Enhanced production of penicillin G acylase from a recombinant Escherichia coli

Penicillin G acylase (pac) gene was cloned into a stable asd ⁺ vector (pYA292) and expressed in Escherichia coli. This recombinant strain produced 1000 units penicillin G acylase g^–1 cell dry wt, which is 23-fold more than that produced by parental Escherichia coli ATCC11105. This enzyme was purified to 16 units mg^–1 protein by a novel two-step process.     

Sequence analysis,cloning and over-expression of an endoxylanase from the alkaliphilic <Emphasis Type="BoldItalic">Bacillus halodurans</Emphasis>

The BhMIR32 xyn11A gene, encoding an extracellular endoxylanase of potential interest in bio-bleaching applications, was amplified from Bacillus halodurans MIR32 genomic DNA. The protein encoded is an endo-1,4-β-xylanase belonging to family 11 of glycosyl hydrolases. Its nucleotide sequence was analysed and the mature peptide was subcloned into pET22b(+) expression vector. The enzyme was over-expressed in a high density Escherichia coli culture as a soluble and active protein, and purified in a single step by immobilised metal ion affinity chromatography with a specific activity of 3073 IU mg⁻¹.     

Improvement of succinate production by overexpression of a cyanobacterial carbonic anhydrase in Escherichia coli

Succinate fermentation was investigated in Escherichia coli strains overexpressing cyanobacterium Anabaena sp. 7120 ecaA gene encoding carbonic anhydrase (CA). In strain BL21 (DE3) bearing ecaA, the activity of CA was 21.8 U mg⁻¹ protein, whereas non-detectable CA activity was observed in the control strain. Meanwhile, the activity of phosphoenolpyruvate carboxylase (PEPC) increased from 0.2 U mg⁻¹ protein to 1.13 U mg⁻¹ protein. The recombinant bearing ecaA reached a succinate yield of 0.39 mol mol⁻¹ glucose at the end of the fermentation. It was 2.1-fold higher than that of control strain which was just 0.19 mol mol⁻¹ glucose. EcaA gene was also introduced into E. coli DC1515, which was deficient in glucose phosphotransferase, lactate dehydrogenase and pyruvate:formate lyase. Succinate yield can be further increased to 1.26 mol mol⁻¹ glucose. It could be concluded that the enhancement of the supply of HCO₃⁻ in vivo by ecaA overexpression is an effective strategy for the improvement of succinate production in E. coli.     

Overexpression, purification and characterization of functional calf purine nucleoside phosphorylase (PNP)

Calf PNP is a ubiquitous enzyme of the salvage metabolic pathway. The procedure for this enzyme production in large quantities is described. The coding sequence of bovine PNP was amplified from the calf spleen cDNA library and was inserted into an expression vector pET28a(+). The construct was transformed into Escherichia coli BL21(DE3) strain. The protein expression efficiencies in the presence and the absence of IPTG were compared. It was found that IPTG is not necessary for obtaining a large quantity of recombinant calf PNP: 35 mg from 1 L cell culture. The enzyme was purified to 92% homogeneity by a two-step procedure consisting of gel filtration and ion exchange chromatography. The purity of recombinant enzyme is sufficient to form well diffracting single crystals.The basic kinetic parameters of recombinant PNP were determined and compared with the parameters of commercially available PNP from calf spleen. The specific activity in 50 mM phosphate buffer with inosine as a variable substrate (30.7 μmol min⁻¹ mg⁻¹) and other kinetic parameters: Michaelis constants, maximal velocities, dissociation and inhibition constants, determined for several typical PNP ligands, are similar to the values published previously for non-recombinant calf spleen PNP. As expected for mammalian PNP, recombinant calf PNP was found to have no substrate activity vs adenosine. The overexpression and purification method of the recombinant calf PNP provides significant amounts of the enzyme, which can successfully replace the non-recombinant PNP.     

