Quinoprotein D-glucose dehydrogenases inAcinetobacter calcoaceticus LMD 79. 41: Purification and characterization of the membrane-bound enzyme distinct from the soluble enzyme

Acinetobacter calcoaceticus is known to contain soluble and membrane-bound quinoprotein D-glucose dehydrogenases while other oxidative bacteria such asPseudomonas orGluconobacter contain only membrane-bound enzyme. The two different forms were believed to be the same enzyme or interconvertible. Present results show that the two different forms of glucose dehydrogenase are distinct from each other in their enzymatic and immunological properties as well as in their molecular size.The soluble and membrane-bound glucose dehydrogenases were separated after French press-disruption by repeated ultracentrifugation, and then purified to nearly homogeneous state. The soluble enzyme was a polypeptide of 55 Kdaltons, while the membrane-bound enzyme was a polypeptide of 83 Kdaltons which is mainly monomeric in detergent solution. Both enzymes showed different enzymatic properties including substrate specificity, optimum pH, kinetics for glucose, and reactivity for ubiquinone-homologues. Furthermore, the two enzymes could be distinguished immunochemically: the membrane-bound enzyme is cross-reactive with an antibody raised against membrane-bound enzyme purified fromPseudomonas but not with antibody elicited against the soluble enzyme, while the soluble enzyme is not cross-reactive with the antibody of membrane-bound enzyme.Data also suggest that the membrane-bound enzyme functions by linking to the respiratory chain via ubiquinone though the function of the soluble enzyme remains unclear.     

Characterization of a bacterial xylanase resistant to repression by glucose and xylose

Summary Bacillus subtilis CD4, when grown in nutrient broth or minimal medium in presence of xylan, produced extracellular xylanase that hydrolyzed xylan optimally at pH 5. The enzyme was induced by xylan, xylose and glucose. Addition of xylose or glucose in xylan containing medium did not affect enzyme production. The structural gene encoding xylanase was cloned and expressed in E. coli. The recombinant enzyme exhibited similar properties like that of native enzyme including resistance to repression by xylose and glucose.     

Production of dextranase byLipomyces starkeyi

Summary The optimal growth rate ofLipomyces starkeyi, with dextran as sole carbon source, was found within the pH range 2.5–4.0, and temperature between 25–30°C. This yeast was unable to grow above 33°C. Dextranase production optima paralleled growth optima, except at pH 2.5. Decrease in enzyme yield at this pH could not be attributed to poor yeast growth or enzyme stability.     

Enzyme activity engineering based on sequence co-evolution analysis

The utility of engineering enzyme activity is expanding with the development of biotechnology. Conventional methods have limited applicability as they require high-throughput screening or three-dimensional structures to direct target residues of activity control. An alternative method uses sequence evolution of natural selection. A repertoire of mutations was selected for fine-tuning enzyme activities to adapt to varying environments during the evolution. Here, we devised a strategy called sequence co-evolutionary analysis to control the efficiency of enzyme reactions (SCANEER), which scans the evolution of protein sequences and direct mutation strategy to improve enzyme activity. We hypothesized that amino acid pairs for various enzyme activity were encoded in the evolutionary history of protein sequences, whereas loss-of-function mutations were avoided since those are depleted during the evolution. SCANEER successfully predicted the enzyme activities of beta-lactamase and aminoglycoside 3′-phosphotransferase. SCANEER was further experimentally validated to control the activities of three different enzymes of great interest in chemical production: cis-aconitate decarboxylase, α-ketoglutaric semialdehyde dehydrogenase, and inositol oxygenase. Activity-enhancing mutations that improve substrate-binding affinity or turnover rate were found at sites distal from known active sites or ligand-binding pockets. We provide SCANEER to control desired enzyme activity through a user-friendly webserver.     

Factors affecting the use of chloramphenicol acetyltransferase as a marker for Brassica genetic transformation

The CAT gene which codes for the enzyme chloramphenicol acetyltransferase was found to be ineffective as a reporter gene in cells and tissues of Brassica species. High levels of endogenous CAT activity were found to be widespread among this genus and did not appear to be distributed in a tissue- or cell-specific manner. Moreover, the presence of an inhibitor of CAT activity was discovered in Brassica napus and Brassica juncea. This inhibitor appeared to act selectively on bacterial CAT in transgenic plants. These findings provided an explanation for difficulties experienced in the detection of transgenic CAT activity in B. napus.     

CMCase production by Spicellum roseum in liquid and solid culture

Summary CMCase was produced by 7 strains of Spicellum roseum in both liquid and wheat bran solid substrate cultures. No growth occurred above 35°C. Maximum enzyme production occurred at 30°C, whereas best enzyme activity occurred at pH 5.0 and 50°C. In liquid cultures of S. roseum, NRRL strains 13103, 13104, and 13106 produced activities of ca. 1.1, 1.5, and 1.5 mg glucose per hr/ml culture supernate at 1 week and 2.9, 1.5, and 2.1, respectively at 3 weeks compared to Trichoderma reesei NRRL 11236 (MCG77), which produced activities of 2.8 and 1.3 at 1 and 3 weeks.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

Activation of tubulinyl-tyrosine carboxypeptidase by spermine,spermidine and putrescine

