Enzymatic characterization of an extracellular glucoamylase from Tricholoma matsutake and its cloning and secretory expression in Pichia pastoris

ABSTRACT

A glucoamylase from the ectomycorrhizal fungus Tricholoma matsutake (TmGLA) was purified 33.2-fold to homogeneity as a single monomeric glycoprotein with a molecular mass of 63.9 kDa. Maximum activity was observed at 60°C and pH 5.0. The enzyme is active down to 50°C and in the pH range of 4.0–6.0, and its activity is strongly inhibited by Ag⁺. It degrades α-1,4- and α-1,6-glycosidic linkages in various polysaccharides. Its gene (TmGlu1) was cloned using information from the enzyme’s internal amino acid sequences and the whole genome sequence of T. matsutake NBRC 30605. The deduced amino acid sequence showed clear homology with those of GH family 15 proteins. Pichia pastoris transformed with TmGlu1 secreted the active enzyme in a glycosylated form, and its characteristics were the same as the native enzyme.     

A unique β-agarase,AgaA, from a marine bacterium, <Emphasis Type="Italic">Vibrio</Emphasis> sp. strain PO-303

The agaA gene encoding β-agarase-a (AgaA) was cloned from the chromosomal DNA of a marine bacterium, Vibrio sp. strain PO-303. The nucleotide sequence of the agaA gene consists of 2,958 bp and encodes a protein of 985 amino acids with a molecular mass of 106,062 Da. The deduced enzyme protein contains a typical N-terminal signal peptide of 29 amino acid residues, followed by a 266 amino acid sequence that is homologous to catalytic module of family 16 glycoside hydrolases, a bacterial immunoglobulin group 2 (Big-2)-like domain of 52 amino acid residues, two carbohydrate-binding modules of family 6 separated from Big-2-like domain by nine times repeated GDDTDP amino acid sequence. AgaA is the first agarase that was identified to possess a Big-2-like domain. The recombinant AgaA (rAgaA) expressed in Escherichia coli exhibited maximal activity around 40°C and pH 7.5, with a specific activity of 16.4 units mg⁻¹, a K _m of 1.10 mg ml⁻¹, and a V _max of 22.5 μmol min⁻¹ mg⁻¹ for agarose. The rAgaA hydrolyzed neoagarohexaose, but did not act on neoagarotetraose and neoagarobiose.     

Biochemical and phylogenetic characterization of isocitrate dehydrogenase from a hyperthermophilic archaeon, Archaeoglobus fulgidus

A thermostable homodimeric isocitrate dehydrogenase from the hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus was purified and characterized. The mol. mass of the isocitrate dehydrogenase subunit was 42 kDa as determined by SDS-PAGE. Following separation by SDS-PAGE, A. fulgidus isocitrate dehydrogenase could be renatured and detected in situ by activity staining. The enzyme showed dual coenzyme specificity with a high preference for NADP⁺. Optimal temperature for activity was 90° C or above, and a half-life of 22 min was found for the enzyme when incubated at 90° C in a 50 mM Tricine-KOH buffer (pH 8.0). Based on the N-terminal amino acid sequence, the gene encoding the isocitrate dehydrogenase was cloned. DNA sequencing identified the icd gene as an open reading frame encoding a protein of 412 amino acids with a molecular mass corresponding to that determined for the purified enzyme. The deduced amino acid sequence closely resembled that of the isocitrate dehydrogenase from the archaeon Caldococcus noboribetus (59% identity) and bacterial isocitrate dehydrogenases, with 57% identity with isocitrate dehydrogenase from Escherichia coli. All the amino acid residues directly contacting substrate and coenzyme (except Ile-320) in E. coli isocitrate dehydrogenase are conserved in the enzyme from A. fulgidus. The primary structure of A. fulgidus isocitrate dehydrogenase confirmes the presence of Bacteria-type isocitrate dehydrogenases among Archaea. Multiple alignment of all the available amino acid sequences of di- and multimeric isocitrate dehydrogenases from the three domains of life shows that they can be divided into three distinct phylogenetic groups. Received: 6 February 1997 / Accepted: 12 June 1997     

