracellular carbon and energy storage compounds which accumulate as granules in the cytoplasm of the cells. The biosynthesis of PHAs, in the thermophilic bacterium T. thermophilus grown in a mineral medium supplemented with sodium gluconate as sole carbon source has been recently reported. Here, we report the purification at apparent homogeneity of a #x003B2;-ketoacyl-CoA thiolase from T. thermophilus, the first enzyme of the most common biosynthetic pathway for PHAs. B-Ketoacyl-CoA thiolase appeared as a single band of 45.5-kDa molecular mass on SDS/PAGE. The enzyme was purified 390-fold with 7% recovery. The native enzyme is a multimeric protein of a molecular mass of approximately of 182 kDa consisting of four identical subunits of 45.5 kDa, as identified by an in situ renaturation experiment on SDS-PAGE. The enzyme exhibited an optimal pH of approximately 8.0 and highest activity at 65 °C for both direction of the reaction. The thiolysis reaction showed a substrate inhibition at high concentrations; when one of the substrates (acetoacetyl CoA or CoA) is varied, while the concentrations of the second substrates (CoA or acetoacetyl CoA respectively) remain constant. The initial velocity kinetics showed a pattern of a family of parallel lines, which is in accordance with a ping-pong mechanism. #x003B2;-Ketothiolase had a relative low Km of 0.25 mM for acetyl-CoA and 11 M and 25 M for CoA and acetoacetyl-CoA, respectively. The enzyme was inhibited by treatment with 1 mM N-ethylmaleimide either in the presence or in the absence of 0.5 mM of acetyl-CoA suggesting that possibly a cysteine is located at/or near the active site of #x003B2;-ketothiolase. (Mol Cell Biochem 269: 27–36, 2005)     

Polyhydroxyalkanoate (PHA) biosynthesis in Thermus thermophilus: Purification and biochemical properties of PHA synthase

The biosynthesis of polyhydroxyalkanoates (PHAs) was studied, for the first time, in the thermophilic bacterium Thermus thermophilus. Using sodium gluconate (1.5% w/v) or sodium octanoate (10 mM) as sole carbon sources, PHAs were accumulated to approximately 35 or 40% of the cellular dry weight, respectively. Gas chromatographic analysis of PHA isolated from gluconate-grown cells showed that the polyester (M_w: 480,000 g.mol^–1) was mainly composed of 3-hydroxydecanoate (3HD) with a molar fraction of 64%. In addition, 3-hydroxyoctanoate (3HO), 3-hydroxyvalerate (3HV) and 3-hydroxybutyrate (3HB) occurred as constituents. In contrast, the polyester (M_w: 391,000 g mol^–1) from octanoate-grown cells was composed of 24.5 mol% 3HB, 5.4 mol% 3HO, 12.3 mol% 3-hydroxynonanoate (3HN), 14.6 mol% 3HD, 35.4 mol% 3-hydroxyundecanoate (3HUD) and 7.8 mol% 3-hydroxydodecanoate (3HDD). Activities of PHA synthase, a -ketothiolase and an NADPH-dependent reductase were detected in the soluble cytosolic fraction obtained from gluconate-grown cells of T. thermophilus. The soluble PHA synthase was purified 4271-fold with 8.5% recovery from gluconate-grown cells, presenting a K_m of 0.25 mM for 3HB-CoA. The optimal temperature of PHA synthase activity was about 70°C and acts optimally at pH near 7.3. PHA synthase activity was inhibited 50% with 25 M CoA and lost all of its activity when it was treated with alkaline phosphatase. PHA synthase, in contrary to other reported PHA synthases did not exhibit a lag phase on its kinetics, when low concentration of the enzyme was used. Incubation of PHA synthase with 1 mM N-ethyl-maleimide inhibits the enzyme 56%, indicating that cysteine might be involved in the catalytic site of the enzyme. Acetyl phosphate (10 mM) activated both the native and the dephosphorylated enzyme. A major protein (55 kDa) was detected by SDS-PAGE. When a partially purified preparation was analyzed on native PAGE the major band exhibiting PHA synthase activity was eluted from the gel and analyzed further on SDS-PAGE, presenting the first purification of a PHA synthase from a thermophilic microorganism.     

