Thermostable aminopeptidase from Pyrococcus horikoshii

From the genome sequence data of the thermophilic archaeon Pyrococcus horikoshii, an open reading frame was found which encodes a protein (332 amino acids) homologous with an endoglucanase from Clostridium thermocellum (42% identity), deblocking aminopeptidase from Pyrococcus furiosus (42% identity) and an aminopeptidase from Aeromonas proteolytica (18% identity). This gene was cloned and expressed in Escherichia coli, and the characteristics of the expressed protein were examined. Although endoglucanase activity was not detected, this protein was found to have aminopeptidase activity to cleave the N-terminal amino acid from a variety of substrates including both N-blocked and non-blocked peptides. The enzyme was stable at 90 degrees C, with the optimum temperature over 90 degrees C. The metal ion bound to this enzyme was calcium, but it was not essential for the aminopeptidase activity. Instead, this enzyme required the cobalt ion for activity. This enzyme is expected to be useful for the removal of N(alpha)-acylated residues in short peptide sequence analysis at high temperatures.     

A β-lactamase produced by a thermophilic Bacillus

Abstract A β-lactamase was purified from a thermophilic Bacillus strain, that had been isolated from a traditional hot bath in the Meknes area (Morocco). The properties of the enzyme were very similar to those of the β-lactamase produced by Bacillus licheniformis 749C but it exhibited a somewhat increased thermostability and a higher activation energy with cefazolin as substrate. These properties were expected for an enzyme produced by a thermophilic strain.     

Purification and characterization of a thermostable carboxypeptidase (carboxypeptidase Taq) from Thermus aquaticus YT-1.

A thermostable carboxypeptidase, which we named carboxypeptidase Taq, was purified from Thermus aquaticus YT-1 and characterized. The molecular weight of the enzyme was estimated to be about 56,000 and 58,000 on SDS-polyacrylamide gel electrophoresis and gel filtration, respectively, indicating that the enzyme has a monomeric structure. The optimum pH of the enzyme was 8.0, and the optimum temperature for the reaction was 80 degrees C. The enzyme activity was dependent on cobalt ion and was inhibited by metal-chelating reagents, indicating that the enzyme is a metalloenzyme. The enzyme had high thermostability independent of cobalt ion; about 90% of its activity remained even after treatment at 80 degrees C for 5 h. The enzyme showed broad substrate specificity, although proline at the C-terminus of peptides was not cleaved. The enzyme released amino acids sequentially from the C-terminus.     

Isolation and characterization of an intracellular aminopeptidase from the extreme thermophilic archaebacterium Sulfolobus solfataricus

An intracellular aminopeptidase (EC 3.4.11.-) was purified from the extreme thermophilic archaebacterium, Sulfolobus solfataricus. The molecular weight of the native enzyme was about 320,000, as calculated by gel-filtration studies, and a subunit Mr of 80,000 was estimated by SDS-polyacrylamide gel electrophoresis. The temperature optimum of the enzyme was at 75 degrees C and the pH optimum was found to be 6.5. The aminopeptidase was highly active against the chromogenic substrates L-Leu-p-NA and L-Ala-p-NA. The enzyme was inhibited by EDTA, but the activity could be partially restored by removal of the EDTA and incubation with Co2+ or Mn2+. Bestatin, a typical inhibitor of aminopeptidase, fully inhibited the enzyme activity, but inhibitors of serine proteinases had no effect. Beside a high thermostability, the enzyme showed a remarkable stability against 6 M urea, organic solvents and acetonitrile.     

Ribulose 1,5-bisphosphate carboxylase from the thermophilic, acidophilic alga, Cyanidium caldarium (Geitler). Purification, characterisation and thermostability of the enzyme.

