Heterologous expression in Escherichia coli of Neurospora crassa neutral trehalase as an active enzyme

Neutral trehalase from Neurospora crassa was expressed in Escherichia coli as a polypeptide of 84 kDa in agreement with the theoretical size calculated from the corresponding cDNA. The recombinant neutral trehalase, purified by affinity chromatography exhibited a specific activity of 80–150 mU/mg protein. Optima of pH and temperature were 7.0 and 30 °C, respectively. The enzyme was absolutely specific for trehalose, and was quite sensitive to incubation at 40 °C. The recombinant enzyme was totally dependent on calcium, and was inhibited by ATP, copper, silver, aluminium and cobalt. K_M was 42 mM, and V_max was 30.6 nmol of glucose/min. The recombinant protein was phosphorylated by cAMP-dependent protein kinase, but not significantly activated. Immunoblotting with polyclonal antiserum prepared against the recombinant protein showed that neutral trehalase protein levels increased during exponential phase of N. crassa growth and dropped at the stationary phase. This is the first report of a neutral trehalase produced in E. coli with similar biochemical properties described for fungi native neutral trehalases, including calcium-dependence.     

Characterization of a novel low molecular weight sucrase from filamentous fungus Termitomyces clypeatus

An extracellular sucrase from the culture filtrate of filamentous basidiomycota Termitomyces clypeatus grown on high sucrose (5%, w/v) was purified by gel filtration chromatography, ion exchange chromatography and HPGPLC. The biochemical properties, molecular weight and conformation of sucrase produced were significantly different from the sucrase earlier purified from sucrose (1%, w/v) medium in the fungus. Purified sucrase was characterized as a low molecular weight protein of 13.5 kDa as approximated by SDS-PAGE and HPGPLC and exhibited predominantly random coil conformation in far-UV CD spectra. The enzyme was optimally active at 47 °C and pH 5.0. K_m and catalytic activity of the enzyme for sucrose were found to be 3.5 mM and 1.06 U/mg/mM, respectively. The enzyme was maximally active towards sucrose than to raffinose and sucrase activity was significantly inhibited by bivalent metal ions and reducing group agents. The results indicated that due to changes in aggregation pattern, molecular organization of purified sucrase, produced in high sucrose medium, was altered and was different from the previously reported enzyme. This is the first report of a sucrase of such low size showing activity.     

Characterization of a extreme thermostable fructose-1,6-bisphosphate aldolase from hyperthermophilic bacterium Aquifex aeolicus

Fructose-1,6-bisphosphate (FBP) aldolase, is a glycolytic enzyme that catalyzes the reversible condensation reaction of FBP to dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P). The aldolase gene from Aquifex aeolicus was subcloned, overexpressed in E. coli and purified to 95% homogeneity. The purified enzyme was activated by high concentrations of NH₄⁺ and low concentrations of Co²⁺. The native molecular weight of the purified FBP aldolase was identified as 67 kDa (dimer) by gel filtration chromatography. The enzyme exhibits optimum pH at 6.5 and temperature at 90 °C. Based on the kinetic characterizations, the apparent K_m was calculated to be 4.4 ± 0.07 mM, while V_max was found to be 100 ± 0.02 μM min⁻¹ mg protein⁻¹. The recombinant protein showed extreme heat stability; no activity loss was observed even at 100 °C for 2 h. In addition, the thermophilic enzyme also showed higher stability against several organic solvents viz. acetonitrile, 1,4-dioxane, and methanol. With higher stability against both heat and organic solvents than any other class II aldolase, the A. aeolicus FBP aldolase is an attractive enzyme for use as a biocatalyst for industrial applications.     

Cloning, expression, and characterization of a new deoxyribose 5-phosphate aldolase from Yersinia sp. EA015

A new deoC gene encoding deoxyribose 5-phosphate aldolase (DERA) was identified in Yersinia sp. EA015 isolated from soil. The DERA gene had an open reading frame (ORF) of 672 base pairs encoding 223 amino acids to yield a protein of molecular mass 24.8 kDa. The amino acid sequence was 94% identical to that of DERA from Yersinia intermedia ATCC 29909. DERA was over-expressed in Escherichia coli and purified using Ni–NTA affinity chromatography. The specific activity was 137 μmol/min/mg. The Michaelis constant (k_m value) of DERA was 9.1 mM. DERA was optimally active at pH 6.0 and 50 °C. DERA was tolerant to a high concentration (300 mM) of acetaldehyde.     

