Isolation and analysis of two cellulase cDNAs from Orpinomyces joyonii

Two cellulase cDNAs, celB29 and celB2, were isolated from a cDNA library derived from mRNA extracted from the anaerobic fungus, Orpinomyces joyonii strain SG4. The nucleotide sequences of celB2 and celB29 and the primary structures of the proteins encoded by these cDNAs were determined. The larger celB29 cDNA was 1966bp long and encoded a 477 amino acid polypeptide with a molecular weight of 54kDa. Analysis of the 1451bp celB2 cDNA revealed an 1164bp open reading frame coding for a 44kDa protein consisting of 388 amino acids. Both deduced proteins had a high sequence similarity in central regions containing putative catalytic domains. Primary structure analysis revealed that CelB29 contained a Thr/Pro-rich sequence that separated the N-terminal catalytic domain from a C-terminal reiterated region of unknown function. Homology analysis showed that both enzymes belong to glycosyl hydrolase family 5 and were most closely related to endoglucanases from the anaerobic fungi Neocallimastic patriciarum, Neocallimastix frontalis and Orpinomyces sp. The classification of CelB29 and CelB2 as endoglucanases was supported by enzyme assays. The cloned enzymes had high activities towards barley beta-glucan, lichenan and carboxymethylcellulose (CMC), but not Avicel, laminarin, pachyman, xylan and pullulan. In addition, CelB29 and CelB2 showed activity against p-nitrophenyl-beta-D-cellobioside (pNP-G(2)) to p-nitrophenyl-beta-D-cellopentaoside (pNP-G(5)) but not p-nitrophenyl-beta-D-glucopyranoside (pNP-G(1)) with preferential activity against p-nitrophenyl-beta-D-cellotrioside (pNP-G(3)). Based on these results, we proposed that CelB29 and CelB2 are endoglucanases with broad substrate specificities for short- and long-chain beta-1,4-glucans.     

Interaction of the rumen fungus Orpinomyces joyonii with Megasphaera elsdenii and Eubacterium limosum

The degradation and fermentation of microcrystalline cellulose were studied in monoculture of the polycentric anaerobic fungus Orpinomyces joyonii and in co-cultures with the rumen bacteria Megasphaera elsdenii and Eubacterium limosum. More than 25% of cellulose hydrolysis products (glucose and cellodextrins) were released by the fungus into the medium after 8 d of cultivation. These products were metabolized by bacteria in mixed cultures. In co-culture with the fungus M. elsdenii and E. limosum . increased the extent of microcrystalline cellulose degradation by 10·12% and 7·96%, respectively. Biomass yield in co-cultures was increased by 89·9% and 59·4% for M. elsdenii and E. limosum . Y_cellulose for fungus alone was 52·29 g dry matter mol^-1 glucose. These values were 64·93 and 55·92 g mol^-1 glucose unit in co-culture with M. elsdenii and E. limosum , respectively.     

Purification and characterization of Escherichia coli endonuclease III from the cloned nth gene

The gene which codes for endonuclease III of Escherichia coli has been sequenced. The nth gene was previously subcloned and defined as the gene which led to overproduction of endonuclease III when present on a multicopy plasmid and which created a deficiency in endonuclease III activity when mutated. The nth gene was sequenced and translated into a predicted polypeptide. The molecular weight (23,546), the amino-terminal amino acid sequence, and the amino acid composition of the polypeptide predicted from the nucleotide sequence are excellent agreement with those same properties determined for the purified protein. Thus, the nth gene is the structural gene for endonuclease III. Inspection of the nucleotide sequence reveals that there is an open reading frame immediately upstream of the nth gene, suggesting that it might be part of an operon. There is a region of dyad symmetry which could form a hairpin stem and loop structure if transcribed into RNA characteristic of a rho-dependent terminator downstream from the nth gene. The nth gene of Escherichia coli has been cloned onto a lambda PL expression vector which yields approximately 300-fold overproduction of endonuclease III. We have purified the enzyme to apparent homogeneity using two chromatographic steps. Our purification scheme allowed the preparation of 117 mg of protein from 190 g of E. coli with a 70% yield. The purified protein has both AP endonuclease activity and DNA N-glycosylase activity. The protein has a Stokes radius of 2.25 nm, a sedimentation coefficient of 2.65 S, a molecular weight of 26,300 in the native state and 27,300 in the denatured state, and a frictional ratio of 1.13.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of glutathione reductase encoded by a cloned and over-expressed gene in Escherichia coli.