Novel thermostable ssDNA-binding proteins from Thermus thermophilus and T. aquaticus-expression and purification

Single-stranded DNA-binding proteins (SSBs) play essential roles in DNA replication, recombination, and repair in bacteria, archaea, and eukarya. We report here the identification, expression, and purification of the SSB-like proteins of the thermophilic bacteria Thermus thermophilus and T. aquaticus. The nucleotide (nt) sequence revealed that T. thermophilus SSB (TthSSB) and T. aquaticus (TaqSSB) consist of 264 and 266 amino acids, respectively, and have a molecular weight of 29.87 and 30.03kDa, respectively. The homology between these protein, is very high-82% identity and 90% similarity. They are the largest known prokaryotic SSB proteins. TthSSB and TaqSSB monomers have two putative ssDNA-binding sequences: N-terminal (located in the region from amino acids 1 to 123) and C-terminal (located between amino acids 124 and 264 or 266 in TthSSB and TaqSSB, respectively). PCR-derived DNA fragment containing the complete structural gene for TthSSB or TaqSSB protein was cloned into an expression vector. The clones expressing SSB-like proteins were selected and cloned DNA fragments were verified to be authentic by sequencing several clones. The purification was carried out using reduction of contamination by the host protein with heat treatment, followed by QAE-cellulose and ssDNA-cellulose column chromatography. We found our expression and purification system to be quite convenient and efficient, and will use it for production of thermostable SSB-proteins for crystallography study. We have applied the use of TthSSB and TaqSSB protein to increase the amplification efficiency with a number of diverse templates. The use of SSB protein may prove to be generally applicable in improving the PCR efficiency.     

Improved detergent-based recovery of polyhydroxyalkanoates (PHAs)

Extracting polyhydroxyalkanoate (PHA) polymer from bacterial cells often involves harsh conditions, including use of environmentally harmful solvents. We evaluated different detergents under various conditions to extract PHA from Ralstonia eutropha and Escherichia coli cells. Most detergents tested recovered highly pure PHA polymer from cells in amounts that depended on the percentage of polymer present in the cell. Detergents such as linear alkylbenzene sulfonic acid (LAS-99) produced a high yield of high purity polymer, and less detergent was needed compared to the amount of SDS to produce comparable yields. LAS-99 also has the advantage of being biodegradable and environmentally safe. Chemical extraction of PHA with detergents could potentially minimize or eliminate the need to use harsh organic solvents, thus making industrial PHA production a cleaner technology process.     

Efficient selection for thermostable protease in Thermus thermophilus

An efficient procedure was established to select for thermostable proteases in an extreme thermophile, Thermus thermophilus. A non-protease-secreting mutant derived from T. thermophilus TH125 was used as host and the expression plasmid for aqualysin I from T. aquaticus YT-1 was constructed as a source of thermostable protease. T. thermophilus cells harboring the recombinant plasmid produced active aqualysin I into the medium and were able to grow on a minimal medium containing milk casein as the sole source of carbon and nitrogen.     

Production of polyhydroxyalkanoates from whey by Thermus thermophilus HB8

The thermophilic bacterium Thermus thermophilus HB8 is able to utilize lactose from whey-based media for the biosynthesis of polyhydroxyalkanoates (PHAs) under nitrogen limitation. T. thermophilus can utilize both, glucose and galactose, the products of lactose hydrolysis. When T. thermophilus HB8 was grown in culture media containing 24% (v/v) whey, PHA was accumulated up to 35% (w/w) of its biomass after 24 h of cultivation. The effect of initial phosphate concentration on the PHA production was also investigated. Using an initial phosphate concentration of 50 mM the PHA accumulation was enhanced. Analysis of the produced PHA from T. thermophilous HB8 grown in whey-based media revealed a novel heteropolymer consisting of the short chain length 3-hydroxyvalerate (3HV; 38 mol%) and the medium chain length, 3-hydroxyheptanoate (3HHp; 9.89 mol%), 3-hydroxynanoate (3HN; 16.59 mol%) and 3-hydroxyundecanoate (3HU; 35.42 mol%). Despite the low molecular weight of the produced PHA by T. thermophilus, whey could be an excellent substrate for the production of heteropolymers with unique properties.     