High-level expression, purification and characterization of recombinant Aspergillus oryzae alkaline protease in Pichia pastoris

The alkaline protease gene from Aspergillus oryzae was cloned, and then it was successfully expressed in the heterologous Pichia pastoris GS115 with native signal peptide or α-factor secretion signal peptide. The yield of the recombinant alkaline protease with native signal peptide was about 1.5-fold higher than that with α-factor secretion signal peptide, and the maximum yield of the recombinant alkaline protease was 513 mg/L, which was higher than other researches. The recombinant alkaline protease was purified by ammonium sulfate precipitation, ion exchange chromatography and gel filtration chromatography. The purified recombinant alkaline protease showed on SDS–PAGE as a single band with an apparent molecular weight of 34 kDa. The recombinant alkaline protease was identical to native alkaline protease from A. oryzae with regard to molecular weight, optimum temperature for activity, optimum pH for activity, stability to pH, and similar sensitivity to various metal ions and protease inhibitors. The native enzyme retained 61.18% of its original activity after being incubated at 50 °C for 10 min, however, the recombinant enzyme retained 56.22% of its original activity with same disposal. The work demonstrates that alkaline protease gene from A. oryzae can be expressed largely in P. pastoris without affecting its enzyme properties and the recombinant alkaline protease could be widely used in various industrial applications.     

Purification and characterization of a thermophilic alkaline xylanase from thermoalkaliphilic Bacillus sp. strain TAR-1

Thermoalkaliphilic Bacillus sp. strain TAR-1 isolated from soil produced an extracellular xylanase. The enzyme (xylanase R) was purified to homogeneity by ammonium sulfate fractionation and anion-exchange chromatography. The molecular mass of xylanase R was 40 kDa and the isoelectric point was 4.1. The enzyme was most active over the range of pH 5.0 to 10.0 at 50°C. The optimum temperatures for activity were 75°C at pH 7.0 and 70°C at pH 9.0. Xylanase R was stable up to 65°C at pH 9.0 for 30 min in the presence of xylan. Mercury(ll) ion at 1 mM concentration abolished all the xylanase activity. The predominant products of xylan-hydrolysate were xylobiose, xylotriose, and higher oligosaccharides, indicating that xylanase R was an endo-acting enzyme. Xylanase R had a K_m of 0.82 mg/ml and a V_max of 280 μmol min⁻¹ mg⁻¹ for xylan at 50°C and pH 9.0.     

Optimization of a Thermostable Lipase from Bacillus stearothermophilus P1: Overexpression, Purification, and Characterization

An expression library was generated from a partial NcoI and HindIII digest of genomic DNA from the thermophilic bacterium, Bacillus stearothermophilus P1. The DNA fragments were cloned into the expression vector pQE-60 and transformed into Escherichia coli M15[EP4]. Sequence analysis of a lipase gene showed an open reading frame of 1254 nucleotides coding a 29-amino-acid signal sequence and a mature sequence of 388 amino acids. The expressed lipase was isolated and purified to homogeneity in a single chromatographic step. The molecular mass of the lipase was determined to be approximately 43 kDa by SDS-PAGE and mass spectrometry. The purified lipase had an optimum pH of 8.5 and showed maximal activity at 55°C. It was highly stable in the temperature range of 30–65°C. The highest activity was found with p-nitrophenyl ester-caprate as the synthetic substrate and tricaprylin as the triacylglycerol. Its activity was strongly inhibited by 10 mM phenylmethanesulfonyl fluoride and 1-hexadecanesulfonyl chloride, indicating that it contains a serine residue which plays a key role in the catalytic mechanism. In addition, it was stable for 1 h at 37°C in 0.1% Chaps and Triton X-100.     

Purification of the extracellular pectinolytic enzyme from the fungus Rhizopus oryzae NBRC 4707

The pectinolytic enzyme from the solid-state culture of Rhizopus oryzae NBRC 4707 was purified to homogeneity by column chromatography on CM-Toyopearl 650 M and hydroxylapatite. The molecular weight of the enzyme was estimated by SDS-polyacrylamide gel electrophoresis to be 31,000 and was reduced to 29,700 after treatment with endoglycosidase H. Maximal activity was observed near pH 4.5 at 45°C. The enzyme was shown to be endopolygalacturonase, as judged from the formation of oligogalacturonides as its reaction products. The addition of purified enzyme, as expected, enhanced the formation of lactic acid and ethanol in potato pulp grown with R. oryzae.     