An expression vector, pKGR, for the gor gene from Escherichia coli encoding glutathione reductase was constructed by subcloning of an AvaII fragment of the Clarke & Carbon bank plasmid pGR [Greer & Perham (1986) Biochemistry 25, 2736-2742] into the plasmid pKK223-3. The expression of glutathione reductase from the plasmid pKGR was found to have been successfully placed under the control of the tac promoter. Transformation of E. coli cells with this plasmid resulted in 100-200-fold increase in glutathione reductase activity in cell-free extracts. A rapid purification procedure for the enzyme, based on affinity chromatography on Procion Red HE-7B-CL-Sepharose 4B, was developed. The purified enzyme was homogeneous as judged by SDS/polyacrylamide-gel electrophoresis, and all its properties were consistent with the DNA sequence of the gene [Greer & Perham (1986) Biochemistry 25, 2736-2742] and with those previously reported for E. coli glutathione reductase [Mata, Pinto & Lopez-Barea (1984) Z. Naturforsch. C. Biosci. 39, 908-915]. These experiments have enabled an investigation of the protein chemical and mechanistic properties of the enzyme by site-directed mutagenesis.     

Dihydroorotase from Escherichia coli. Purification and characterization

Dihydroorotase (4,5-L-dihydroorotate amidohydrolase (EC 3.5.2.3], which catalyzes the reversible cyclization of N-carbamyl-L-aspartate to dihydro-L-orotate, has been purified to homogeneity from an over-producing strain of Escherichia coli. Treatment of 70 g of frozen cell paste produces about 7 mg of pure enzyme, a yield of about 35%. The native molecular weight, determined by equilibrium sedimentation, is 80,900 +/- 4,300. The subunit molecular weight, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 38,400 +/- 2,600, and by amino acid analysis is 41,000. The enzyme is thus a dimer and contains 0.95 +/- 0.08 tightly bound zinc atoms per subunit when isolated by the described procedure, which would remove any loosely bound metal ions. Isoelectric focusing under native conditions yields a major species at isoelectric point 4.97 +/- 0.27 and a minor species at 5.26 +/- 0.27; dihydroorotase activity is proportionately associated with both bands. The enzyme has a partial specific volume of 0.737 ml/g calculated from the amino acid composition and a specific absorption at 278 nm of 0.638 for a 1 mg/ml solution. At 30 degrees C, the Michaelis constant and kcat for dihydro-DL-orotate (at pH 8.0) are 0.0756 mM and 127 s-1, respectively; for N-carbamyl-DL-aspartate (at pH 5.80), they are 1.07 mM and 195 s-1.     

Purification and characterization of a DNA-dependent ATPase from Escherichia coli.

A DNA-dependent ATPase has been isolated and purified from an Escherichia coli cell-free extract. The ATPase has the following characteristics: preferential dependence on single-stranded DNA, specificity for ATP hydrolysis, Km value of 1.4 X 10-4 M for ATP, and molecular weight of approximately 69,000. The ATPase can be shown to bind to single stranded DNA. The resemblance between this ATPase and that isolated from vaccinia cores is discussed.     

Purification and characterization of the D-alanyl-D-alanine-adding enzyme from Escherichia coli

The Escherichia coli D-alanyl-D-alanine-adding enzyme, which catalyzes the final cytoplasmic step in the biosynthesis of the bacterial peptidoglycan precursor UDP-N-acetylmuramyl-L-Ala-gamma-D-Glu-meso-diaminopimelyl-D-Ala-D- Ala, has been purified to homogeneity from an E. coli strain that harbors a recombinant plasmid bearing the structural gene for this enzyme, murF. The enzyme is a monomer of molecular weight 49,000, and it has a turnover number of 784 min-1 for ATP-driven amide bond formation. Experiments monitoring the fate of radiolabeled UDP-N-acetylmuramyl-L-Ala-gamma-D-Glu-meso-2,6-diaminopimelate and D-trifluoroalanine proved that the preceding enzyme in the D-alanine branch pathway, D-alanine:D-alanine ligase (ADP), is capable of synthesizing fluorinated dipeptides, which the D-Ala-D-Ala-adding enzyme can then incorporate to form UDP-N-acetylmuramyl-L-Ala-gamma-D-Glu-meso-2,6-diaminopimelyl-D-++ +trifluoroAla-D- trifluoroAla.     

