Enzymatic analysis of a thermostabilized mutant of an Escherichia coli hygromycin B phosphotransferase

An Escherichia coli hygromycin B phosphotransferase (HPH) and its thermostabilized mutant protein, HPH5, containing five amino acid substitutions, D20G, A118V, S225P, Q226L, and T246A (Nakamura et al., J. Biosci. Bioeng., 100, 158-163 (2005)), obtained by an in vivo directed evolution procedure in Thermus thermophilus, were produced and purified from E. coli recombinants, and enzymatic comparisons were performed. The optimum temperatures for enzyme activity were 50 and 55 degrees C for HPH and HPH5 respectively, but the thermal stability of the enzyme activity and the temperature for protein denaturation of HPH5 increased, from 36 and 37.2 degrees C of HPH to 53 and 58.8 degrees C respectively. Specific activities and steady-state kinetics measured at 25 degrees C showed only slight differences between the two enzymes. From these results we concluded that HPH5 was thermostabilized at the protein level, and that the mutations introduced did not affect its enzyme activity, at least under the assay conditions.     

High pressure homogenization inactivation of Escherichia coli and Staphylococcus aureus in phosphate buffered saline,milk and apple juice

High pressure homogenization (HPH) offers new opportunities for food pasteurization/sterilization. Escherichia coli and Staphylococcus aureus suspended in phosphate buffered saline (PBS) buffer, milk and apple juice at initial concentration of ~10⁶ log₁₀ CFU per ml were subjected to HPH treatments up to 200 MPa with inlet temperatures at 4–40°C. After HPH at 200 MPa with the inlet temperature at 40°C, the count of E. coli suspended in PBS, milk and apple juice reduced by 3·42, 3·67 and 3·19 log₁₀ CFU per ml respectively while the count of S. aureus decreased by 2·21, 1·02 and 2·33 log₁₀ CFU per ml respectively suggesting that S. aureus was more resistant. The inactivation data were well fitted by the polynomial equation. Milk could provide a protective effect for S. aureus against HPH. After HPH at 200 MPa with the inlet temperature at 20°C, the cell structure of E. coli was destroyed, while no obvious damages were found for S. aureus.     

Characterization and a point mutational approach of a psychrophilic lipase from an arctic bacterium,Bacillus pumilus

A bacterium with lipolytic activity was isolated from the Chukchi Sea within the Arctic Ocean. The lipase BpL5 from the isolate, Bacillus pumilus ArcL5, belongs to subfamily 4 of lipase family I. The optimum pH and temperature of the recombinant enzyme BpL5, as expressed in Escherichia coli, were 9.0 and 20 °C, respectively. The enzyme retained 85 % of its activity at 5 °C. There was a significant difference between temperatures for maximal activity (20 °C) and for protein denaturation (approx. 45 °C). The enzyme preferred middle-chain (C8) p-nitrophenyl substrates. Two mutants, S139A and S139Y, were rationally designed based on the 3D-structure model, and their activities were compared with that of the wild type. The both mutants showed significantly improved activity against tricaprylin.     

Characterization of a thermostable lipase showing loss of secondary structure at ambient temperature

A gene encoding extracellular lipase was cloned and characterized from metagenomic DNA extracted from hot spring soil. The recombinant gene was expressed in E. coli and expressed protein was purified to homogeneity using hydrophobic interactions chromatography. The mature polypeptide consists of 388 amino acids with apparent molecular weight of 43 kDa. The enzyme displayed maximum activity at 50°C and pH 9.0. It showed thermal stability up to 40°C without any loss of enzyme activity. Nearly 80% enzyme activity was retained at 50°C even after incubation for 75 min. However above 50°C the enzyme displayed thermal instability. The half life of the enzyme was determined to be 5 min at 60°C. Interestingly the CD spectroscopic study carried out in the temperature range of 25–95°C revealed distortion in solution structure above 35°C. However the intrinsic tryptophan fluorescence spectroscopic study revealed that even with the loss of secondary structure at 35°C and above the tertiary structure was retained. With p-nitrophenyl laurate as a substrate, the enzyme exhibited a K _m , V _max and K _cat of 0.73 ± 0.18 μM, 239 ± 16 μmol/ml/min and 569 s⁻¹ respectively. Enzyme activity was strongly inhibited by CuCl₂, HgCl₂ and DEPC but not by PMSF, eserine and SDS. The protein retained significant activity (~70%) with Triton X-100. The enzyme displayed 100% activity in presence of 30% n-Hexane and acetone.     