Sustainable synthesis and applications of polyhydroxyalkanoates (PHAs) from biomass

Polyhydroxyalkanoates (PHAs) belong to group of biopolymers that have in recent times received growing research interest as a result of being eco-friendly and close characteristics with petrochemical based plastics. Alternatives to utilization of synthetic plastics are being explored since synthetic plastics are non-recyclable and non-biodegradable in nature. One of the innovations of Green Chemistry is utilization of renewable feedstocks such as biomass to achieve sustainable development with future circular economy. Bio-based products are of great interest to sustainable development as a result of diminishing fossil fuel reserves and rising environmental concerns. This review summarizes the productions of PHAs from renewable feedstocks such as lignocellulose, crude glycerol, levulinic acid (LA), palm-oil mill effluents (POME) and waste oils. The production of bio-based polymers has become much more professional and differentiated in recent years. Presently, there are bio-based alternatives for practically every application, therefore, this review presents applications of PHA in bio-refinery, medical sectors, agriculture sector, construction industry, and in packaging industry. The cost analysis of PHA from renewable sources with commercially available ones and potential to attain circular economy were also stressed. The reasons for this shift are connected to the non-renewability of fossil-based resources, the deteriorating environmental impacts, and the lack of biodegradability of the petroleum-produced materials.     

Crystal structures of thermostable xylose isomerases from Thermus caldophilus and Thermus thermophilus: possible structural determinants of thermostability.

The crystal structures of highly thermostable xylose isomerases from Thermus thermophilus (TthXI) and Thermus caldophilus (TcaXI), both with the optimum reaction temperature of 90 degrees C, have been determined by X-ray crystallography. The model of TcaXI has been refined to an R-factor of 17.8 % for data extending to 2.3 A and that of TthXI to 17.1 % for data extending to 2.2 A. The tetrameric arrangement of subunits characterized by the 222-symmetry and the tertiary fold of each subunit in both TcaXI and TthXI are basically the same as in other xylose isomerases. Each monomer is composed of two domains. Domain I (residues 1 to 321) folds into the (beta/alpha)8-barrel. Domain II (residues 322 to 387), lacking beta-strands, makes extensive contacts with domain I of an adjacent subunit. Each monomer of TcaXI contains ten beta-strands, 15 alpha-helices, and six 310-helices, while that of TthXI contains ten beta-strands, 16 alpha-helices, and five 310-helices. Although the electron density does not indicate the presence of bound metal ions in the present models of both TcaXI and TthXI, the active site residues show the conserved structural features. In order to understand the structural basis for thermostability of these enzymes, their structures have been compared with less thermostable XIs from Arthrobacter B3728 and Actinoplanes missouriensis (AXI and AmiXI), with the optimum reaction temperatures of 80 degrees C and 75 degrees C, respectively. Analyses of various factors that may affect protein thermostability indicate that the possible structural determinants of the enhanced thermostability of TcaXI/TthXI over AXI/AmiXI are (i) an increase in ion pairs and ion-pair networks, (ii) a decrease in the large inter-subunit cavities, (iii) a removal of potential deamidation/isoaspartate formation sites, and (iv) a shortened loop.     

Recovery of medium-chain-length polyhydroxyalkanoates (PHAs) through enzymatic digestion treatments and ultrafiltration

Medium-chain-length (mcl) polyhydroxyalkanoates (PHAs) are biodegradable polyesters accumulated intracellularly as energy resources by bacterial species such as Pseudomonas putida. The most popular method for PHA recovery in the downstream processing is solvent extraction using chloroform and methanol. An alternate method is bioseparation using enzymatic digestion process which eliminates the need for hazardous solvents. This research focuses on an attempt to optimize the recovery of PHAs by solubilisation of non-PHA granules through enzymatic treatments such as; Alcalase (to digest the denatured proteins), sodium dodecyl sulfate (SDS) to assist solubilisation, ethylene diamine tetra acetic acid (EDTA) to complex divalent cations and lysozyme to digest the peptidoglycan wall enveloping the cell. The experiment was designed through Taguchi's design of experiment (DOE) using Qualitek-4 software. The results show that Alcalase enzyme used had the most significant effect on the treatment process and contributed to about 71.5% in terms of process factor importance among the different factors on treatment performance for PHA recovery. It is desired to recover the PHA granules in water suspension after the enzymatic treatment by removing the solubilised non-PHA cell material through crossflow ultrafiltration system and purified through continuous diafiltration process. Final purity of PHA in water suspension obtained using GC analysis is 92.6%, with a nearly 90% recovery, thus concluding that this method is indeed a suitable alternative.     