Cloning of an alkaline phosphatase gene from the moderately thermophilic bacterium<Emphasis Type="Italic"> Meiothermus ruber</Emphasis> and characterization of the recombinant enzyme

A gene that codes for an alkaline phosphatase was cloned from the thermophilic bacterium Meiothermus ruber, and its nucleotide sequence was determined. The deduced amino acid sequence indicates that the enzyme precursor including the putative signal sequence is composed of 503 amino acid residues and has an estimated molecular mass of 54,229 Da. Comparison of the peptide sequence with that of the prototype alkaline phosphatase from Escherichia coli revealed conservation of the regions in the vicinity of the corresponding phosphorylation site and metal binding sites. The protein was expressed in E. coli and its enzymatic properties were characterized. In the absence of exogenously added metal ions, activity was negligible; to obtain maximal activity, addition of free Mg²⁺ ions was required. Zn²⁺ ions had an inhibitory effect on the activity of the M. ruber enzyme. The pH and temperature optima for activity were found to be 11.0 and 62°C, respectively. The enzyme was moderately thermostable: it retained about 50% activity after incubation for 6 h at 60°C, whereas at 80°C it was completely inactivated within 2 h. The Michaelis constant for cleavage of 4-nitrophenylphosphate was 0.055 mM. While having much in common with other alkaline phosphatases, the M. ruber enzyme presents some unique features, such as a very narrow pH range for activity and an absolute requirement for magnesium for activity.Communicated by G. P. Georgiev     

Purification and characterization of a fibrinolytic subtilisin-like protease of Bacillus subtilis TP-6 from an Indonesian fermented soybean, Tempeh

We have isolated a bacterium (TP-6) from the Indonesian fermented soybean, Tempeh, which produces a strong fibrinolytic protease and was identified as Bacillus subtilis. The protease (TPase) was purified to homogeneity by ammonium sulfate fractionation and octyl sepharose and SP sepharose chromatography. The N-terminal amino acid sequence of the 27.5 kDa enzyme was determined, and the encoding gene was cloned and sequenced. The result demonstrates that TPase is a serine protease of the subtilisin family consisting of 275 amino acid residues in its mature form. Its apparent K _m and V _max for the synthetic substrate N-succinyl-Ala-Ala-Pro-Phe-pNA were 259 μM and 145 μmol mg⁻¹ min⁻¹, respectively. The fibrinogen degradation pattern generated by TPase as a function of time was similar to that obtained with plasmin. In addition, N-terminal amino acid sequence analysis of the fibrinogen degradation products demonstrated that TPase cleaves Glu (or Asp) near hydrophobic acids as a P1 site in the α- and β-chains of fibrinogen to generate fragments D′, E′, and D′ similar to those generated by plasmin. On plasminogen-rich fibrin plates, TPase did not seem to activate fibrin clot lysis. Moreover, the enzyme converted the active plasminogen activator inhibitor-1 to the latent form.Seong-Bo Kim and Dong-Woo Lee contributed equally to the work.     

Purification, characterization, and molecular cloning of organic-solvent-tolerant cholesterol esterase from cyclohexane-tolerant Burkholderia cepacia strain ST-200

Extracellular cholesterol esterase of Burkholderia cepacia strain ST-200 was purified from the culture supernatant. Its molecular mass was 37 kDa. The enzyme was stable at pH 5.5–12 and active at pH 5.5–6, showing optimal activity at pH 7.0 at 45°C. Relative to the commercially available cholesterol esterases, the purified enzyme was highly stable in the presence of various water-miscible organic solvents. The enzyme preferentially hydrolyzed long-chain fatty acid esters of cholesterol, except for that of cholesteryl palmitate. The enzyme exhibited lipolytic activity toward various p-nitrophenyl esters. The hydrolysis rate of p-nitrophenyl caprylate was enhanced 3.5- to 7.2-fold in the presence of 5–20% (vol/vol) water-miscible organic solvents relative to that in the absence of organic solvents. The structural gene encoding the cholesterol esterase was cloned and sequenced. The primary translation product was predicted to be 365 amino acid residues. The mature product is composed of 325 amino acid residues. The amino acid sequence of the product showed the highest similarity to the lipase LipA (87%) from B. cepacia DSM3959.     