Purification and characterization of a beta-lactamase from Haemophilus ducreyi in Escherichia coli

A pCb plasmid encoding a beta-lactamase from Haemophilus ducreyi was transferred to Escherichia coli, purified, and characterized. The beta-lactamase could be isolated from a culture filtrate and further purified by ammonium sulfate and chelating Sepharose fast flow loaded with Zn(2+). The purified enzyme resulted in a major band at approximately 30-kDa on SDS-PAGE and its pI was determined to be 5.4. The beta-lactamase could hydrolyze both penicillin antibiotics including ampicillin, benzylpenicillin, and carbenicillin as well as cephalosporin antibiotics including nitrocefin, cephalothin, cephaloridine, and cefoperazone. However, benzylpenicillin was the best substrate. The enzyme activity was inhibited by clavulanic acid but not by boric acid, cefotaxime, ethylenediaminetetraacetic acid, or phenylmethylsulfonyl fluoride. The sequence of the beta-lactamase gene was also determined. It confirmed that the enzyme belonged to a class A beta-lactamase which had 99% identity to the ampicillin resistance transposon Tn3 of pBR322. Two nucleotides were different between the E. coli (Tn3) and H. ducreyi (pCb) genes that affected the amino-acid sequence. The valine at position 82 (ABL 84) was changed to isoleucine and the alanine at position 182 (ABL 184) was changed to valine. Genetic homogeneity among beta-lactamases is remarkable. Amino acid sequencing of some beta-lactamases has shown that substitution of only a few amino acids in the bla gene leads to high-level resistance against specific cephalosporins.     

Fructose bisphosphatase from Escherichia coli. Purification and characterization

Escherichia coli fructose-1,6-bisphosphatase has been purified for the first time, using a clone containing an approximately 50-fold increased amount of the enzyme. The procedure includes chromatography in phosphocellulose followed by substrate elution and gel filtration. The enzyme has a subunit molecular weight of approximately 40,000 and in nondenaturing conditions is present in several aggregated forms in which the tetramer seems to predominate at low enzyme concentrations. Fructose bisphosphatase activity is specific for fructose 1,6-bisphosphate (Km of approximately 5 microM), shows inhibition by substrate above 0.05 mM, requires Mg2+ for catalysis, and has a maximum of activity around pH 7.5. The enzyme is susceptible to strong inhibition by AMP (50% inhibition around 15 microM). Phosphoenolpyruvate is a moderate inhibitor but was able to block the inhibition by AMP and may play an important role in the regulation of fructose bisphosphatase activity in vivo. Fructose 2,6-bisphosphate did not affect the rate of reaction.     

A recombinant cellulolytic Escherichia coli: Cloning of the cellulase gene and characterization of a bifunctional cellulase

A genomic library of Bacillus subtilis CD4 was constructed in Escherichia coli JM83. A clone designated as E. coli pBcelR was identified which formed blue colony in presence of 5-bromo-4-chloro-3-indolyl--d-glucopyranoside (X-Glu) and hydrolysed carboxymethyl cellulose (CMC). The clone E. coli (pBcelR) expressed both cellobiase and endoglucanase activities and contained an insert of 1.2 kb. E. coli pBcelR encoded a protein of 12.9 kDa which was endowed with bifunctional (endoglucanase and cellobiase) activities. In recombinant E. coli, the encoded protein and enzyme activity were localized in periplasm. Recombinant E. coli pBcelR utilized CMC, cellobiose and soluble cellulose as sole carbon source.     

