An efficient purification with a high recovery of the inulin fructotransferase of Arthrobacter sp. A-6 from recombinant Escherichia coli

Inulin fructotransferase (IFTase, EC 2.4.1.93) of Arthrobacter sp. A-6 was purified from a cell extract of the recombinant Escherichia coli DH5 /pDFE cells carrying the IFTase gene using heat treatment followed by gel filtration. The enzyme was purified 45-fold to apparent homogeneity with a recovery of 79%. SDS-PAGE yielded a single protein band of M _r 46.5 kDa. The recombinant IFTase had a similar thermostability as the original enzyme from Arthrobacter sp. A-6.     

Maize recombinant C4-pyruvate,orthophosphate dikinase: Expression in Escherichia coli, partial purification, and characterization of the phosphorylatable protein

The gene for C₄-pyruvate,orthophosphate dikinase (PPDK) from maize (Zea mays) was cloned into an Escherichia coli expression vector and recombinant PPDK produced in E. coli cells. Recombinant enzyme was found to be expressed in high amounts (5.3 U purified enzyme-activity liter^-1 of induced cells) as a predominantly soluble and active protein. Biochemical analysis of partially purified recombinant PPDK showed this enzyme to be equivalent to enzyme extracted from illuminated maize leaves with respect to (i) molecular mass, (ii) specific activity, (iii) substrate requirements, and (iv) phosphorylation/inactivation by its bifunctional regulatory protein.Abbreviations DTT- dithiothreitol - FPLC- fast-protein liquid chromatography - HAP- hydroxyapatite - IPTG- isopropyl--thiogalactoside - MOPS- 3-(N-morpholino)propanesulfonic acid - PCR- polymerase chain reaction - PEP- phosphoenolpyruvate - PMSF- phenylmethylsufonyl fluoride - PPDK- pyruvate,orthophosphate dikinase - RP- regulatory protein     

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.     

Lamivudine production via enantioselective deamination by thermostable Bacillus caldolyticus cytidine deaminase

To decrease the costs of producing the anti-HIV drug, lamivudine, an enzymatic conversion process was developed instead of the traditional chemical method. Thermostable cytidine deaminase was over-produced by cloning the cdd gene into E. coli JF611/pCJH53 from Bacillus caldolyticus. The purified cytidine deaminase was recovered from the lysate of the recombinant E. coli JF611/pCJH53 by removing heat-denatured proteins and eluting sequential chromatography. When the enzyme was used to deaminate (–)--l-(2R, 5S)- and (+)--d-(2S, 5R)-1, 3-oxathiolanyl-cytosine, about 68% of the (+)--d-(2S, 5R)-1, 3-oxathiolanyl-cytosine was deaminated into the corresponding (+)-thiauridine maximally.     

Cloning and high expression of glutaryl 7-aminocephalosporanic acid acylase gene from Pseudomonas diminuta

The gene coding for the glutaryl 7-aminocephalosporanic acid (GL 7-ACA) acylase from Pseudomonas diminuta KAC-1 was cloned and expressed in Escherichia coli. The acylase gene was composed of 2160 base pairs and encoded a polypeptide of 720 amino acid residues. The E. coli BL21 carrying pET2, the plasmid construct for high expression of GL 7-ACA acylase gene, produced this enzyme at approx. 30% of the total proteins with 3.2 units activity mg protein^–1. Growth at temperature below 31 °C and deletion of signal peptide increased the processing of precursor acylase to active enzyme in the recombinant E. coli cells.     

Plasmid pAO1 of Arthrobacter oxidans encodes 6-hydroxy-D-nicotine oxidase: cloning and expression of the gene in Escherichia coli

Summary The 160 kb plasmid pAO1 from Arthrobacter oxidans (Brandsch and Decker 1984) was subcloned in Escherichia coli with the aid of the plasmid vectors pUR222 and pBR322. Screening of the recombinant clones for enzyme activity revealed that the flavoenzyme 6-hydroxy-d-nicotine oxidase (6-HDNO), one of the enzymes of the nicotine-degradative pathway in A. oxidans, is encoded on pAO1. Immunoprecipitation of ³⁵S-methionine-labelled E. coli cells with 6-HDNO-specific antiserum and expression of recombinant plasmid DNA in E. coli maxicells revealed that 6-HDNO is made as a 52,000 dalton protein, approximately 4,500 daltons larger than 6-HDNO from A. oxidans. The 6-HDNO activity was constitutively expressed in E. coli cells, possibly from an A. oxidans promoter, as shown by subcloning of the 6-HDNO gene in pBR322, using the expression vector pKK223-3 and the promoter probe vector pCB192.     