Rotihibin A,a Novel Plant Growth Regulator,from Streptomyces sp.

A thermophilic Thermoactinomyces sp. E79 producing a highly thermostable alkaline protease was isolated from soil. The protease, produced extracellularly by Thermoactinomyces sp. E79, was purified by DEAE-Sepharose CL-6B and Butyl-Toyopearl 650M column chromatography. The relative molecular mass was estimated to be 31,000 by SDS–polyacrylamide gel electrophoresis. Enzyme activity was inhibited by phenylmethylsulfonyl fluoride, suggesting the enzyme to be a serine protease. The optimum temperature for the enzyme activity was 85°C, and about 50% of the original activity remained after incubation at 90°C for 10 min in the presence of Ca^{2 +} . The optimum pH for the enzyme activity was 11.0 and the enzyme was fairly stable from pH 5.0 to 12.0. The gene for this thermostable alkaline protease was cloned in Escherichia coli and the expressed intracellular enzyme was activated by heat treatment. Sequence analysis showed an open reading frame of 1,152 base pairs, coding for a poiypeptide of 384 amino acids. The polypeptide was composed of a signal sequence (25 amino acids), a prosequence (81 amino acids), and a mature protein of 278 amino acids. The deduced amino acid sequence of the mature protease had high similarity with thermitase, a serine protease from Thermoactinomyces vulgaris, and the extent of sequence identity was 76%.     

A β-1,4-endoglucanase-encoding gene from Cellulomonas pachnodae

 A gene library of Cellulomonas pachnodae was constructed in Escherichia coli and was screened for endoglucanase activity. Five endoglucanase-positive clones were isolated that carried identical DNA fragments. The gene, designated cel6A, encoding an endoglucanase enzyme, belongs to the glycosyl hydrolase family 6 (cellulase family B). The recombinant Cel6A had a molecular mass of 53 kDa, a pH optimum of 5.5, and a temperature optimum of 50–55 °C. The recombinant endoglucanase Cel6A bound to crystalline cellulose and beech litter. Based on amino acid sequence similarity, a clear cellulose-binding domain was not distinguished. However, the regions in the Cel6A amino acid sequence at the positions 262–319 and 448–473, which did not show similarity to any of the known family-6 glycosyl hydrolases, may be involved in substrate binding. Received: 14 January 1999 / Received revision: 29 March 1999 / Accepted: 6 April 1999     

Extremely high alkaline protease from a deep-subsurface bacterium, Alkaliphilus transvaalensis

A new high-alkaline protease (ALTP) was purified to homogeneity from a culture of the strictly anaerobic and extremely alkaliphilic Alkaliphilus transvaalensis. The molecular mass was 30 kDa on sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The enzyme showed the maximal caseinolytic activity higher than pH 12.6 in KCl–NaOH buffer at 40°C. Hydrolysis of the oxidized insulin B-chain followed by mass spectrometric analysis of the cleaved products revealed that as many as 24 of the total 29 peptide bonds are hydrolyzed in a block-cutting manner, suggesting that ALTP has a widespread proteolytic functions. Calcium ion had no effect on the activity and stability of ALTP, unlike known subtilisins. The deduced amino acid sequence of the enzyme comprised 279 amino acids plus 97 prepropeptide amino acids. The amino acid sequence of mature ALTP was confirmed by capillary liquid chromatography coupled to tandem mass spectrometry, which was the 93% coverage of the deduced amino acid sequence. The mature enzyme showed moderate homology to subtilisin LD1 from the alkaliphilic Bacillus sp. strain KSM-LD1 with 64% identity, and both enzymes formed a new subcluster at an intermediate position among true subtilisins and high-alkaline proteases in a phylogenetic tree of subtilase family A. ALTP is the first high-alkaline protease reported from a strict anaerobe in this family.     