Purification and characterization of a glycine betaine binding protein from Escherichia coli

A major component of the Escherichia coli response to elevated medium osmolarity is the synthesis of a periplasmic protein with an Mr of 31,000. The protein was absent in mutants with lambda placMu insertions in the proU region, a locus involved in transport of the osmoprotectant glycine betaine. This periplasmic protein has now been purified to homogeneity. Antibody directed against the purified periplasmic protein crossreacts with the fusion protein produced as a result of the lambda placMu insertion, indicating that proU is the structural gene specifying the 31-kDa protein. The purified protein binds glycine betaine with high affinity but has no affinity for either proline or choline, clarifying the role of proU in osmoprotectant transport. The amino-terminal sequence of the mature glycine betaine binding protein is Ala-Asp-Leu-Pro-Gly-Lys-Gly-Ile-Thr-Val-Asn-Pro.     

Purification and characterization of a periplasmic oligopeptide binding protein from Escherichia coli

We have purified and characterized an oligopeptide binding protein released from the periplasm of Escherichia coli W by mild osmotic shock. The purified protein was greater than 97% homogeneous as determined by either sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr = 60,000) or isoelectric focusing (pI = 5.95). The binding protein has a Stokes radius of 30 A and a sedimentation coefficient (s(0)20,w) of 4.6 S. Based on these hydrodynamic studies, the native protein has a molecular weight of 56,000. The tripeptide, Ala-Phe-[3H]Gly, which is transported via the shock-sensitive sensitive oligopeptide permease, binds to the purified protein in dilute solution with a Kd of 0.1 microM and a stoichiometry of approximately 1 to 1. Results from this study support the hypothesis that this periplasmic oligopeptide binding protein functions in the initial recognition of peptide substrates for the oligopeptide permease system.     

Purification and characterization of the d-xylose isomerase gene from Escherichia coli

A DNA fragment containing both the Escherichia coli d-xylose isomerase (d-xylose ketol-isomerase, EC 5.3.1.5) gene and the d-xylulokinase (ATP: d-xylulose 5-phosphotransferase, EC 2.7.1.17) gene has been cloned on an E. coli plasmid. The d-xylose isomerase gene was separated from the d-xylulokinase gene by the construction of a new deletion plasmid, pLX7. The d-xylose isomerase gene cloned on pLX7 was found still to be an intact gene. The precise location of the d-xylose isomerase gene on the plasmid pLX7 was further determined by the construction of two more plasmids, pLX8 and pLX9. This is believed to be the first d-xylose isomerase gene that has been isolated and extensively purified from any organism. d-Xylose isomerase, the enzyme product of the d-xylose isomerase gene, is responsible for the conversion of d-xylose to d-xylulose, as well as d-glucose to d-fructose. It is widely believed that yeast cannot ferment d-xylose to ethanol primarily because of the lack of d-xylose isomerase in yeast. d-Xylose isomerase (also known as d-glucose isomerase) is also used for the commercial production of high-fructose syrups. The purification of the d-xylose isomerase gene may lead to the following industrial applications: (1) cloning and expression of the gene in yeast to make the latter organism capable of directly fermenting d-xylose to ethanol, and (2) cloning of the gene on a high-copy-number plasmid in a proper host to overproduce the enzyme, which should have a profound impact on the high-fructose syrup technology.     

Purification and characterization of aminoimidazole ribonucleotide synthetase from Escherichia coli

Aminoimidazole ribonucleotide (AIR) synthetase has been purified 15-fold to apparent homogeneity from Escherichia coli which contains a multicopy plasmid containing the purM, AIR synthetase, gene. The protein is a dimer composed of two identical subunits of Mr 38,500. The N-terminal sequence, amino acid composition, and steady-state kinetics of the protein have been determined. AIR synthetase has been shown to catalyze the transfer of the formyl oxygen of [18O]formylglycinamide ribonucleotide to Pi.     

Purification and characterization of protease III from Escherichia coli.