Purification and characterization of NADPH-dependent aldo–keto reductase specific for β-keto esters from Penicillium citrinum,and production of methyl (S)-4-bromo-3-hydroxybutyrate

A novel -keto ester reductase (KER) was purified to homogeneity from recombinant Escherichia coli (pTrcKER) cells, which efficiently expressed the ker gene cloned from Penicillium citrinum IFO4631. The enzyme was monomeric and had a molecular mass of 37 kDa. It catalyzed the reduction of some -keto esters, especially alkyl 4-halo-3-oxobutyrates. However, it did not catalyze the reverse reaction, the dehydrogenation of alkyl 4-halo-3-hydroxybutyrates and other alcohols. The enzyme required NADPH as a cofactor and showed no activity with NADH. Therefore, it was defined as a NADPH-dependent aldo–keto reductase (AKR3E1), belonging to the AKR superfamily. The enzyme stereospecifically produced methyl (S)-4-bromo-3-hydroxybutyrate from its keto derivative with high stereospecificity (97.9% enantiomer excess). E. coli cells expressing KER and glucose dehydrogenase in the water/butyl acetate two-phase system achieved a high productivity of (S)-4-bromo-3-hydroxybutyrate (277 mM, 54 mg/ml) in the organic solvent layer.     

Cold-active DnaK of an Antarctic psychrotroph Shewanella sp. Ac10 supporting the growth of dnaK-null mutant of Escherichia coli at cold temperatures

Shewanella sp. Ac10 is a psychrotrophic bacterium isolated from the Antarctica that actively grows at such low temperatures as 0°C. Immunoblot analyses showed that a heat-shock protein DnaK is inducibly formed by the bacterium at 24°C, which is much lower than the temperatures causing heat shock in mesophiles such as Escherichia coli. We found that the Shewanella DnaK (SheDnaK) shows much higher ATPase activity at low temperatures than the DnaK of E. coli (EcoDnaK): a characteristic of a cold-active enzyme. The recombinant SheDnaK gene supported neither the growth of a dnaK-null mutant of E. coli at 43°C nor phage propagation at an even lower temperature, 30°C. However, the recombinant SheDnaK gene enabled the E. coli mutant to grow at 15°C. This is the first report of a DnaK supporting the growth of a dnaK-null mutant at low temperatures.     

Expression of a thermostable restriction endonuclease in recombinant Escherichia coli cells and optimization of fermentation conditions

Taq I restriction endonuclease gene of the thermophilic eubacterium Thermus aquaticus YT-1 (ATCC 25104) was successfully cloned and expressed in recombinant Escherichia coli cells under the control of the lac promoter/operator system. Higher Taq I endonuclease specific activities and biomass yields were obtained from E. coli ER2508(pUCTaq) cells when they were induced at the late-exponential phase of their growth. Taq I endonuclease expression was found to be host strain-dependent such that, among the three different strains examined, E. coli XL1(pUCTaq) produced the highest specific Taq I endonuclease activities for longer induction periods. Decreasing the inducer concentration from 1 to 0.1 mM not only improved the specific enzyme activity yields but also is more economical, considering the high cost of isopropyl--D-thiogalactopyranoside (IPTG). The optimum culture temperature was found to be 37 °C. Taq I endonuclease specific activity recovered from E. coli XL1(pUCTaq) cells was 935 U/mg under optimum conditions.     