Glucose-1-phosphatase (AgpE) from Enterobacter cloacae displays enhanced phytase activity

Using a screening procedure developed for detection of phytate hydrolysing enzymes, the gene agpE encoding glucose-1-phosphatase was cloned from an Enterobacter cloacae VKPM B2254 plasmid library. Sequence analysis revealed 78% identity on nucleotide and 79% identity on peptide level to Escherichia coli glucose-1-phosphatase characterising the respective gene product as a representative of acid histidine phosphatases harbouring the RH(G/N)RXRP motif. The purified recombinant protein displayed maximum specific activity of 196 U mg⁻¹ protein against glucose-1-phosphate but was also active against other sugar phosphates and p-nitrophenyl phosphate. High-performance ion chromatography of hydrolysis products revealed that AgpE can act as a 3-phytase but is only able to cleave off the third phosphate group from the myo-inositol sugar ring. Based on sequence comparison and catalytic behaviour against phytate, we propose to classify bacterial acid histidine phosphatases/phytases in the three following subclasses: (1) AppA-related phytases, (2) PhyK-related phytases and (3) Agp-related phytases. A distinguished activity of 32 U mg⁻¹ of protein towards myo-inositol-hexa-phosphate, which is two times higher than that of E. coli Agp, suggests that possibly functional differences in terms of phytase activity between Agp- and AppA-like acid histidine phosphatases are fluent. Electronic supplementary material Supplementary material is available for this article at and accessible for authorised users.     

An Uniquely Purified HCV NS3 Protease and NS4A21–34 Peptide Form a Highly Active Serine Protease Complex in Peptide Hydrolysis

The N-terminal domain of the hepatitis C virus (HCV) polyprotein containing the NS3 protease (residues 1027 to 1206) was expressed in Escherichia coli as a soluble protein under the control of the T7 promoter. The enzyme has been purified to homogeneity with cation exchange (SP-Sepharose HR) and heparin affinity chromatography in the absence of any detergent. The purified enzyme preparation was soluble and remained stable in solution for several weeks at 4°C. The proteolytic activity of the purified enzyme was examined, also in the absence of detergents, using a peptide mimicking the NS4A/4B cleavage site of the HCV polyprotein. Hydrolysis of this substrate at the expected Cys–Ala scissile bond was catalyzed by the recombinant protease with a pseudo second-order rate constant (k_cat/K_M) of 205 and 196,000 M⁻¹ s⁻¹, respectively, in the absence and presence of a central hydrophobic region (sequence represented by residues 21 to 34) of the NS4A protein. The rate constant in the presence of NS4A peptide cofactor was two orders of magnitude greater than reported previously for the NS3 protease domain. A significantly higher activity of the NS3 protease–NS4A cofactor complex was also observed with a substrate mimicking the NS4B/5A site (k_cat/K_M of 5180 ± 670 M⁻¹ s⁻¹). Finally, the optimal formation of a complex between the NS3 protease domain and the cofactor NS4A was critical for the high proteolytic activity observed.     

Molecular cloning and expression of the pyranose 2-oxidase cDNA from<Emphasis Type="Italic"> Trametes ochracea</Emphasis> MB49 in<Emphasis Type="Italic"> Escherichia coli</Emphasis>

A cDNA of a structural gene encoding pyranose 2-oxidase (P2O) from Trametes ochracea strain MB49 was cloned into Escherichia coli strain BL21(DE3) on a multicopy plasmid under the control of the trc promoter. Synthesis of P2O was studied in batch cultures in LB or M9-based mineral medium at 28°C. While there was a low specific activity of P2O in LB medium, the enzyme was synthesised constitutively in mineral medium and represented 3% of the cell soluble protein (0.3 U mg^–1). The effect of isopropyl -d-thiogalactoside on the expression of P2O was studied in mineral medium at 25 and 28°C. The synthesis of P2O at 28°C corresponded to 39% of the cell soluble protein but the major portion of P2O (93%) was in the form of non-active inclusion bodies (activity of P2O equalled 0.19 U mg^–1). At 25°C, the amount of P2O represented 14% of the cell soluble protein and the activity of P2O was 1.1 U mg^–1. The soluble enzyme represented 70% of the total amount of P2O.     