An an initial stage in the study of proteins from thermophilic algae, the enzyme ribulose 1,5-bisphosphate carboxylase 2-phospho-D-glycerate carboxylyase (dimerizing, EC 4.1.1.39) was purified 11-fold from the thermophilic alga Cyandium caldarium, with a 24% recovery. This purified enzyme appeared homogeneous on polyacrylamide gels and could be dissociated into two subunit types of molecular weights 55,000 and 14,900. The optimal assay temperature was 42.5 degrees C, whilst enzyme purified from Chlorella spp. showed maximum activity at 35 degrees C. The thermostability of Cyanidium ribulose 1,5-bisphosphate carboxylase was considerably greater than that of the Chlorella enzyme, and the presence of Mg2+ and HCO-3 further enhanced this heat stability. A break in the Arrhenius plot occured at 20 degrees C for Chlorella ribulose 1,5-bisphosphate carboxylase and 36 degrees C for the enzyme from Cyanidium. It is suggested that the thermostability of Cyanidium ribulose 1,5-bisphosphate carboxylase is a result of an inherent stability of the enzyme molecule which permits efficient CO2 fixation at high temperatures but results in low activity in the mesophilic temperature range.     

Bivalent cations and amino-acid composition contribute to the thermostability of Bacillus licheniformis xylose isomerase.

Comparative analysis of genome sequence data from mesophilic and hyperthermophilic micro-organisms has revealed a strong bias against specific thermolabile amino-acid residues (i.e. N and Q) in hyperthermophilic proteins. The N + Q content of class II xylose isomerases (XIs) from mesophiles, moderate thermophiles, and hyperthermophiles was examined. It was found to correlate inversely with the growth temperature of the source organism in all cases examined, except for the previously uncharacterized XI from Bacillus licheniformis DSM13 (BLXI), which had an N + Q content comparable to that of homologs from much more thermophilic sources. To determine whether BLXI behaves as a thermostable enzyme, it was expressed in Escherichia coli, and the thermostability and activity properties of the recombinant enzyme were studied. Indeed, it was optimally active at 70-72 degrees C, which is significantly higher than the optimal growth temperature (37 degrees C) of B. licheniformis. The kinetic properties of BLXI, determined at 60 degrees C with glucose and xylose as substrates, were comparable to those of other class II XIs. The stability of BLXI was dependent on the metallic cation present in its two metal-binding sites. The enzyme thermostability increased in the order apoenzyme < Mg2+-enzyme < Co2+-enzyme approximately Mn2+-enzyme, with melting temperatures of 50.3 degrees C, 53.3 degrees C, 73.4 degrees C, and 73.6 degrees C. BLXI inactivation was first-order in all conditions examined. The energy of activation for irreversible inactivation was also strongly influenced by the metal present, ranging from 342 kJ x mol(-1) (apoenzyme) to 604 kJ x mol(-1) (Mg2+-enzyme) to 1166 kJ x mol(-1) (Co2+-enzyme). These results suggest that the first irreversible event in BLXI unfolding is the release of one or both of its metals from the active site. Although N + Q content was an indicator of thermostability for class II XIs, this pattern may not hold for other sets of homologous enzymes. In fact, the extremely thermostable alpha-amylase from B. licheniformis was found to have an average N + Q content compared with homologous enzymes from a variety of mesophilic and thermophilic sources. Thus, it would appear that protein thermostability is a function of more complex molecular determinants than amino-acid content alone.     

High-temperature alkaline alpha-amylase from Bacillus iicheniformis TCRDC-B13

A bacterial strain, Bacillus licheniformis, has been isolated and identified which produces high-temperature alkaline alpha-amylase. Cultural conditions, such as types of carbon and nitrogen sources, temperature, pH, and time of reaction, have been optimized for production of alpha-amylase in shake flask and fermenter. The enzyme produced was quite active even at 100 degrees C; however, it showed optimum activity at 90 degrees C. It exhibited optimum activity in the broad pH range 5.5-10. The effects of Na(+) and Ca(2+) ions on enzyme activity was also studied.     