Production of polyhydroxyalkanoates (PHAs) with canola oil as carbon source

Wautersia eutropha was able to synthesize medium chain length polyhydroxyalkanoates (PHAs) when canola oil was used as carbon source. W. eutropha was cultivated using fructose and ammonium sulphate as carbon and nitrogen sources, respectively, for growth and inoculum development. The experiments were done in a laboratory scale bioreactor in three stages. Initially, the biomass was adapted in a batch culture. Secondly, a fed-batch was used to increase the cell dry weight and PHA concentration to 4.36 g L(-1) and 0.36 g L(-1), respectively. Finally, after the addition of canola oil as carbon source a final concentration of 18.27 g L(-1) PHA was obtained after 40 h of fermentation. With canola oil as carbon source, the polymer content of the cell dry matter was 90%. The polymer was purified from dried cells and analyzed by FTIR, NMR and DSC using PHB as reference. The polymer produced by W. eutropha from canola oil had four carbon monomers in the structure of the PHA and identified by 1H and 13C NMR analysis as 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), 3-hydroxyoctanoate (3HO), and 3-hydroxydodecanoate (3HDD).     

Identification of extracellular lipases/esterases produced by Thermus thermophilus HB27: partial purification and preliminary biochemical characterisation

Thermus thermophilus HB27 produces important levels of extracellular lipolytic activity when grown for 30 h at 70 degrees C in a complex medium. A method to detect esterase activity in these samples after non-reducing SDS-polyacrylamide electrophoresis was developed. The method, that implies the renaturalisation of the enzymes in the SDS-gels by washing with Triton X-100 at high temperatures, allowed detecting three esterases with different molecular weights (108, 62 and 34 kDa, respectively). The electrophoretic mobility determined under different experimental conditions suggested that the 34- and 108 kDa-esterases might correspond with two oligomeric states of a sole enzyme (monomer and trimer). Dissociation of the trimer into the monomer started when the samples were heated at temperatures higher than 60 degrees C in the presence of sodium dodecyl sulphate. Evidences were found that indicated the independent nature of the 62 kDa-esterase. A method to purify these enzymes from postincubates of T. thermophilus HB27 was developed following three steps: sodium cholate treatment, ethanol/ether precipitation and hydrophobic chromatography. In this way, an enzyme solution was obtained that contained the identified esterases/lipases. The partially purified enzymes showed an optimum of activity for the hydrolysis of p-nitrophenyl laurate at alkaline pH values and 80 degrees C, a high thermal stability and were very stable in the presence of high concentrations of isopropanol.     

Beta-glycosidase of Thermus thermophilus KNOUC202: gene and biochemical properties of the enzyme expressed in Escherichia coli

The beta-glycosidase gene of Thermus thermophilus KNOUC202 was cloned, expressed in Escherichia coli JM109(DE3), and the enzyme was purified and characterized. The gene (KNOUC202/beta-gly) was composed of 1296 bp encoding a beta-glycosidase (KNOUC202beta-glycosidase) of 431 a.a., belonging to the family 1 of glycosyl hydrolase. The gene was expressed as monomer of 430 a.a. with amino terminal methionine excised in E. coli JM109(DE3). The enzyme hydrolyzed beta-glycosides whose glycone are galactose, glucose and fucose well, however showed no or very low activity on beta-D-glycosides whose glycone are disaccharides and xylose. kcat of the enzyme for the hydrolysis of p-Nph-beta-D-Glcp was lower than those for p-Nph-beta-D-Galp and ONPG, however K(m) for p-Nph-beta-D-Glcp was highly lower than those for p-Nph-beta-D-Galp and ONPG resulting in the catalytic efficiency(k(cat)/K(m)) for the hydrolysis of p-Nph-beta-D-Glcp much higher than those for p-Nph-beta-D-Galp and ONPG. Optimum pH and optimum temperature of the enzyme were pH 5.4 and 90 degrees C. The enzyme has high thermostability, not losing its activity at 80 degrees C for 2 h in 0.05 M Na-phosphate buffer of pH 6.8 with T(m) of 100.0 +/- 0.031 degrees C in 0.02 M Tris-HCl buffer of pH 8.2. The beta-glycosidase produced a disaccharide composed of galactose as transglycosylation byproduct during hydrolysis of lactose.     