Alternative production process strategies in E. coli improving protein quality and downstream yields

Process strategies for production of recombinant rhamnulose 1-phosphate aldolase (RhuA) in Escherichia coli were found to have an important impact on downstream processing when preparing the enzyme for its use as immobilized biocatalyst. First, a continuous inducer feed was implemented in substrate limited fed-batch cultures to overexpress RhuA with a hexa histidine-tag (6xHis-tag) at its N-terminus. The final specific RhuA level was 180 mg g⁻¹ DCW, but the final specific enzyme activity (1.7 AU mg⁻¹ RhuA) was considerably lower than expected. Only 55% of immobilization yield was achieved when immobilized metal affinity chromatography (IMAC) was used to purify and immobilize RhuA from cellular lysate in a single step. Western blot analyses showed that only 20% of overexpressed RhuA kept the whole 6xHis-tag at the end of the culture due to partial proteolysis. Two different growth strategies improved protein quality and immobilization yield:

(i) Temperature reduction to 28 °C in substrate limited operation decreased proteolysis and allowed higher specific activities, 210 mg g⁻¹ DCW. The enzyme activity increased to 4 AU mg⁻¹ RhuA and purification-immobilization yield to 93%.

(ii) A novel fed-batch operational procedure, working at high glucose concentration was implemented. High aldolase levels, 233 mg g⁻¹ DCW, were reached at the end of the culture. The final enzyme activity was also higher than 4 AU mg⁻¹ RhuA, and 95% of immobilization yield was achieved.

For both cases, Western blot analyses showed that 80–100% of overexpressed RhuA kept the whole 6xHis-tag at the end of the culture, confirming that recombinant protein quality had been improved.     

Overexpression,Purification, and Characterization of the Recombinant Leucine Aminopeptidase II of <Emphasis Type="Italic">Bacillus stearothermophilus</Emphasis>

For expression of Bacillus stearothermophilus NCIB 8924 leucine aminopeptidase II (LAP II) in Escherichia coli regulated by a T5 promoter, the gene was amplified by polymerase chain reaction and cloned into expression vector pQE-32 to generate pQE-LAPII. The His₆-tagged enzyme was overexpressed in IPTG-induced E. coli M15 (pQE-LAPII) as a soluble protein and was purified to homogeneity by nickel-chelate chromatography to a specific activity of 425 U/mg protein with a final yield of 76%. The subunit molecular mass of the purified protein was estimated to be 44.5 kDa by SDS-PAGE. The temperature and pH optima for the purified protein were 60°C and 8.0, respectively. Under optimal condition, the purified enzyme showed a marked preference for Leu-p-nitroanilide, followed by Arg- and Lys-derivatives. The His₆-tagged enzyme was stimulated by Co²⁺ ions, but was strongly inhibited by Cu²⁺ and Hg²⁺ and by the chelating agents, DTT and EDTA. The EDTA-treated enzyme could be reactivated with Co²⁺ ions, indicating that it is a cobalt-dependent exopeptidase. Taking the biochemical characteristics together, we found that the recombinant LAP II exhibits no important differences from those properties described for the native enzyme. Received: 16 August 2002 / Accepted: 4 September 2002     

Isolation, Characterization, and Heterologous Expression of a Carboxylesterase of Pseudomonas aeruginosa PAO1