Production,purification and characterisation of a novel halostable xylanase from Bacillus sp. NTU-06

Bacillus sp. NTU-06 was used to produce xylanase, which is an important industrial enzyme used in the pulp and paper industry. The enzyme was purified by fast protein liquid chromatography (FPLC) and had a molecular mass of 24 kDa. The enzyme was active over a concentration range of 0–20% sodium chloride in culture broth, although its activity was optimal in 5% sodium chloride. A salinity stability test showed that 43% of the enzyme activity was retained after 4 h in 20% sodium chloride. Xylanase activity was maximal at pH 8.0 and 40°C. The enzyme was somewhat thermostable, retaining 20% of the original activity after incubation at 70°C for 4 h. The xylanase had K_m and V_max values of 3.45 mg mL⁻¹ and 387.3 µmol min⁻¹mg⁻¹, respectively. The deduced internal amino acid sequence of Bacillus sp. NTU-06 xylanase resembled the sequence of beta-1,4-endoxylanase, which is a member of glycoside hydrolase family 11. Some of the novel characteristics that make this enzyme potentially effective in xylan biodegradation are discussed.     

Characterization of a novel dye-linked <Emphasis Type="SmallCaps">l</Emphasis>-proline dehydrogenase from an aerobic hyperthermophilic archaeon, <Emphasis Type="Italic">Pyrobaculum calidifontis</Emphasis>

The activity of a dye-linked l-proline dehydrogenase (dye-l-proDH) was found in the crude extract of an aerobic hyperthermophilic archaeon, Pyrobaculum calidifontis JCM 11548, and was purified 163-fold through four sequential chromatography steps. The enzyme has a molecular mass of about 108 kDa and is a homodimer with a subunit molecular mass of about 46 kDa. The enzyme retained more than 90% of its activity after incubation at 100 °C for 120 min (pH 7.5) or after incubation at pHs 4.5–9.0 for 30 min at 50 °C. The enzyme catalyzed l-proline dehydrogenation to Δ¹-pyroline-5-carboxylate using 2,6-dichloroindophenol (DCIP) as the electron acceptor and the Michaelis constants for l-proline and DCIP were 1.67 and 0.026 mM, respectively. The prosthetic group on the enzyme was identified as flavin adenine dinucleotide by high-performance liquid chromatography. The subunit N-terminal amino acid sequence was MYDYVVVGAG. Using that sequence and previously reported genome information, the gene encoding the enzyme (Pcal_1655) was identified. The gene was then cloned and expressed in Escherichia coli and found to encode a polypeptide of 415 amino acids with a calculated molecular weight of 46,259. The dye-l-proDH gene cluster in P. calidifontis inherently differs from those in the other hyperthermophiles reported so far.     

3-Methylaspartate ammonia-lyase from a facultative anaerobe,strain YG-1002

3-Methylaspartase was purified 24-fold and crystallized from the crude extract of the cells of a facultative anaerobic bacterium from soil, strain YG-1002. The molecular mass of the native enzyme was about 84 kDa and that of the subunit was about 42 kDa. The pH optimum for the deamination reaction of (2S, 3S)-3-methylaspartic acid and those for the amination reaction of mesaconic acid were 9.7 and 8.5; its optimum temperature was 50°C. The enzyme was stable at pH 5.5–11.0 and up to 50°C. The enzyme required both divalent and monovalent cations such as Mg²⁺ and K⁺. The enzyme was inhibited by sulfhydryl reagents, metal-chelating reagents and some divalent cations. The enzyme catalyzed the reversible amination/deamination reactions between several 3-substituted (S)-aspartic acids and their corresponding fumaric acid derivatives. The enzyme preferentially acted on (2S, 3S)-3-methylaspartic acid and mesaconic acid in the deamination and the amination reactions respectively. The enzyme showed high similarities in several enzymological properties and N-terminal amino acid sequence with 3-methylaspartase from an obligate anaerobic bacteriumClostridium tetanomorphum.     