Efficient production of thermostable Thermus thermophilus xylose isomerase in Escherichia coli and Bacillus brevis

Summary The xylose (glucose) isomerase from the thermophile Thermus thermophilus seems to have potential for the development of new isomerization processes using high temperatures and slightly acidic pH. The isomerase has an optimum temperature at 95° C, and is also very stable at high temperatures. The optimum pH is around 7.0, close to where by-product formation is minimal. Since Thermus produces only a little of this useful isomerase, the production of the cloned gene in Escherichia coli and Bacillus brevis were compared. Especially B. brevis was able to produce the isomerase effeciently, more than 1 g/l, in spite of the high G + content (67%) of the Thermus gene, and the presence of codons not frequently used in E. coli or B. brevis.Offprint requests to: S. Udaka     

A DING phosphatase in Thermus thermophilus

Summary. Phosphate transport in bacteria occurs via a phosphate specific transporter system (PSTS) that belongs to the ABC family of transporters, a multisubunit system, containing an alkaline phosphatase. DING proteins were characterized due to the N-terminal amino acid sequence DINGG GATL, which is highly conserved in animal and plant isolates, but more variable in microbes. Most prokaryotic homologues of the DING proteins often have some structural homology to phosphatases or periplasmic phosphate-binding proteins. In E. coli, the product of the inducible gene DinG, possesses ATP hydrolyzing helicase enzymic activity. An alkaline phosphorolytic enzyme of the PSTS system was purified to homogeneity from the thermophilic bacterium Thermus thermophilus. N-terminal sequence analysis of this protein revealed the same high degree of similarity to DING proteins especially to the human synovial stimulatory protein P205, the steroidogenesis-inducing protein and to the phosphate ABC transporter, periplasmic phosphate-binding protein, putative (P. fluorescens Pf-5). The enzyme had a molecular mass of 40 kDa on SDS/PAGE, exhibiting optimal phosphatase activity at pH 12.3 and 70 °C. The enzyme possessed characteristics of a DING protein, such as ATPase, ds endonuclease and 3′ phosphodiesterase (3′-exonuclease) activities and binding to linear dsDNA, displaying helicase activity on supercoiled DNA. Purification and biochemical characterization of a T. thermophilus DING protein was achieved. The biochemical properties, N-terminal sequence similarities of this protein implied that the enzyme belongs to the PSTS family and might be involved in the DNA repair mechanism of this microorganism. Authors’ address: Assist. Prof. A. A. Pantazaki, Laboratory of Biochemistry, Department of Chemistry, Aristotle University of Thessaloniki, Thessaloniki 54124, Greece     

Directed evolution of thermostable kanamycin-resistance gene: a convenient selection marker for Thermus thermophilus.

The whole-genome sequencing of an extreme thermophile, Thermus thermophilus, is now in progress. Like other genome projects, major concern is shifting from the sequence itself to post-sequencing research such as functional or structural genomics. Under such circumstances, the demand for convenient genetic-engineering tools is increasing. In this study we have increased the thermostability of a kanamycin-resistance gene product using strategies based on directed evolution in T. thermophilus to the upper limit of its growth temperature. The most thermostable mutant has 19 amino-acid substitutions, whereby the thermostability is increased by 20 degrees C, but the enzymatic activity is not significantly changed. Most of the mutated residues are located on the surface of the protein molecule, and, interestingly, five of the 19 substitutions are those to proline residues. The evolved kanamycin-resistance gene products could be used as selection markers at the optimum growth temperature of T. thermophilus. The development of such a convenient genetic-engineering tool would facilitate post-sequencing research on T. thermophilus.     