Optimization of culture conditions for the production of thermostable polygalacturonase by <Emphasis Type="Italic">Penicillium</Emphasis> SPC-F 20

Penicillium strain isolated from citrus fruit was found to produce thermostable polygalacturonases. Optimization of process parameters resulted in high levels of enzyme production after 3 days of incubation at a pH of 5.0 at 30 degrees C in the presence of 1% pectin. The optimum temperature for enzyme activity was 60 degrees C and a pH of 5.5 was found to be the optimal pH. The enzyme showed a high level of thermostability in the presence of substrate with a residual activity of 48% after 2 h of incubation at 60 degrees C. A thermostable nature with a high pH range for activity makes it an industrially important enzyme.     

Characterization of Amylolytic Enzymes, Having Both alpha-1,4 and alpha-1,6 Hydrolytic Activity, from the Thermophilic Archaea Pyrococcus furiosus and Thermococcus litoralis

Extracellular pullulanases were purified from cell-free culture supernatants of the marine thermophilic archaea Thermococcus litoralis (optimal growth temperature, 90 degrees C) and Pyrococcus furiosus (optimal growth temperature, 98 degrees C). The molecular mass of the T. litoralis enzyme was estimated at 119,000 Da by electrophoresis, while the P. furiosus enzyme exhibited a molecular mass of 110,000 Da under the same conditions. Both enzymes tested positive for bound sugar by the periodic acid-Schiff technique and are therefore glycoproteins. The thermoactivity and thermostability of both enzymes were enhanced in the presence of 5 mM Ca, and under these conditions, enzyme activity could be measured at temperatures of up to 130 to 140 degrees C. The addition of Ca also affected substrate binding, as evidenced by a decrease in K(m) for both enzymes when assayed in the presence of this metal. Each of these enzymes was able to hydrolyze, in addition to the alpha-1,6 linkages in pullulan, alpha-1,4 linkages in amylose and soluble starch. Neither enzyme possessed activity against maltohexaose or other smaller alpha-1,4-linked oligosaccharides. The enzymes from T. litoralis and P. furiosus appear to represent highly thermostable amylopullulanases, versions of which have been isolated from less-thermophilic organisms. The identification of these enzymes further defines the saccharide-metabolizing systems possessed by these two organisms.     

Production and properties of a bacterial thermostable exo-inulinase

Inulinase and Invertase Activities, Thermophilic Bacilli, Enzyme Thermostability Enzyme production of newly isolated thermophilic inulin-degrading Bacillus sp. 11 strain was studied by batch cultivation in a fermentor. The achieved inulinase and invertase activities after a short growth time (4.25 h) were similar or higher compared to those reported for other mesophilic aerobic or anaerobic thermophilic bacterial producers and yeasts. The investigated enzyme belonged to the exo-type inulinases and splitted-off inulin, sucrose and raffinose. It could be used at temperatures above 65 degrees C and pH range 5.5-7.5. The obtained crude enzyme preparation possessed high thermostability. The residual inulinase and invertase activities were 92-98% after pretreatment at 65 degrees C for 60 min in the presence of substrate inulin.     

Cloning of genes, purification and properties investigation of recombinant DNA ligases from the thermophilic archaeon Pyrococcus abyssi and Methanobacterium thermoautotrophicum

The genes encoding of DNA ligases from the thermophilic archaeon Pyrococcus abyssi (PabDNA ligase) and Methanobacterium thermoautotrophicum (MthDNA ligase) were cloned and expressed in Escherichia coli. The activity of purified enzymes was studied by ligation of two oligonucleotides, one of which had preformed hairpin structure. In the used system the maximal output of reaction products for both DNA ligases was observed near 70 degrees C that is explained by substrate thermostability. At stoichiometric ratio of enzymes and substrate the output of a product reaches of plateau at 70-75% of theoretical ones. Investigated DNA ligases showed different thermostability. The half-time life of PabDNA ligase was about 60 min at 90 degrees C. MthDNA ligase was completely inactivated at this temperature during 10 min. Recombinant DNA ligases from P. abyssi and M. thermoautotrophicum possessed high stability during a storage at 4 degrees C.     

Why is one Bacillus alpha-amylase more resistant against irreversible thermoinactivation than another?