We purified to homogeneity an intracellular esterase from the opportunistic pathogen Pseudomonas aeruginosa PAO1. The enzyme hydrolyzes p-nitrophenyl acetate and other acetylated substrates. The N-terminal amino acid sequence was analyzed and 11 residues, SEPLILDAPNA, were determined. The corresponding gene PA3859 was identified in the P. aeruginosa PAO1 genome as the only gene encoding for a protein with this N-terminus. The encoding gene was cloned in Escherichia coli, and the recombinant protein expressed and purified to homogeneity. According to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis and analytical gel filtration chromatography, the esterase was found to be a monomer of approximately 24 kDa. The experimentally determined isoelectric point was 5.2 and the optimal enzyme activity was at 55°C and at pH 9.0. The esterase preferentially hydrolyzed short-chain fatty acids. It is inhibited by phenylmethylsulfonyl fluoride (PMSF) but not by ethylendiaminotetraacetic acid (EDTA). Native enzyme preparations typically showed a Michaelis constant (K_m) and V_max of 0.43 mM and 12,500 U mg^–1, respectively, using p-nitrophenyl acetate as substrate. Homology-based database searches clearly revealed the presence of the consensus GXSXG signature motif that is present in the serine-dependent acylhydrolase protein family.     

Antagonistic effect of enterocin CCM 4231 from Enterococcus faecium on “bryndza”, a traditional Slovak dairy product from sheep milk

“Bryndza” is a traditional Slovak dairy product (type of soft cheese) made from sheep cheese which was ripened for 14 days. Because its manufacture, transporting and/or storing represent conditions which facilitate contamination, the effect of enterocin CCM4231 in “bryndza” was investigated with the aim to reduce the contaminant agents. “Bryndza” was divided into equal portions (50 g). The experimental sample (ES) as well as the control sample one (C1) were inoculated with Listeria innocua Li1 strain. The other control samples C2 and C3 were without Li1 strain. C3 control was selected as a reference control. ES and C2 portions were treated with purified enterocin CCM4231 in a concentration of 6400 AU/ml. Before the experimental inoculation, “bryndza” was checked for the presence of contaminant agents. The experiment lasted 1 week and the samples were stored in the refrigerator at 4 °C. Sampling was performed on day 1, on day 4 and on day 7. The control samples C2 and C3 were checked only on day 1 and then after 1 week. The following contaminant agents were detected in “bryndza” before its experimental inoculation with L. innocua Li1 strain: Escherichia coli in the amount 10³ cfu/ml/g, Staphylococcus aureus (10² cfu/ml/g) and enterococci (10⁴ cfu/ml/g). In the control sample C2, the number of E. coli was reduced to 10² cfu/ml/g. Enterococci and staphylococci were totally eliminated there. Concerning C3 control, natural decrease of bacteria was found and/or their unchanged counts. The value of pH (5) was stable during the whole experiment. In the experimental sample inoculated with Li1 strain, its counts were decreased immediately after enterocin CCM4231 addition approximately by one order of magnitude. This inhibitory effect was also detectable on day 4 by the difference of one order of magnitude between ES and C1. On day 7, 10³ cfu/ml/g of Li1 strain were detected in both samples (ES, C1). The difference by one order of magnitude indicated, an inhibitory effect of enterocin CCM4231 in “bryndza”. However, bacteriocin activity was not determined by laboratory analyses.     

Bacterial Expression and Purification of the Arabidopsis NADPH–Cytochrome P450 Reductase ATR2

An N-terminally modified form of the Arabidopsis NADPH–cytochrome P450 ATR2 (ATR2mod) was expressed from the tactac promoter in Escherichia coli to obtain high yields of the enzyme. The N-terminal modification eliminates the predicted chloroplast transit peptide of ATR2 allowing for more efficient expression. ATR2mod was purified from membrane extracts using a 2′,5′-ADP–agarose affinity column. The specific activity of the purified ATR2mod for cytochrome c reduction was 9.4 μmol min⁻¹ mg⁻¹ and the K_m for cytochrome c reduction was 15 ± 2 μM. The purified NADPH–cytochrome P450 reductase was able to support function of CYP79B2.     