Purification and Characterization of the NAD-Dependent Glutamate Dehydrogenase in the Ectomycorrhizal FungusLaccaria bicolor(Maire) Orton

The NAD-dependent glutamate dehydrogenase (GDH) (EC 1.4.1.2) fromLaccaria bicolorwas purified 410-fold to apparent electrophoretic homogeneity with a 40% recovery through a three-step procedure involving ammonium sulfate precipitation, anion-exchange chromatography on DEAE–Trisacryl, and gel filtration. The molecular weight of the native enzyme determined by gel filtration was 470 kDa, whereas sodium dodecyl sulfate–polyacrylamide gel electrophoresis gave rise to a single band of 116 kDa, suggesting that the enzyme is composed of four identical subunits. The enzyme was specific for NAD(H). The pH optima were 7.4 and 8.8 for the amination and deamination reactions, respectively. The enzyme was found to be highly unstable, with virtually no activity after 20 days at −75°C, 4 days at 4°C, and 1 h at 50°C. The addition of ammonium sulfate improved greatly the stability of the enzyme and full activity was still observed after several months at −75°C. NAD-GDH activity was stimulated by Ca²⁺and Mg²⁺but strongly inhibited by Cu²⁺and slightly by the nucleotides AMP, ADP, and ATP. The Michaelis constants for NAD, NADH, 2-oxoglutarate, and ammonium were 282 μM, 89 μM, 1.35 mM, and 37 mM, respectively. The enzyme had a negative cooperativity for glutamate (Hill number of 0.3), and itsK_mvalue increased from 0.24 to 3.6 mM when the glutamate concentration exceeded 1 mM. These affinity constants of the substrates, compared with those of the NADP-GDH of the fungus, suggest that the NAD-GDH is mainly involved in the catabolism of glutamate, while the NADP-GDH is involved in the catalysis of this amino acid.     

Biochemical and molecular characterisation of a thermoactive, alkaline and detergent-stable lipase from a newly isolated Staphylococcus aureus strain

A newly soil-isolated Staphylococcus aureus strain secretes a non-induced lipase in the culture medium. The extracellular lipase from S. aureus (SAL3) is purified to homogeneity. The purified enzyme is a tetrameric protein (180 kDa) corresponding to the association of four lipase molecules. The 15 N-terminal amino acid residues showed a high degree of homology with other staphylococcal lipase sequences. The part of the gene encoding the mature SAL3 is cloned and sequenced. The deduced polypeptide sequence, corresponding to the mature SAL3, was very similar to the mature Staphylococcus simulans lipase sequence with two additional amino acid residues (LK) at the N-terminus of SAL3. The lipase activity is maximal at pH 9.5 and 55 °C. The specific activity of about 4200 U/mg or 3500 U/mg was measured using tributyrin or olive oil emulsion as substrate, respectively, at pH 9.5 and 55 °C.In contrast to other staphylococcal lipases previously characterised, SAL3 is found to be stable between pH 5 and 12 after 24 h incubation. The enzyme retained 50% of its activity after 60 min incubation at 60 °C. This novel lipase is able to hydrolyse its substrate in presence of various oxidizing agents as well as some surfactants and some commercial detergents, then SAL3 can be considered as a good candidate for industrial and biotechnological applications.     

Purification and characterization of carbonyl reductase from Geotrichum candidum

Geotrichum candidum is well known for the reduction of prochiral ketones to chiral alcohol with high yield and excellent enantioselectivity. Carbonyl reductase from G. candidum was purified by ammonium sulphate precipitation, anion exchange and hydrophobic interaction chromatographies. Gel filtration chromatography together with SDS-PAGE revealed this protein to be a dimer of 60 kDa subunits. Maximum enzyme activity was found in acetate buffer at pH 5.4 with t_1/2 of 7.13 h at 30 °C and t_1/2 of 2.8 h at 65 °C. The enzyme was inhibited by p-hydroxymercuribenzoate and hydroxylamine indicating the involvement of thiol and carbonyl groups in the reduction reaction catalyzed by the enzyme. Chelating agents also reduced the enzyme activity indicating the requirement of metal ions as cofactors. The purified carbonyl reductase was found to be highly selective for ketones containing naphthyl ring, whereas aryl or hetero-aryl ketones showed very less or no activity at all.     