Half-lives of Bacillus alpha-amylases at 90 degrees C and pH 6.5 greatly increase in the series from Bacillus amyloliquefaciens to Bacillus stearothermophilus to Bacillus licheniformis, e.g. the difference in thermostability between the first and the third enzymes exceeds 2 orders of magnitude. This stabilization is achieved by lowering the rate constant of monomolecular conformational scrambling, which is the cause of irreversible thermoinactivation of B. amyloliquefaciens and B. stearothermophilus alpha-amylases, so that for B. licheniformis alpha-amylase, another process, deamidation of Asn/Gln residues, emerges as the cause of inactivation. The extra thermostability of the thermophilic enzyme was found to be mainly due to additional salt bridges involving a few specific lysine residues (Lys-385 and Lys-88 and/or Lys-253). These stabilizing electrostatic interactions reduce the extent of unfolding of the enzyme molecule at high temperatures, consequently making it less prone to forming incorrect (scrambled) structures and thus decreasing the overall rate of irreversible thermoinactivation. The implications of these findings for protein engineering are discussed.     

Characterization of a novel zinc-containing, lysine-specific aminopeptidase from the hyperthermophilic archaeon Pyrococcus furiosus

Cell extracts of the proteolytic, hyperthermophilic archaeon Pyrococcus furiosus contain high specific activity (11 U/mg) of lysine aminopeptidase (KAP), as measured by the hydrolysis of L-lysyl-p-nitroanilide (Lys-pNA). The enzyme was purified by multistep chromatography. KAP is a homotetramer (38.2 kDa per subunit) and, as purified, contains 2.0 +/- 0.48 zinc atoms per subunit. Surprisingly, its activity was stimulated fourfold by the addition of Co2+ ions (0.2 mM). Optimal KAP activity with Lys-pNA as the substrate occurred at pH 8.0 and a temperature of 100 degrees C. The enzyme had a narrow substrate specificity with di-, tri-, and tetrapeptides, and it hydrolyzed only basic N-terminal residues at high rates. Mass spectroscopy analysis of the purified enzyme was used to identify, in the P. furiosus genome database, a gene (PF1861) that encodes a product corresponding to 346 amino acids. The recombinant protein containing a polyhistidine tag at the N terminus was produced in Escherichia coli and purified using affinity chromatography. Its properties, including molecular mass, metal ion dependence, and pH and temperature optima for catalysis, were indistinguishable from those of the native form, although the thermostability of the recombinant form was dramatically lower than that of the native enzyme (half-life of approximately 6 h at 100 degrees C). Based on its amino acid sequence, KAP is part of the M18 family of peptidases and represents the first prokaryotic member of this family. KAP is also the first lysine-specific aminopeptidase to be purified from an archaeon.     

Purification and some properties of a thermostable DNA polymerase from a Thermotoga species

A stable DNA polymerase (EC 2.7.7.7) has been purified from the extremely thermophilic eubacterium Thermotoga sp. strain FjSS3-B.1 by a five-step purification procedure. First, the crude extract was treated with polyethylenimine to precipitate nucleic acids. The endonuclease activity coprecipitated. DEAE-Sepharose, CM-Sephrarose, and hydroxylapatite column chromatography were used to purify the preparation. As a final step on a small scale, preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was used. The purified DNA polymerase exhibited a molecular weight of 85,000, as determined by both SDS-polyacrylamide gel electrophoresis and size-exclusion chromatography. Its pH optimum was in the range pH 7.5-8. When assayed over the temperature range 30-80 degrees C, the maximum activity in a 30-min assay was at 80 degrees C. The enzyme was moderately thermostable and exhibited half-lives of 3 min at 95 degrees C and 60 min at 50 degrees C in the absence of substrate. Several additives such as Triton X-100 enhanced thermostability. During storage at 4 degrees C and -70 degrees C, the stability of the enzyme was improved by the addition of gelatin.     