Changes in Hypocholesterolemic Activity in Rats by Various Konnyaku Powder Treatments

Galactosylsucroses contained in soybeans are not digestible. Thus we wished to detect α-galactosidase (EC 3.2.1.22) in intestinal bacteria. The strain of E. coli in the title was found to produce considerably this enzyme adaptively. We could prepare rather pure solution of the enzyme from the sonicate of the strain. It was purified about 142-fold. It showed optimum pH and temperature at 6.8 and 37°C, respectively, with the substrate p-nitrophenyl-α-d-galactoside (PNPG). Dilute enzyme solutions were very unstable even at 0–5°C. However, concentrated solutions were considerably stable. The Michaelis constant (m) was 1.07 × 10^?4, 2.33 × 10^?3, and 3.65 × 10^?2 for PNPG, melibiose, and raffinose, respectively. The maximum velocity (mole/min/mg protein) was 2.72 × 10^?5, 2.67 × 10^?5, and 2.04×l0^?5, respectively for the same three substrates. This enzyme had a weak transferase action.     

Hyperthermostable acetyl xylan esterase

An esterase which is encoded within a Thermotoga maritima chromosomal gene cluster for xylan degradation and utilization was characterized after heterologous expression of the corresponding gene in Escherichia coli and purification of the enzyme. The enzyme, designated AxeA, shares amino acid sequence similarity and its broad substrate specificity with the acetyl xylan esterase from Bacillus pumilus, the cephalosporin C deacetylase from Bacillus subtilis, and other (putative) esterases, allowing its classification as a member of carbohydrate esterase family 7. The recombinant enzyme displayed activity with p-nitrophenyl-acetate as well as with various acetylated sugar substrates such as glucose penta-acetate, acetylated oat spelts xylan and DMSO (dimethyl sulfoxide)-extracted beechwood xylan, and with cephalosporin C. Thermotoga maritima AxeA represents the most thermostable acetyl xylan esterase known to date. In a 10 min assay at its optimum pH of 6.5 the enzyme's activity peaked at 90°C. The inactivation half-life of AxeA at a protein concentration of 0.3 µg µl⁻¹ in the absence of substrate was about 13 h at 98°C and about 67 h at 90°C. Differential scanning calorimetry analysis of the thermal stability of AxeA corroborated its extreme heat resistance. A multi-phasic unfolding behaviour was found, with two apparent exothermic peaks at approximately 100–104°C and 107.5°C. In accordance with the crystal structure, gel filtration analysis at ambient temperature revealed that the enzyme has as a homohexameric oligomerization state, but a dimeric form was also found.     

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     

Effect of the chitin binding domain deletion from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> subsp. <Emphasis Type="Italic">kurstaki</Emphasis> chitinase Chi255 on its stability in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Bacillus thuringiensis subsp. kurstaki BUPM255 secretes a chitobiosidase Chi255 having an expected molecular weight of 70.665 kDa. When the corresponding gene, chi255, was expressed in E. coli, the active form, extracted from the periplasmic fraction of E. coli/pBADchi255, was of about 54 kDa, which suggested that Chi255 was excessively degraded by the action of E. coli proteases. Therefore, in vitro progressive C-terminal Chi255 deleted derivatives were constructed in order to study their stability and their activity in E. coli. Interestingly, when the chitin binding domain (CBD) was deleted from Chi255, an active form (Chi2555Δ5) of expected size of about 60 kDa was extracted from the E. coli periplasmic fraction, without the observation of any proteolytic degradation. Compared to Chi255, Chi255Δ5 exhibited a higher chitinase activity on colloidal chitin. Both of the enzymes exhibit activities at broad pH and temperature ranges with maximal enzyme activities at pH 5 and pH 6 and at temperatures 50°C and 40°C, respectively for Chi255 and Chi255Δ5. Thus, it was concluded that the C-terminal deletion of Chi255 CBD might be a nice tool for avoiding the excessive chitinase degradation, observed in the native chitinase, and for improving its activity.     