An endoproteolytic enzyme of Escherichia coli, designated protease III, has been purified about 9,600-fold to homogeneity with a 6% yield. The purified enzyme consists of a single polypeptide chain of Mr 110,000 and is most active at pH 7.4. Protease III is very sensitive to metal-chelating agents and reducing agents. The EDTA-inactivated enzyme can be reactivated by Zn2+, Co2+ or Mn2+. Protease III is devoid of activity toward aminopeptidase, carboxypeptidase, or esterase substrates but rapidly degrades small proteins. When fragments of beta-galactosidase are used as substrates for protease III, the enzyme preferentially degrades proteins with molecular weights of less than 7,000. Protease III cleaves the oxidized insulin B chain at two sites with an initial rapid cleavage at Tyr-Leu (16-17) and a second slower cut at Phe-Tyr (25-26).     

Purification and characterization of 3-dehydroquinase from Escherichia coli.

A procedure has been developed for the purification of 3-dehydroquinase from Escherichia coli. Homogeneous enzyme with specific activity 163 units/mg of protein was obtained in 19% overall yield. The subunit Mr estimated from polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate was 29,000. The native Mr, estimated by gel permeation chromatography on Sephacryl S-200 (superfine) and on TSK G3000SW, was in the range 52,000-58,000, indicating that the enzyme is dimeric. The catalytic properties of the enzyme have been determined and shown to be very similar to those of the biosynthetic 3-dehydroquinase component of the arom multifunctional enzyme of Neurospora crassa.     

Purification and characterization of phosphopantetheine adenylyltransferase from Escherichia coli.

Phosphopantetheine adenylyltransferase (PPAT) catalyzes the penultimate step in coenzyme A (CoA) biosynthesis: the reversible adenylation of 4'-phosphopantetheine yielding 3'-dephospho-CoA and pyrophosphate. Wild-type PPAT from Escherichia coli was purified to homogeneity. N-terminal sequence analysis revealed that the enzyme is encoded by a gene designated kdtB, purported to encode a protein involved in lipopolysaccharide core biosynthesis. The gene, here renamed coaD, is found in a wide range of microorganisms, indicating that it plays a key role in the synthesis of 3'-dephospho-CoA. Overexpression of coaD yielded highly purified recombinant PPAT, which is a homohexamer of 108 kDa. Not less than 50% of the purified enzyme was found to be associated with CoA, and a method was developed for its removal. A steady state kinetic analysis of the reverse reaction revealed that the mechanism of PPAT involves a ternary complex of enzyme and substrates. Since purified PPAT lacks dephospho-CoA kinase activity, the two final steps of CoA biosynthesis in E. coli must be catalyzed by separate enzymes.     

Purification and characterization of a protease-resistant cellulase from Aspergillus niger

An endo-β-1,4-glucanase (EC 3.2.1.4) was purified from a culture filtrate of Aspergillus niger IFO31125 by column chromatography through TSK-gel DEAE-3SW and TSK-gel DEAE-5PW, and by gel filtration through TSK-gel G2000SW by high performance liquid chromatography. The enzyme was estimated to have a molecular weight of about 40 kDa by both gel filtration and SDS-polyacrylamide gel electrophoresis, and appeared to consist of a monomeric protein. It contained 8.9% carbohydrate. The optimal pH for activity was 6.0–7.0, and the stable pH range was 5.0–10.0. The optimum temperature at pH 6.0 was around 70°C. The enzyme was very thermally stable and no loss of original activity was found on incubation at 60°C for 2 h. The enzyme efficiently hydrolyzed carboxymethylcellulose and lichenan, but crystalline forms of cellulose, curdlan, laminarin, cellobiose, p-nitrophenyl-β-d-glucopyranoside and p-nitrophenyl-β-d-cellobioside were barely hydrolyzed. The activity of the enzyme was inhibited by Hg²⁺ and Cu²⁺ but was not affected by other inhibitors of thiol enzymes such as p-chloromercuribenzoate and N-ethylmaleimide. N-Bromosuccinimide showed a strong inhibitory effect, suggesting that a tryptophan residue is essential for the activity of the enzyme. The N-terminal amino acid sequence of the enzyme showed considerable homology to those of endo-β-1,4-glucanases from some other microorganisms, including Sclerotinia sclerotiorum and Schizophyllum commune. The enzyme had very strong protease-resistance, and showed no loss of activity when incubated with proteases such as Savinase at 40°C, even for 2 weeks.