Identification of archaeon-producing hyperthermophilic α-amylase and characterization of the α-amylase

The extremely thermophilic anaerobic archaeon strain, HJ21, was isolated from a deep-sea hydrothermal vent, could produce hyperthermophilic alpha-amylase, and later was identified as Thermococcus from morphological, biochemical, and physiological characteristics and the 16S ribosomal RNA gene sequence. The extracellular thermostable alpha-amylase produced by strain HJ21 exhibited maximal activity at pH 5.0. The enzyme was stable in a broad pH range from pH 5.0 to 9.0. The optimal temperature of alpha-amylase was observed at 95 degrees C. The half-life of the enzyme was 5 h at 90 degrees C. Over 40% and 30% of the enzyme activity remained after incubation at 100 degrees C for 2 and 3 h, respectively. The enzyme did not require Ca(2+) for thermostability. This alpha-amylase gene was cloned, and its nucleotide sequence displayed an open reading frame of 1,374 bp, which encodes a protein of 457 amino acids. Analysis of the deduced amino acid sequence revealed that four homologous regions common in amylases were conserved in the HJ21 alpha-amylase. The molecular weight of the mature enzyme was calculated to be 51.4 kDa, which correlated well with the size of the purified enzyme as shown by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Identification, purification, and characterization of a thermophilic imidase from pig liver

This study investigates thermophilic imidase activity of the liver. We demonstrate that imidase catalyzes the hydrolysis of imides at a temperature substantially higher than that of its native environment. Then, a thermophilic imidase is purified to homogeneity from pig liver, and its thermoproperties are studied. About 2500-fold of purification and 15% yield of imidase activity are obtained after ammonium sulfate precipitation, octyl, DEAE, chelation, and gel filtration chromatography. While avoiding heat treatment for the protein purification, this study also indicates that only one enzyme is responsible for the imidase activity. This homogenous enzyme prefers to catalyze hydrolysis of imides at above 60 degrees C rather than at the body temperature of a pig. Although stable at below 50 degrees C, imidase quickly loses its activity at above 65 degrees C. Thus, the temperature effect on imidase activity is limited mainly by its thermostability. Substrate specificity of imidase is also temperature dependent. Our results demonstrate that the hydrolysis of physiological substrates is the most temperature dependent and that of hydantoins is the least temperature dependent. When increasing the reaction temperature from 25 to 60 degrees C, specific activities increase 50- and 60-fold for dihydrouracil and dihydrothymine, respectively. The temperature effect on the K(m) and V(max) of imidase is substrate dependent.     

Thermostable lipase of Bacillus Stearothermophilus: high-level production, purification, and calcium-dependent thermostability

An efficient expression system was developed for the production of the thermostable lipase from Bacillus stearothermophilus L1 in an Escherichia coli system. A structural gene corresponding to mature lipase was subcloned in the pET-22b(+) expression vector and its expression was induced by IPTG at 30 degrees C in E. coli cells. The lipase activity in a cell-free extract was as high as 448,000 units/g protein, which corresponds to as much as 26% of the total cellular protein and is 77 times higher than that of E. coli RR1/pLIP1. Based on its pI (7.4) and pH stability data reported previously, the L1 lipase was efficiently purified to homogeneity with CM (at pH 6.0) and DEAE (at pH 8.8) column chromatographies with a recovery yield of 62%. The specific activity of the purified enzyme was 1700 units/mg protein when olive oil emulsion was used as a substrate. Its optimum temperature for the hydrolysis of olive oil was 68 degrees C and it was stable up to 55 degrees C for 30 min-incubation. The thermostability increased by about 8-10 degrees in the presence of calcium ions. This calcium-dependent thermostability was confirmed by the tryptophan fluorescence emission kinetics showing that the enzyme starts to unfold at 66 degrees C in the presence of calcium ions but at 58 degrees C in the absence of calcium ions, implying that the calcium ions bind to the thermostable enzyme and stabilize the protein tertiary structure even at such high temperatures.     