Identification and characterization of a novel cold-adapted esterase from a metagenomic library of mountain soil

A novel lipolytic enzyme was isolated from a metagenomic library after demonstration of lipolytic activity on an LB agar plate containing 1% (w/v) tributyrin. A novel esterase gene (estIM1), encoding a lipolytic enzyme (EstIM1), was cloned using a shotgun method from a pFosEstIM1 clone of the metagenomic library, and the enzyme was characterized. The estIM1 gene had an open reading frame (ORF) of 936 base pairs and encoded a protein of 311 amino acids with a molecular mass 34 kDa and a pI value of 4.32. The deduced amino acid sequence was 62% identical to that of an esterase from an uncultured bacterium (ABQ11271). The amino acid sequence indicated that EstIM1 was a member of the family IV of lipolytic enzymes, all of which contain a GDSAG motif shared with similar enzymes of lactic acid microorganisms. EstIM1 was active over a temperature range of 1–50°C, at alkaline pH. The activation energy for hydrolysis of p-nitrophenyl propionate was 1.04 kcal/mol, within a temperature range of 1–40°C. The activity of EstIM1 was about 60% of maximal even at 1°C, suggesting that EstIM1 is efficiently cold-adapted. Further characterization of this cold-adapted enzyme indicated that the esterase may be very valuable in industrial applications.     

Purification and properties of an alkaline protease from alkalophilic Bacillus sp. KSM-K16

Alkaline protease (EC 3.4.21.14) activity, suitable for use in detergents, was detected in the alkaline culture medium of Bacillus sp. KSM-K16, which was originally isolated from soil. The enzyme, designated M protease, was purified to homogeneity from the culture broth by column chromatographies. The N-terminal amino acid sequence was Ala-Gln-Ser-Val-Pro-Trp-Gly-Ile-Ser-Arg-Val-Gln-Ala-Pro-Ala-Ala-His-Asn-Arg-Gly-Leu-Thr-Gly. The molecular mass of the protease was 28 kDa, and its isoelectric point was close to pH 10.6. Maximum activity toward casein was observed at 55°C and at pH 12.3 in 50 mM phosphate/NaOH buffer. The activity was inhibited by phenylmethylsulfonyl flouride and chymostatin. The enzyme was very stable in long-term incubation with liquid detergents at 40°C. The enzyme cleaved the oxidized insulin B chain initially at Leu¹⁵-Tyr¹⁶ and efficiently at ten more sites. Among various oligopeptidyl p-nitro-anilides (pNA) tested, N-succinyl-Ala-Ala-Pro-Phe-pNA was efficiently hydrolyzed by M protease. M protease was precipitated in (NH₄)₂SO₄-saturated acetate buffer (pH 5.0) as plank-like cyrstals.     

Cloning of the Escherichia coli endo-1,4-D-glucanase gene and identification of its product

A plasmid (pYP17) containing a genomic DNA insert from Escherichia coli K-12 that confers the ability to hydrolyze carboxymethylcellulose (CMC) was isolated from a genomic library constructed in the cosmid vector pLAFR3 in E. coli DH5α. A small 1.65-kb fragment, designated bcsC (pYP300), was sequenced and found to contain an ORF of 1,104 bp encoding a protein of 368 amino acid residues, with a calculated molecular weight of 41,700 Da. BcsC carries a typical prokaryotic signal peptide of 21 amino acid residues. The predicted amino acid sequence of the BcsC protein is similar to that of CelY of Erwinia chrysanthemi, CMCase of Cellulomonas uda, EngX of Acetobacter xylinum, and CelC of Agrobacterium tumefaciens. Based on these sequence similarities, we propose that the bcsC gene is a member of glycosyl hydrolase family 8. The apparent molecular mass of the protein, when expressed in E. coli, is approximately 40 kDa, and the CMCase activity is found mainly in the extracellular space. The enzyme is optimally active at pH 7 and a temperature of 40° C. Received: 6 February 1998 / Accepted: 6 November 1998     

Purification and characterization of a novel halostable cellulase from Salinivibrio sp. strain NTU-05