Preliminary characterization and crystal structure of a thermostable cytochrome P450 from Thermus thermophilus

The second structure of a thermophile cytochrome P450, CYP175A1 from the thermophilic bacterium Thermus thermophilus HB27, has been solved to 1.8-A resolution. The overall P450 structure remains conserved despite the low sequence identity between the various P450s. The CYP175A1 structure lacks the large aromatic network found in the only other thermostable P450, CYP119, thought to contribute to thermal stability. The primary difference between CYP175A1 and its mesophile counterparts is the investment of charged residues into salt-link networks at the expense of single charge-charge interactions. Additional factors involved in the thermal stability increase are a decrease in the overall size, especially shortening of loops and connecting regions, and a decrease in the number of labile residues such as Asn, Gln, and Cys.     

Disruption of Thermus thermophilus genes by homologous recombination using a thermostable kanamycin-resistant marker

Genes of an extremely thermophilic bacterium, Thermus thermophilus, were disrupted by homologous recombination using a recently developed, thermostable kanamycin-resistant marker. First, the trpE gene was disrupted with various constructions of DNA. The transformation efficiency was exponentially increased as the length of the homologous regions flanking the marker gene increased above the minimum length (200-300 bp). We then disrupted five genes of the nucleotide excision repair system and examined their phenotypes. The convenience and high reliability of this method should prompt its application to the high-throughput systematic disruption of the genes of this thermophilic bacterium.     

Molecular characterization of a novel thermostable mannose-6-phosphate isomerase from Thermus thermophilus

Mannose-6-phosphate isomerase catalyzes the interconversion of mannose-6-phosphate and fructose-6-phosphate. The gene encoding a putative mannose-6-phosphate isomerase from Thermus thermophilus was cloned and expressed in Escherichia coli. The native enzyme was a 29 kDa monomer with activity maxima for mannose 6-phosphate at pH 7.0 and 80 °C in the presence of 0.5 mM Zn²⁺ that was present at one molecule per monomer. The half-lives of the enzyme at 65, 70, 75, 80, and 85 °C were 13, 6.5, 3.7, 1.8, and 0.2 h, respectively. The 15 putative active-site residues within 4.5 Å of the substrate mannose 6-phosphate in the homology model were individually replaced with other amino acids. The sequence alignments, activities, and kinetic analyses of the wild-type and mutant enzymes with amino acid changes at His50, Glu67, His122, and Glu132 as well as homology modeling suggested that these four residues are metal-binding residues and may be indirectly involved in catalysis. In the model, Arg11, Lys37, Gln48, Lys65 and Arg142 were located within 3 Å of the bound mannose 6-phosphate. Alanine substitutions of Gln48 as well as Arg142 resulted in increase of K_m and dramatic decrease of k_cat, and alanine substitutions of Arg11, Lys37, and Lys65 affected enzyme activity. These results suggest that these 5 residues are substrate-binding residues. Although Trp13 was located more than 3 Å from the substrate and may not interact directly with substrate or metal, the ring of Trp13 was essential for enzyme activity.     

Recognition sites of tRNA by a thermostable tRNA(guanosine-2'-)-methyltransferase from Thermus thermophilus HB27

Recognition sites of tRNA by tRNA(guanosine-2'-)-methyltransferase (Gm-methylase) [EC 2.1.1.34] from an extreme thermophile, Thermus thermophilus HB27, were studied by two independent methods--fragment reactions and footprinting analyses, using yeast tRNA(Phe) and Escherichia coli tRNA(fMet) as substrates. None of the tRNA-derived oligonucleotides which have the G-G sequence but are not long enough to form the "stem-loop" structure could be methylated by Gm-methylase. The 5'-half fragments having the intact D-"stem-loop" structure served as substrates for Gm-methylase, with a similar Vmax but 6-8 times larger Km, as compared with the intact tRNAs. The results of footprinting analyses were consistent with the foregoing findings. Gm-methylase protected only the D-loop region of tRNA from RNase T1 attack, but other parts of tRNA extending from the amino acid stem to the T arm became more sensitive to RNase T1, suggesting a considerable change of tRNA tertiary structure due to complex formation with Gm-methylase. These results indicate that a D-"stem-loop" structure is a prerequisite for recognition by Gm-methylase.