Thermostable xylanase from Marasmius sp.: purification and characterization

We have screened 766 strains of fungi from the BIOTEC Culture Collection (BCC) for xylanases working in extreme pH and/or high temperature conditions, the so-called extreme xylanases. From a total number of 32 strains producing extreme xylanases, the strain BCC7928, identified by using the internal transcribed spacer (ITS) sequence of rRNA to be a Marasmius sp., was chosen for further characterization because of its high xylanolytic activity at temperature as high as 90 degrees C. The crude enzyme possessed high thermostability and pH stability. Purification of this xylanase was carried out using an anion exchanger followed by hydrophobic interaction chromatography, yielding the enzyme with >90% homogeneity. The molecular mass of the enzyme was approximately 40 kDa. The purified enzyme retained broad working pH range of 4-8 and optimal temperature of 90 degrees C. When using xylan from birchwood as substrate, it exhibits Km and Vmax values of 2.6 +/- 0.6 mg/ml and 428 +/- 26 U/mg, respectively. The enzyme rapidly hydrolysed xylans from birchwood, beechwood, and exhibited lower activity on xylan from wheatbran, or celluloses from carboxymethylcellulose and Avicel. The purified enzyme was highly stable at temperature ranges from 50 to 70 degrees C. It retained 84% of its maximal activity after incubation in standard buffer containing 1% xylan substrate at 70 degrees C for 3 h. This thermostable xylanase should therefore be useful for several industrial applications, such as agricultural, food and biofuel.     

Influence of divalent cations on the structural thermostability and thermal inactivation kinetics of class II xylose isomerases

The effects of divalent metal cations on structural thermostability and the inactivation kinetics of homologous class II d-xylose isomerases (XI; EC 5.3.1.5) from mesophilic (Escherichia coli and Bacillus licheniformis), thermophilic (Thermoanaerobacterium thermosulfurigenes), and hyperthermophilic (Thermotoga neapolitana) bacteria were examined. Unlike the three less thermophilic XIs that were substantially structurally stabilized in the presence of Co2+ or Mn2+ (and Mg2+ to a lesser extent), the melting temperature [(Tm) approximately 100 degrees C] of T. neapolitana XI (TNXI) varied little in the presence or absence of a single type of metal. In the presence of any two of these metals, TNXI exhibited a second melting transition between 110 degrees C and 114 degrees C. TNXI kinetic inactivation, which was non-first order, could be modeled as a two-step sequential process. TNXI inactivation in the presence of 5 mm metal at 99-100 degrees C was slowest in the presence of Mn2+[half-life (t(1/2)) of 84 min], compared to Co2+ (t(1/2) of 14 min) and Mg2+ (t(1/2) of 2 min). While adding Co2+ to Mg2+ increased TNXI's t(1/2) at 99-100 degrees C from 2 to 7.5 min, TNXI showed no significant activity at temperatures above the first melting transition. The results reported here suggest that, unlike the other class II XIs examined, single metals are required for TNXI activity, but are not essential for its structural thermostability. The structural form corresponding to the second melting transition of TNXI in the presence of two metals is not known, but likely results from cooperative interactions between dissimilar metals in the two metal binding sites.     

Isolation, purification and investigation of physico-chemical properties and specificity of Leu-Gly-Gly-amino peptidase]

A highly purified (237-fold) preparation of extracellular Leu-Gly-Gly aminopeptidase was isolated from the 716 strain of mould Aspergillis flavus. The enzyme was found electrophoretically and enzymatically homogeneous, using Leu-beta-naphthylimide as substrate. The pH optimum is 8.60; the temperature optimum is about 50 degrees C. The enzyme was inhibited by EDTA and completely reactivated by Co2+ ions; Ca2+ and Mn2+ ions considerably restored the enzyme activity. The enzyme showed the optimal activity during the cleavage of substrates, containing N-terminal leucine. Mild hydrolysis of leucine-free tripeptides and dipeptides with N-terminal glycine and alanine was observed. The enzyme was found to be stereospecific in some respects. Peptides with a blocked terminal NH2-group are not hydrolyzed by the enzyme.