“Action potentials” in NEUROSPORA CRASSA, a mycelial fungus

Occasional spontaneous “action potentiais” are found in mature hyphae of the fungus Neurospora crassa. They can arise either from low-level sinusoidal oscillations of the membrane potential or from a linear slow depolarization which accelerates into a rapid upstroke at a voltage 5–20 mV depolarized from the normal resting potential (near − 180 mV). The “action potentiais” are long-lasting, 1–2 min and at the peak reach a membrane potential near −40 mV. A 2− to 8−fold increase of membrane conductance accompanies the main depolarization, but a slight decrease of membrane conductance occurs during the slow depolarization. Two plausible mechanisms for the phenomenon are (a) periodic increases of membrane permeability to inorganic ions, particularly H⁺ or Cl^- and (b) periodic decreases in activity of the major electrogenic pump (H⁺) of the Neurospora membrane, coupled with a nonlinear (inverse sigmoid) current-voltage relationship.Identification of action potential-like disturbances in fungi means that such behavior has now been found in all major biologic taxa which have been probed with suitable electrodes. As yet there is no obvious function for the events in fungi.     

Purification and properties of a novel raw starch degrading-cyclodextrin glycosyltransferase from Klebsiella pneumoniae AS- 22

A novel raw starch degrading α-cyclodextrin glycosyltransferase (CGTase; E.C. 2.4.1.19), produced by Klebsiella pneumoniae AS-22, was purified to homogeneity by ultrafiltration, affinity and gel filtration chromatography. The specific cyclization activity of the pure enzyme preparation was 523 U/mg of protein. No hydrolysis activity was detected when soluble starch was used as the substrate. The molecular weight of the pure protein was estimated to be 75 kDa with SDS-PAGE and gel filtration. The isoelectric point of the pure enzyme was 7.3. The enzyme was most active in the pH range 5.5–9.0 whereas it was most stable in the pH range 6–9. The CGTase was most active in the temperature range 35–50°C. This CGTase is inherently temperature labile and rapidly loses activity above 30°C. However, presence of soluble starch and calcium chloride improved the temperature stability of the enzyme up to 40°C. In presence of 30% (v/v) glycerol, this enzyme was almost 100% stable at 30°C for a month. The K_m and k_cat values for the pure enzyme were 1.35 mg ml⁻¹ and 249 μM mg⁻¹ min⁻¹, respectively, with soluble starch as the substrate. The enzyme predominantly produced α-cyclodextrin without addition of any complexing agents. The conditions employed for maximum α-cyclodextrin production were 100 g l⁻¹ gelatinized soluble starch or 125 g l⁻¹ raw wheat starch at an enzyme concentration of 10 U g⁻¹ of starch. The α:β:γ-cyclodextrins were produced in the ratios of 81:12:7 and 89:9:2 from gelatinized soluble starch and raw wheat starch, respectively.     

Acid dissociation constants and pH values for standard “bes” and “tricine” buffer solutions in 30, 40, and 50 mass% dimethyl sulfoxide/water between 25 and −25 °C

Information is given concerning two standard buffer solutions suitable as pH references in 30, 40, and 50 mass% dimethyl sulfoxide (DMSO)/H₂O mixed solvents at subzero temperatures from −20 to 0 °C, with the intention of establishing a pH (designated pH^*) scale. The two buffers selected were the ampholytes N,N-bis(2-hydroxyethyl)-2-aminoethane sulfonic acid (“bes”) and N-tris(hydroxymethyl)methylglycine (“tricine”), and the reference standard consisted of equal molal quantities of the buffer and its respective sodium salt. The assignment of pH^* values was based on measurements of the emf of cells without liquid junction of the type: Pt;H₂(g,1 atm) ¦Bes, Na Besate, NaCl ¦ AgCl;Ag and Pt;H₂(g,1 atm) ¦Tricine, Na Tricinate, NaCl ¦AgCl;Ag and the pH^* was derived from a determination of K₂, the equilibrium constant for the dissociation process (Buffer)^±/ai (Buffer)⁻ + H⁺.     