First characterisation of the active oligomer form of sulfur oxygenase reductase from the bacterium Aquifex aeolicus

Sulfur oxygenase reductase (SOR) enzyme is responsible for the initial oxidation step of elemental sulfur in archaea. Curiously, Aquifex aeolicus, a hyperthermophilic, chemolithoautotrophic and microaerophilic bacterium, has the SOR-encoding gene in its genome. We showed, for the first time the presence of the SOR enzyme in A. aeolicus, its gene was cloned and recombinantly expressed in Escherichia coli and the protein was purified and characterised. It is a 16 homo-oligomer of approximately 600 kDa that contains iron atoms indispensable for the enzyme activity. The optimal temperature of SOR activity is 80°C and it is inactive at 20°C. Studies of the factors involved in getting the fully active molecule at high temperature show clearly that (1) incubation at high temperature induces more homogeneous form of the enzyme, (2) conformational changes observed at high temperature are required to get the fully active molecule and (3) acquisition of an active conformation induced by the temperature seems to be more important than the subunit number. Differences between A. aeolicus SOR and the archaea SORs are described.     

A unique highly thermostable 2-phosphoglycerate forming glycerate kinase from the hyperthermophilic archaeon <Emphasis Type="Italic">Pyrococcus horikoshii</Emphasis>: gene cloning,expression and characterization

A glycerate kinase (GK) gene (PH0495) from the hyperthermophilic archaeon Pyrococcus horikoshii, was cloned and expressed in Escherichia coli. The recombinant protein was purified to homogeneity by affinity chromatography and ion exchange chromatography. The enzyme was likely a homodimer based on SDS-PAGE (47 kDa) and gel filtration chromatography (100 kDa) analysis. A radioisotope-labeling examination method was initially used for the enzymatic activity detection, and the enzyme (GK_ph) was found to catalyze the formation of 2-phosphoglycerate using d-glycerate as the substrate. The enzyme exhibited unique phosphoryl donor specificity with maximal activity towards pyrophosphate. The temperature and pH optima of the enzyme were 45°C and 7.0, respectively, and about half of the maximal activity remained at 100°C. The enzyme was highly thermostable with almost no loss of activity at 90°C for 12 h. Based on sequence alignment and structural comparison it was assigned to group I of the trichotomy of GKs. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A thermoactive glucoamylase with biotechnological relevance from the thermoacidophilic Euryarchaeon <Emphasis Type="Italic">Thermoplasma acidophilum</Emphasis>

A gene encoding an intracellular glucoamylase was identified in the genome of the extreme thermoacidophilic Archaeon Thermoplasma acidophilum. The gene taGA, consisting of 1,911 bp, was cloned and successfully expressed in Escherichia coli. The recombinant protein was purified 22-fold to homogeneity using heat treatment, anion-exchange chromatography, and gel filtration. Detailed analysis shows that the glucoamylase, with a molecular weight of 66 kDa per subunit, is a homodimer in its active state. Amylolytic activity was measured over a wide range of temperature (40–90°C) and pH (pH 3.5–7) and was maximal at 75°C and at acidic condition (pH 5). The recombinant archaeal glucoamylase uses a variety of polysaccharides as substrate, including glycogen and amylose. Maximal activity was measured towards amylopectin with a specific activity of 4.2 U/mg and increased almost threefold in the presence of manganese. Calcium ions have a pronounced effect on enzyme stability; in the presence of 5 mM CaCl₂, the half-life increased from 15 min to 2 h at 80°C.     