Extracellular production of active vibriolysin engineered by random mutagenesis in Escherichia coli

Vibriolysin, an extracellular protease of Vibrio proteolyticus, is synthesized as a preproenzyme. The N-terminal propeptide functions as an intramolecular chaperone and an inhibitor of the mature enzyme. Extracellular production of recombinant vibriolysin has been achieved in Bacillus subtilis, but not in Escherichia coli, which is widely used as a host for the production of recombinant proteins. Vibriolysin is expressed as an inactive form in E. coli possibly due to the inhibitory effect of the N-terminal propeptide. In this study, we isolated the novel vibriolysin engineered by in vivo random mutagenesis, which is expressed as active mature vibriolysin in E. coli. The Western blot analysis showed that the N-terminal propeptide of the engineered enzyme was processed and degraded, confirming that the propeptide inhibits the mature enzyme. Two mutations located within the engineered vibriolysin resulted in the substitution of stop codon for Trp at position 11 in the signal peptide and of Val for Ala at position 183 in the N-terminal propeptide (where position 1 is defined as the first methionine). It was found that the individual mutations are related to the enzyme activity. The novel vibriolysin was extracellularly overproduced in BL21(DE3) and purified from the culture supernatant by ion-exchange chromatography followed by hydrophobic-interaction chromatography, resulting in an overall yield of 2.2 mg/L of purified protein. This suggests that the novel engineered vibriolysin is useful for overproduction in an E. coli expression system.     

Cloning, sequencing, characterization, and expression of a new α-amylase isozyme gene (amy3) from Pseudomonas sp.

A new gene encoding an -amylase has been cloned, sequenced and expressed in E. coli from an alkaliphilic Pseudomonas sp. KFCC10818. The structural gene is 1356 base pairs long and encodes a protein of 452 amino acids. The recombinant -amylase has been purified and biochemically characterized. Molecular mass of the protein deduced from SDS-PAGE was 50 kDa. The enzyme showed an activity optimum at pH 8 and at 40 °C with complete stability at pH 13 for 3 h. The enzyme released maltose and maltotriose on hydrolysis of soluble starch. Amylose was hydrolysed over 5 times faster than amylopectin by the enzyme while the hydrolysis of cyclodextrin or pullulan was negligible.     

Characterization of the trehalosyl dextrin-forming enzyme from the thermophilic archaeon <Emphasis Type="Italic">Sulfolobus solfataricus</Emphasis> ATCC 35092

The trehalosyl dextrin-forming enzyme (TDFE) mainly catalyzes an intramolecular transglycosyl reaction to form trehalosyl dextrins from dextrins by converting the -1,4-glucosidic linkage at the reducing end to an -1,1-glucosidic linkage. In this study, the treY gene encoding TDFE was PCR cloned from the genomic DNA of Sulfolobus solfataricus ATCC 35092 to an expression vector with a T7 lac promoter and then expressed in Escherichia coli. The recombinant TDFE was purified sequentially by using heat treatment, ultrafiltration, and gel filtration. The obtained recombinant TDFE showed an apparent optimal pH of 5 and an optimal temperature of 75°C. The enzyme was stable in a pH range of 4.5–11, and the activity remained unchanged after a 2-h incubation at 80°C. The transglycosylation activity of TDFE was higher when using maltoheptaose as substrate than maltooligosaccharides with a low degree of polymerization (DP). However, the hydrolysis activity of TDFE became stronger when low DP maltooligosaccharides, such as maltotriose, were used as substrate. The ratios of hydrolysis activity to transglycosylation activity were in the range of 0.2–14% and increased when the DP of substrate decreased. The recombinant TDFE was found to exhibit different substrate specificity, such as its preferred substrates for the transglycosylation reaction and the ratio of hydrolysis to transglycosylation of the enzyme reacting with maltotriose, when compared with other natural or recombinant TDFEs from Sulfolobus.     

Identification and immunoprotective analysis of an in vivo-induced Edwardsiella tarda antigen