A halostable cellulase with a molecular mass of 29 kDa was purified from culture supernatants of the halophilic bacterium Salinivibrio sp. NTU-05 by way of the Fast Protein Liquid Chromatography method and the biochemical properties of the halostable cellulase was studied. The enzyme was active over a range of 0–25% sodium chloride examined in culture broth. The optimum cellulase activity was observed at 5% sodium chloride. Results from the salinity stability test indicated 24% of enzyme activity was retained at 25% sodium chloride for 4 h. The enzyme was also shown to be slightly thermostable with 40% residual activity under 60 °C for 4 h. The enzyme has a K_m of 3.03 mg/ml and a V_max of 142.86 mol/min/mg when tested using carboxymethyl-cellulose (CMC). The enzyme activity increased in the presence of K⁺, Mg²⁺, Na⁺ ions and decreased when Hg²⁺ ions were present. The deduced internal amino acid sequence of the Salinivibrio sp. NTU-05 cellulase showed similarity to the sequence of the glycoside hydrolase family protein. These are some of the novel characteristics that make this enzyme have potential applications in cellulose biodegradation.     

Phylogenetic Analysis and Biochemical Characterization of a Thermostable Dihydropyrimidinase from Alkaliphilic Bacillus sp. TS-23

Two degenerate primers established from the alignment of highly conserved amino acid sequences of bacterial dihydropyrimidinases (DHPs) were used to amplify a 330-bp gene fragment from the genomic DNA of Bacillus sp. TS-23 and the amplified DNA was successfully used as a probe to clone a dhp gene from the strain. The open reading frame of the gene consisted of 1422 bp and was deduced to contain 472 amino acids with a molecular mass of 52 kDa. The deduced amino acid sequence exhibited greater than 45% identity with that of prokaryotic d-hydantoinases and eukaryotic DHPs. Phylogenetic analysis showed that Bacillus sp. TS-23 DHP is grouped together with Bacillus stearothermophilus d-hydantoinase and related to dihydroorotases and allantoinases from various organisms. His₆-tagged DHP was over-expressed in Escherichia coli and purified by immobilized metal affinity chromatography to a specific activity of 3.46 U mg⁻¹ protein. The optimal pH and temperature for the purified enzyme were 8.0 and 60 °C, respectively. The half-life of His₆-tagged DHP was 25 days at 50 °C. The enzyme activity was stimulated by Co²⁺ and Mn²⁺ ions. His₆-tagged DHP was most active toward dihydrouracil followed by hydantoin derivatives. The catalytic efficiencies (k_cat/K_m) of the enzyme for dihydrouracil and hydantoin were 2.58 and 0.61 s⁻¹ mM⁻¹, respectively.     

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

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

Recombinant Human Cytomegalovirus Protease with a C-Terminal (His)6Extension: Purification, Autocatalytic Release of the Mature Enzyme, and Biochemical Characterization

Human cytomegalovirus protease (CMV PR) is a target for the development of antiviral therapeutics. To obtain large amounts of native protease, a 268-amino-acid polypeptide with a hexahistidinyl tag at the C terminus was expressed inEscherichia coli.The first 262 amino acids of the recombinant protein were identical to the amino acid sequence of native CMV PR, except for mutations introduced at the internal cleavage site to eliminate autoproteolysis at that site. The hexahistidinyl tag was placed downstream of amino acid 262 of the native CMV PR sequence. In this design, the Ala-Ser bond at amino acids 256–257 constitutes a site naturally cleaved by the protease during capsid maturation. The 268-amino-acid polypeptide with the (His)₆tag was expressed at high levels inE. colias inclusion bodies. After solubilization of the inclusion bodies, the protease was purified to homogeneity by a single step using Ni²⁺affinity chromatography. The protease was refolded to an active enzyme using dialysis which leads to effective autocleavage of the Ala-Ser bond at amino acids 256–257 to remove 12 amino acids including the (His)₆tag from the C terminus of the protein. This strategy yielded large amounts of highly purified CMV PR with the native N terminus and C terminus. Approximately 40 mg of purified CMV PR was obtained per liter of cell culture using this strategy. The enzymatic activity of CMV PR purified from inclusion bodies and refolded to an active enzyme was similar to the enzymatic activity of CMV PR expressed as a soluble protein inE. coli.In addition, the refolded CMV PR could be crystallized for X-ray diffraction.