Purification and biochemical characterization of a broad substrate specificity thermostable alkaline protease from Aspergillus nidulans

Aspergillus nidulans PW1 produces an extracellular carboxylesterase activity that acts on several lipid esters when cultured in liquid media containing olive oil as a carbon source. The enzyme was purified by gel filtration and ion exchange chromatography. It has an apparent MW and pI of 37 kDa and 4.5, respectively. The enzyme efficiently hydrolyzed all assayed glycerides, but showed preference toward short- and medium-length chain fatty acid esters. Maximum activity was obtained at pH 8.5 at 40°C. The enzyme retained activity after incubation at pHs ranging from 8 to11 for 12 h at 37°C and 6 to 8 for 24 h at 37°C. It retained 80% of its activity after incubation at 30 to 70°C for 30 min and lost 50% of its activity after incubation for 15 min at 80°C. Noticeable activation of the enzyme is observed when Fe²⁺ ion is present at a concentration of 1 mM. Inhibition of the enzyme is observed in the presence of Cu²⁺, Fe³⁺, Hg²⁺, and Zn²⁺ ions. Even though the enzyme showed strong carboxylesterase activity, the deduced N-terminal amino acid sequence of the purified protein corresponded to the protease encoded by prtA gene.     

Purification and characterization of methyl parathion hydrolase from <Emphasis Type="Italic">Burkholderia cepacia</Emphasis> capable of degrading organophosphate insecticides

Methyl parathion hydrolase (MPH) from a methyl parathion-degrading Burkholderia cepacia indigenous to Thailand was purified to apparent homogeneity by three steps of column chromatography using Resource S, Sephadex G100, and Octyl Sepharose 4FF columns. Its molecular mass was determined to be 35 kDa, and the pI to be 8.5. The recombinant plasmid pGT1, containing the MPH-encoding gene, mpdB, cloned into pGEX-4T-2 was over-expressed in Escherichia coli as GST-MPH fusion protein. The recombinant MPH was purified to homogeneity by a single step, using GSTPrep FF affinity column, with the molecular mass identical to that of the native enzyme. The purified enzyme had the specific activity of about 1,600 unit mg⁻¹ protein and the yield of about 75%, a 39-fold increase in recovery compared to that of the native enzyme. The optimal temperature and pH were 25°C and 9.0, respectively. The MPH was stable, with its activity unchanged for 48 h at 4°C, and reduced to 50% after 5 h and to 45% after 48 h at 25°C. The enzyme activity remained 80–90% after 8–15 h at pH 6–7. Cd²⁺, Co²⁺, and Zn²⁺ ions at the concentration of 1 mM enhanced the activity; while sodium dodecyl sulfate (SDS), dithiothreitol (DTT) and ethylenediaminetetraacetate (EDTA) reduced it. The enzyme also showed cross reactivity with other insecticides within the organophosphate group, and the kinetic parameters for individual substrates were investigated. Since MPH from B. cepacia has wide potential applications in detoxification and detection of organophosphate compounds, this study provides important basis for its future use.     

Cloning, expression and characterization of a thermostable exo-β-D-glucosaminidase from the hyperthermophilic archaeon Pyrococcus horikoshii

An exo-β-d-glucosaminidase gene (PH0511) was cloned from the hyperthermophilic archaeon, Pyrococcus horikoshii, and expressed in Escherichia coli. The purified protein showed a strong exo-β-d-glucosaminidase activity by TLC analysis. DTT (50 mM) had little effect on its homodimeric structure during SDS-PAGE. The enzyme was optimally active at 90°C (over 20 min) and pH 6. It had a half-life of 9 h at 90°C and is the most thermostable glucosaminidase described up to now. The activity was not inhibited by ethanol, 2-propanol, DMSO, PEG-400, denaturing agents SDS (5%, w/v), urea, guanidine hydrochloride (5 M) and Mg²⁺, Mn²⁺, Co²⁺, Ca²⁺, Sr²⁺, Ni²⁺ (at up to 10 mM).     