Edwardsiella tarda is a severe aquaculture pathogen that can infect many important fish species cultured worldwide. The aim of this study was to evaluate the vaccine potential of an E. tarda antigen, Eta21, which was identified from a pathogenic E. tarda strain via the method of in vivo-induced antigen technology (IVIAT). Eta21 is 510-amino acid in length and shares 58% sequence identity with a putative peptidase of several bacterial species. eta21 was subcloned into Escherichia coli, and recombinant Eta21 was purified as a histidine-tagged protein. When used as a subunit vaccine, purified recombinant Eta21 was effective against lethal E. tarda challenge in a Japanese flounder model. In order to improve the immunoprotective efficacy of Eta21, the chimera AgaV-Eta21 was constructed, which consists of Eta21 fused in-frame to the secretion domain of AgaV, an extracellular β-agarase. E. coli DH5α harboring plasmid pTAET21, which constitutively expresses agaV-eta21, was able to produce and secret AgaV-Eta21 into the extracellular milieu. Vaccination of Japanese flounder with live DH5α/pTAET21 elicited immunoprotection that is significantly higher in level than that induced by vaccination with purified recombinant Eta21. Vaccination with DH5α/pTAET21 and recombinant Eta21 both induced the production of specific serum antibodies at four to eight weeks post-vaccination. Taken together, these results demonstrate that Eta21, especially that delivered by DH5α/pTAET21, is an effective vaccine candidate against E. tarda infection.     

Transfer and expression of a Bacillus licheniformis α-amylase gene in Zymomonas mobilis

The gene from Bacillus licheniformis coding for a thermostable -amylase was subcloned into the broad-host-range plasmid pKT210 in Escherichia coli. The recombinant plasmid pGNB6 was transferred into Zymomonas mobilis ATCC 31821 by conjugation. Plasmid pGNB6 was stably maintained in E. coli and unstable in Z. mobilis. The amylase gene was expressed in Z. mobilis at a lower level (25%) than in E. coli and regulation of enzyme biosynthesis was different in the host cells. Almost all the -amylase activity was recovered in the culture medium of Z. mobilis. This enzyme localization seemed to be the result of protein secretion rather than cell lysis. Integration of the amylase gene into a cryptic plasmid of Z. mobilis was observed. The amylase gene was still expressed, although at a lower level, and the -amylase activity, associated with a protein of molecular mass 62,000 daltons, was immunologically identical in Z. mobilis, E. coli and B. licheniformis.     

Characterization of alginate lyase (ALYII) from Pseudomonas sp. OS-ALG-9 expressed in recombinant Escherichia coli

An alginate lyase named ALYII was purified to homogeneity from Escherichia coli JM109 carrying a recombinant plasmid, pJK26 harbouring the alyII gene from Pseudomonas sp. OS-ALG-9 by column chromatography with DEAE-cellulose, CM-Sephadex C-50, butyl-Toyopearl 650 M and isoelectric focusing. The molecular size of the purified ALYII was estimated to be 79 kDa by SDS-PAGE and its pI was 8.3. The enzyme was most active at pH 7.0 and 30 °C. Its activity was completely inhibited by Hg²⁺. The enzyme was poly -D-1, 4-mannuronate-specific rather than -D-1, 4-guluronate-specific and it showed a promotion effect in alginate degradation by combination with ALY, an another poly -D-1, 4-mannuronate-specific alginate lyase from the same strain.     

Cloning,expression and characterization of a beta-agarase gene from a marine bacterium,Pseudomonas sp. SK38

A gene (pagA) encoding -agarase from Pseudomonas sp. SK38 was cloned and expressed in Escherichia coli. The structural gene consists of 1011 bp encoding 337 amino acids with a predicted molecular weight of 37326 and has a signal peptide of 18 amino acids. The deduced amino acid sequence showed 57% and 58% homology to -agarase from Pseudoalteromonas atalntica and Aeromonas sp., respectively. The recombinant enzyme was purified and biochemically characterized. The enzyme had maximum activity at pH 9 and 30 °C. It was stable at pHs from 8 to 9 and below 37 °C.     

Cloning and expression in Escherichia coli of the Clostridium thermoaceticum gene encoding thermostable formyltetrahydrofolate synthetase