Gene cloning,expression, and characterization of a novel trehalose synthase from <Emphasis Type="Italic">Arthrobacter aurescens</Emphasis>

Trehalose synthase (TreS) is an intramolecular transglycosylase. It specially catalyzes the conversion of maltose and trehalose. In this study, a novel treS gene, which had a length of 1,797 bp and encoded 598 amino acids, was cloned from Arthrobacter aurescens CGMCC 1.1892 and expressed in Escherichia coli. Thin layer chromatography results indicated that it could catalyze the conversion between maltose and trehalose in one step. However, the ion chromatography results showed that, as a byproduct, about 13% glucose was also produced. The purified recombinant enzyme had a molecular weight of 68 kDa and showed its optimal activity at 35 °C and pH 6.5. This enzyme was not thermostable, and its activity was increased by 1 mM Mg²⁺, Mn²⁺, and Ca²⁺ while strongly inhibited by 5 mM Cu²⁺ and SDS.     

Biochemical characterization of Rhodococcus erythropolis N′4 nitrile hydratase acting on 4-chloro-3-hydroxybutyronitrile

The Rhodococcus erythropolis strain (N′4) possesses the ability to convert 4-chloro-3-hydroxybutyronitrile into the corresponding acid. This conversion was determined to be performed by its nitrile hydratase and amidase. Ammonium sulfate fractionation, DEAE ion exchange chromatography, and phenyl chromatography were used to partially purify nitrile hydratase from cell-free extract. A SDS-PAGE showed that the partially purified enzyme had two subunits and gel filtration chromatography showed that it consisted of four subunits of α₂β₂. The purified enzyme had a high specific activity of 860 U mg⁻¹ toward methacrylonitrile. The enzyme was found to have high activity at low temperature range, with a maximum activity occurring at 25 °C and be stable in the presence of organic acids at higher temperatures. The enzyme exhibited a preference for aliphatic saturated nitrile substrates over aliphatic unsaturated or aromatic ones. It was inhibited by sulfhydryl, oxidizing, and serine protease inhibitors, thus indicating that essential cysteine and serine residues can be found in the active site.The purified nitrile hydratase was able to convert 4-chloro-3-hydroxybutyronitrile into the corresponding amide at 15 °C. GC analysis showed that the initial conversion rate of the reaction was 215 mg substrate consumed min⁻¹ mg⁻¹. This demonstrated that this enzyme could be used in conjunction with a stereoselective amidase to synthesize ethyl (S)-4-chloro-3-hydroxybutyrate, an intermediate for a hypercholesterolemia drug, Atorvastatin.     

A new recombinant phosphotriesterase homology protein from Geobacillus caldoxylosilyticus TK4: An extremely thermo- and pH-stable esterase

Phosphotriesterase homology protein (PHP) is a member of a recently discovered family of proteins related to phosphotriesterase. Phosphotriesterase is a hydrolytic, bacterial enzyme with unusual substrate specificity for synthetic organophosphate triesters, common constituents of chemical warfare agents and agricultural pesticides. PHP may belong to the family of proteins from which phosphotriesterase evolved. The PHP gene from the thermophilic bacterium Geobacillus caldoxylosilyticus TK4 was cloned and overexpressed in Escherichia coli with 6×His tag in the N-terminal. The recombinant protein was purified with nickel affinity chromatography and characterized in detail. The enzyme did not have any activity against paraoxon. The highest activities were observed with p-nitrophenyl acetate (pNPA) and p-nitrophenyl butyrate. pH and temperature optima were determined as 8.0 and 50 °C, respectively, with pNPA. The enzyme activity was not largely affected by the incubation of the enzyme at 50 °C in the different buffer solutions (pHs between 3.0 and 9.0) for 7 days. After the incubation at 90 °C for 7 days, G. caldoxylosilyticus TK4 PHP retained 62% of its original activity. The enzyme was also resistant to some metal ions and organic solvents. These results suggest that this is the first reported PHP having an extremely pH- and thermo-stable esterase activity.     

Characterization of phenoloxidase activity in Sydney rock oysters (Saccostrea glomerata)

Phenoloxidase (PO) activity was studied in Sydney rock oysters (Saccostrea glomerata). As in other molluscs, PO was found to exist as a pro-enzyme (proPO) in hemocytes. ProPO could be activated to PO by exogenous proteases (trypsin and chymotrypsin), exposure of hemocytes to pathogen-associated molecular patterns (PAMPs) and by the detergents, Triton X-100 and sodium dodecyl sulphate (SDS). Inhibition studies confirmed the proPO activating system of Sydney rock oysters is a proteinase cascade in which Ca²⁺ dependent serine proteinases proteolytically convert proPO into active PO. Activated PO was found to be a tyrosinase-like enzyme that is responsible for both monophenolase and diphenolase activity. The bifunctional PO had higher affinity for the monophenol, hydroquinine monomethyl ether (4HA) (K_m = 4.45 ± 1.46 mM) than for the diphenol, l-DOPA (K_m = 10.27 ± 1.33 mM). Maximum enzyme activity was evident at 37 °C, pH 8 and at salinities of between 30 and 37 ppt. Melanogenesis catalysed by the active enzyme is a composite of eumelanin and the product of a sclerotin pathway combining DOPA decarboxylase with PO activity.     