Characterization of a thermostable cyclodextrin glucanotransferase from <Emphasis Type="Italic">Pyrococcus furiosus</Emphasis> DSM3638

A gene that encodes the enzyme Pyrococcus furiosus cyclodextrin glucanotransferase (PFCGT) was cloned in Escherichia coli. PFCGT was highly expressed in recombinant E. coli after compensation for codon usage bias using the pRARE plasmid. Purified PFCGT was extremely thermostable with an optimal temperature and pH of 95°C and 5.0, respectively, retaining 97% of its activity at 100°C. Incubation at 60°C for 20 min during the purification process led to a 1.5-fold increase in enzymatic activity. A time course assay of the PFCGT reaction with starch indicated that cyclic α-1,4-glucans with DPs greater than 20 were produced at the beginning of the incubation followed by an increase in β-CD. The major final product of PFCGT cyclization was β-CD, and thus the enzyme is a β-CGTase.     

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.     

改造大肠杆菌卟啉代谢途径对重组过氧化物酶活性的影响

【目的】原核表达某些需辅因子的外源蛋白时往往酶活偏低,为提高酶活和减少外加辅因子的成本,我们尝试在大肠杆菌中表达外源过氧化氢-过氧化物酶的同时提高大肠杆菌中与该酶辅因子相关的合成代谢。【方法】本研究克隆了中度嗜盐菌Halomonas elongata DSM2581的过氧化氢-过氧化物酶CAT-POD(catalase-peroxidase)编码基因kat G的ORF,构建原核表达载体p ET28a-kat G,实现了CAT-POD在大肠杆菌中的重组表达。由于CAT-POD活性依赖其活性中心血红素,而血卟啉是血红素的骨架,通过构建原核表达载体p UC19-tac-hem A,将编码5-氨基乙酰丙酸合成酶的hem A基因在大肠杆菌中过量表达,提高卟啉的含量,从而提高重组蛋白CAT-POD的酶活。【结果】最终的CAT酶活达到了377 U/m L,为对照组的7.5倍。【结论】本研究为工业生产高活性CAT-POD提供了有效的方案,也为体外重组表达含辅因子的蛋白提供可借鉴的思路。     

Characterization of a thermostable glucose dehydrogenase with strict substrate specificity from a hyperthermophilic archaeon Thermoproteus sp. GDH-1

A hyperthermophilic archaeon was isolated from a terrestrial hot spring on Kodakara Island, Japan and designated as Thermoproteus sp. glucose dehydrogenase (GDH-1). Cell extracts from cells grown in medium supplemented with glucose exhibited NAD(P)-dependent glucose dehydrogenase activity. The enzyme (TgGDH) was purified and found to display a strict preference for d-glucose. The gene was cloned and expressed in Escherichia coli, resulting in the production of a soluble and active protein. Recombinant TgGDH displayed extremely high thermostability and an optimal temperature higher than 85 °C, in addition to its strict specificity for d-glucose. Despite its thermophilic nature, TgGDH still exhibited activity at 25 °C. We confirmed that the enzyme could be applied for glucose measurements at ambient temperatures, suggesting a potential of the enzyme for use in measurements in blood samples.     

Development of a high-throughput screening method for recombinant Escherichia coli with intracellular dextransucrase activity

To efficiently engineer intracellular dextransucrase (DSase) expression in Escherichia coli, a high-throughput screening method was developed based on the polymer-forming activity of the enzyme. Recombinant E. coli containing the Leuconostoc citreum DSase (LcDS) gene was grown on Luria-Bertani agar plates, containing 2% sucrose, at 37°C for 8 h. The plates were then evenly overlaid with 0.6% soft agar, containing 1.2 mg/ml D-cycloserine, and incubated at 30°C to allow gradual cell disruption until a dextran polymer grew through the overlaid layer. A significant correlation between dextran size and enzyme activity was established and applied for screening truncated mutants with LcDS activity.