Formyltetrahydrofolate synthetase (FTHFS) (EC 6.3.4.3), a thermostable protein of four identical subunits from Clostridium thermoaceticum was cloned into Escherichia coli SK1592. The clone (CRL47) contained a 9.5 kb EcoRI fragment of C. thermoaceticum DNA ligated into pBR322. It produced catalytically active, thermostable FTHFS, that was not found in E. coli SK1592 containing native pBR322. The identity of the expressed enzyme was confirmed by specific binding of rabbit polyclonal anti-FTHFS serum produced against C. thermoaceticum FTHFS. The specific activities (mol·min^-1·mg^-1) of FTHFS in cell free extracts of CRL47 were 28–89 when assayed at 50°C and pH8. This was from 3–10-fold higher than in C. thermoaceticum extracts. FTHFS was purified to homogeneity from CRL47. The purified enzyme behaved during electrophoresis and gel chromatography and it had similar specific activity and thermostability as the enzyme purified from C. thermoaceticum.Abbreviations FTHFS formyltetrahydrofolate synthetase - kb kilobase - H₄-folate tetrahydrofolate - SDS sodium dodecyl sulfate A preliminary account of this work was presented at the annual meeting of the American Society for Microbiology, Atlanta, GA, 1987 (C. R. Lovell, A. Przybyla and L. G. Ljungdahl, Abstr. Annu. Meet. Am. Soc. Microbiol. 1987, K126, p. 223).     

High-level expression of the 1,3-propanediol oxidoreductase from klebsiella pneumoniae in escherichia coli

As one of four key enzymes in glycerol dismutation process, 1,3-propanediol oxidoreductase (EC.1.1.1.202) is important in converting glycerol to 1,3-propanediol in Klebsiella pneumoniae. The dhaT gene encoding 1,3-propanediol oxidoreductase was amplified by polymerase chain reaction (PCR) using the genome DNA of K. pneumoniae as template, and then cloned into cloning vector pMD18-T. After DNA sequence was determined, the dhaT gene was subcloned into Escherichia coli expression vector pET-22b (+) and pET-28a (+). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that both the recombinant E. coli BL21 (DE3) (pET-22b (+)-dhaT) and E. coli BL21(DE3)(pET-28a (+)-dhaT) expressed predicted 42-kDa 1,3-propanediol oxidoreductase after induced by isopropyl-β-d-thiogalactopyranoside (IPTG), and the recombinant enzyme of E. coli BL21 (DE3) (pET-28a (+)-dhaT) was mostly in soluble form, and exhibited high activity (96.8 U/mL culture). The recombinant enzyme was purified and biochemically characterized. The apparent K _m values of the enzyme for 1,3-propanediol and NAD⁺ were 8.5 and 0.21 mM, respectively. The enzyme had maximum activity at pH 9.5 and 30°C.     

Cloning and expression of sucrose phosphorylase gene from Bifidobacterium longum in E. coli and characterization of the recombinant enzyme

A DNA fragment, which complemented the growth of E. coli both on M9 medium containing raffinose and on LB medium containing ampicillin, IPTG and 5-bromo-4-chloro-3-indoxyl--d-galactoside, was isolated from the genomic library of Bifidobacterium longum SJ32, which had been digested with EcoRI. In the cloned DNA fragment, a gene encoding a sucrose phosphorylase (splP) and a partially cloned putative sucrose regulator gene (splR) were identified using the deletion analysis and sequence analysis. A 56 kDa protein was synthesized in E. coli and partially purified by DEAE-ion exchange chromatography. The partially purified enzyme did not react with melibiose, melezitoze and raffinose but did with sucrose. It had transglucosylation activity in addition to hydrolytic activity.     

Molecular cloning and characterization of the gene encoding a fibrinolytic enzyme from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> Strain A1

A fibrinolytic metalloprotease gene from Bacillus subtilis has been cloned in Escheridria coliXL1-Blue and the bacterial expressed enzyme was purified. The nucleotide sequence of the cloned fibrinolytic enzyme gene revealed a single open reading frame of 1023 bp coding for 341 amino acids (M _r 37708.21 Da). N-terminal amino acid sequencing of the fibrinolytic enzyme excreted from E. coli host cells revealed that the mature fibrinolytic enzyme consists of 288 amino acids (M _r 31391.1 Da). The deduced amino acid sequence showed significant homology with Erwina carotovora neutral metalloprotease and Serratia marcescens minor metalloprotease by 65 and 58% amino acid sequence identity, respectively. The protein showed significant alignments with the conserved domain of catalytic activity and the -helix domain in Bacillus anthracisthermolysis metalloprotease. The biochemical properties of the purified enzyme suggested that the enzyme is a fibrinolytic metalloprotease, which has optimal activity at pH 7.0 and 50 °C.