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

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

Purification and characterization of β-agarase from agar-liquefying soil bacterium, Acinetobacter sp., AG LSL-1

The extracellular β-agarase LSL-1 produced by an agar-liquefying, soil bacterium Acinetobacter sp., AG LSL-1 was purified to homogeneity by combination of ion-exchange and size exclusion chromatography with final yield of 44%. The enzyme has a specific activity of 397 U mg⁻¹ protein and with a molecular mass of 100 kDa. The agarase was active in the pH range of 5.0–9.0, optimally at pH 6.0 and temperature between 25 °C and 55 °C and optimal at 40 °C. The enzyme retained 63% of native activity at 50 °C suggesting it is a thermostable. The activity of the agarase was completely inhibited by metal ions, Hg²⁺, Ag⁺ and Cu²⁺, whereas 25–40% of native activity was retained in the presence of Zn²⁺, Sn²⁺ and SDS. Neoagarobiose was the final product of hydrolysis of both agarose and neoagarohexaose by the purified agarase LSL-1. Based on the molecular mass and final products of agarose hydrolysis, the β-agarase LSL-1 may be further grouped under group III β-agarases and may be a member of GH-50 family. This is the first report on the purification and biochemical characterization of β-agarase from an agar-liquefying Acinetobacter species.     

Enrichment of new alkane oxidizing bacterial strains for human drug metabolite production

An extracellular xylanase produced by Streptomyces matensis DW67 was purified from the culture supernatant by ammonium sulfate precipitation, ion exchange and gel filtration chromatography and characterized. The xylanase was purified to 14.5-fold to homogeneity with a recovery yield of 14.1%. The purified xylanase appeared as a single protein band on SDS-PAGE with a molecular mass of 21.2 kDa. However, it had a very low apparent molecular mass of 3.3 kDa as determined by gel filtration chromatography. The N-terminal sequence of first 15 amino acid residues was determined as ATTITTNQTGYDGMY. The optimal temperature and pH for purified xylanase was 65 °C and pH 7.0, respectively. The enzyme was stable within the pH range of 4.5–8.0 and was up to 55 °C. The xylanase showed specific activity towards different xylans and no activity towards other substrates tested. Hydrolysis of birchwood xylan by the xylanase yielded xylobiose and xylotriose as principal products. The enzyme hardly hydrolyzed xylobiose and xylotriose, but it could hydrolyze xylotetraose and xylopentaose to produce mainly xylobiose and xylotriose through transglycosylation. These unique properties of the purified xylanase make this enzyme attractive for biotechnological applications, such as bioblenching in paper and pulp industries, production of xylooligosaccharides. This is the first report of the xylanase from S. matensis.     

Over-expression in E. coli and purification of the human OCTN1 transport protein

The hOCTN1 amplified from skin fibroblast RNA was cloned in pET-28a(+) or in pH6EX3 plasmid. The encoded recombinant hOCTN1 resulted in a 6-His tagged fusion protein with a 34 or 21 amino acid extra N-terminal sequence in the pET-28a(+)-hOCTN1 or in the pH6EX3-hOCTN1 constructs, respectively. Both constructs were used to express the hOCTN1 in Escherichia coli Rosetta(DE3)pLysS. The best over-expression was obtained with the pH6EX3-hOCTN1 after 6 h of induction with IPTG at 28 °C. The expressed protein with an apparent molecular mass of 54 kDa, was collected in the insoluble fraction of the cell lysate. Further improvement was obtained using the E. coli RosettaGami2(DE3)pLysS strain to express the protein encoded by pH6EX3-hOCTN1. After 6 h of induction with IPTG at 28 °C, hOCTN1 accounted for 30% of the total protein in the insoluble pellet. This protein fraction was washed with Triton X-100 and deoxycholate, solubilized with a buffer containing 0.8% Sarkosyl, 3 M urea and applied to a Ni²⁺-chelating chromatography column. The homogeneously purified hOCTN1 was eluted with a buffer containing 50 mM imidazole, 0.1% Triton X-100 and 50 mM 2-mercaptoethanol. A yield of about 3 mg purified protein per liter of cell culture was obtained.