Expression in Escherichia coli and Simple Purification of Human Fhit Protein

The fragile histidine triad (Fhit) protein is a homodimeric protein with diadenosine 5′,5-P¹,P³-triphosphate (Ap₃A) asymmetrical hydrolase activity. We have cloned the human cDNA Fhit in the pPROEX-1 vector and expressed with high yield in Escherichia coli with the sequence Met-Gly-His₆-Asp-Tyr-Asp-Ile-Pro-Thr-Thr followed by a rTEV protease cleavage site, denoted as “H6TV,” fused to the N-terminus of Fhit. Expression of H6TV–Fhit in BL21(DE3) cells for 3 h at 37°C produced 30 mg of H6TV–Fhit from 1 L of cell culture (4 g of cells). The H6TV–Fhit protein was purified to homogeneity in a single step, with a yield of 80%, using nickel-nitrilotriacetate resin and imidazole buffer as eluting agent. Incubation of H6TV–Fhit with rTEV protease at 4°C for 24 h resulted in complete cleavage of the H6TV peptide. There were no unspecific cleavage products. The purified Fhit protein could be stored for 3 weeks at 4°C without loss of activity. The pure protein was stable at −20°C for at least 18 months when stored in buffer containing 25% glycerol. Purified Fhit was highly active, with a K_m value for Ap₃A of 0.9 μM and a k_cat(monomer) value of 7.2 ± 1.6 s⁻¹ (n = 5). The catalytic properties of unconjugated Fhit protein and the H6TV–Fhit fusion protein were essentially identical. This indicates that the 24-amino-acid peptide containing the six histidines fused to the N-terminus of Fhit does not interfere in forming the active homodimers or in the binding of Ap₃A.     

Application of a thermostable glutamate racemase from Bacillus sp. SK-1 for the production of -phenylalanine in a multi-enzyme system

A gene encoding glutamate racemase (GluRA) was found in a thermophilic Bacillus strain named SK-1. The gene was cloned and expressed in Escherichia coli WM335, a -glutamate auxotroph. It consists of 792 bp with a start codon, TTG. The amino acid sequence deduced from the gene indicates that the GluRA has two cysteines and their surrounding regions are well conserved. The GluRA produced in the recombinant E. coli was purified to homogeneity by heat-treatment and Resource Q and Phenyl sepharose column chromatographies. The enzyme, which was determined to be a monomeric protein with a molecular weight of 29,000, did not require a cofactor such as pyridoxal 5′-phosphate, nicotinamide, or flavin for its activity. The enzyme was stable after incubation at 55 °C and retained 60% of its original activity after incubation at 60 °C. It was found to be stable in the region of pH 6.0–11.5. The thermostable GluRA was used as a catalyst in a multi-enzyme system composed of four enzyme reactions for the production of -phenylalanine. By running the multi-enzyme system for 35 h, 58 g l⁻¹ of -phenylalanine was produced with 100% of optical purity from equimolar amount of phenylpyruvate.     

Construction,expression and characterization of fusion enzyme from <Emphasis Type="Italic">Arthrobacter oxydans</Emphasis> dextranase and <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> amylase

An artificial fusion protein of Arthrobacter oxydans dextranase and Klebsiella pneumoniae α-amylase was constructed and expressed in Escherichia coli. Most of the expressed protein existed as an insoluble fraction, which was solubilized with urea. The purified fusion enzyme electrophoretically migrated as a single protein band; M = 137 kDa, and exhibited activities of both dextranase (10.8 U mg⁻¹) and amylase (7.1 U mg⁻¹), which were lower than that of reference dextranase (13.3 U mg⁻¹) and α-amylase (103 U mg⁻¹). The fusion enzyme displayed bifunctional enzyme activity at pH 5–7 at 37°C. These attributes potentially make the fusion enzyme more convenient for use in sugar processing than a two-enzyme system.