Spermine, spermidine and putrescine produce dose dependent stimulation of the invitro tubulinyl-tyrosine carboxypeptidase. Maximal stimulation was obtained with spermine, spermidine or putrescine at 0.06 mM, 1 mM and 6 mM, respectively. At higher concentrations, the enzyme activity was inhibited. The enzyme was also activated by Mg⁺⁺; the concentration formaximal effect was 4–6 mM. The stimulation produced by optimal concentration of each amine was unaffected by Mg⁺⁺ up to 2 mM; higher concentration of Mg⁺⁺ showed inhibitory effect. At optimal Mg⁺⁺ concentration, the carboxypeptidase activity was inhibited by increasing amine concentration. The amines at 0.5 or 5 mM did not produce any effect on the incorporation of tyrosine catalyzed by tubulin tyrosine ligase.     

Regulation of alpha-amylase production inBacillus licheniformis M27 by enzyme end-products in submerged fermentation and its overcoming in solid state fermentation system

Summary Alpha-amylase production byBacillus licheniformis M27 in submerged fermentation was reduced from 480 to 30 units/ml when soluble starch concentration in medium was increased from 0.2 to 1.0%. In contrast, the enzyme production increased by 29 times even with 42 fold increase in the concentration of soluble starch and other starchy substrates in solid state fermentation system. The data establish regulation of the enzyme formation by enzyme end-product in submerged fermentation and ability of solid state fermentation to minimize it significantly. These features were not known earlier.     

Thermostable tryptophan synthase ofBacillus stearothermophilus expressed inEscherichia coli

Summary The tryptophan synthase genes,trpA andtrpB, from a moderate thermophile,Bacillus stearothermophilus IFO13737, were expressed efficiently inEscherichia coli. The recombinant tryptophan synthase amounted to 22% of the soluble cellular protein, and was purified to homogeneity by three steps. The enzyme is more thermostable thanE.coli tryptophan synthase, especially the subunit. The enzyme is also more resistant to sodium dodecylsulfate and methanol thanE.coli enzyme.     

Production of cloned carboxypeptidase G2 by Escherichia coli: Genetic and environmental considerations

Summary The optimum production of cloned carboxypeptidase G₂ from plasmid pNM21 byEscherichia coli was found to be strongly strain- and temperature-dependent. The superior host was strain RV308 and the preferred growth temperature 28°C. Copy number, which decreased during exponential growth of all strains examined, did not relate in these studies to the level of enzyme production: the strain with the highest enzyme yield also having the lowest overall copy number.     

Inhibition of cultured cell growth by tungstate and molybdate

The growth of suspension cultured cells of Nicotiana tabacum (tobacco) was inhibited completely by 100 M tungstate. Even though molybdate reversed the tungstate inactivation of nitrate reductase activity, the growth inhibition was not reversed. The growth inhibition of N. tabacum, Daucus carota, Glycine max and Solanum tuberosum suspension cultured cells by tungstate was similar in media with or without amino acids as a source of reduced nitrogen. Only in the case of G. max was a slight reversal caused by the amino acids. Tungstate was slightly less inhibitory to the growth of a nitrate reductase-lacking mutant N. tabacum line (nia-63) than to the line with nitrate reductase. These results indicate that tungstate must inhibit the cell growth of the four species used, predominantly, in some way other than by inhibiting nitrate reductase activity. Similar studies with molybdate, a sulfate analog which apparently competes with sulfate at the ATP sulfury-lase enzyme, showed that 1 mM concentrations were completely inhibitory to cell growth. The addition of sulfate or cysteine, as a source of reduced sulfur, and amino acids, as a source of reduced nitrogen, in most cases did not reverse the molybdate inhibition appreciably. Some reversal was seen only by sulfate with D. carota cells and by cysteine plus amino acids with D. carota and G. max. These results indicate that selection for tungstate or molybdate resistance will in general not select for higher levels or other alterations in the activity of nitrate reductase or ATP sulfurylase, respectively, since these ions do not inhibit growth by primarily affecting these enzymatic steps in cultured cells of the four species studied.     

Nutritional factors affecting lipase production byRhizopus delemar CDBB H313

Summary Some nutritional factors that affect lipase yields byRhizopus delemar were studied. Dextrin proved to be the best carbon source when used at 1% level. Yeast extract was the best nitrogen source for lipase production. The presence of a lipidic source in the growth medium, at a level not higher than 2% resulted in higher enzyme production. Tween 80 exerted a positive effect on enzyme production, used in a range that goes from 0.02% to 2.00%.     

Production of mold lactase

A process for production of mold lactase was developed. Tests were carried out in pilot and industrial scale with an Aspergillus niger strain selected after screening a number of molds.A computer coupled autoanalyzer system was used for monitoring enzyme formation in the pilot fermentor. Lactase production was investigated using different pH- and temperature-profiles. A. niger lactase has an acid pH optimum, a high temperature optimum and good stability. It does not require any metal ions. It is suitable for immobilization for hydrolysis of lactose in acid whey.Three-fold enhancement of lactase production was obtained by mutagenizing A. niger using NTG as mutagenic agent.The lactases produced by the mutants have the same pH and temperature optima and stability but the growth properties of the mutants were different from those of the original strain.Sufficient specific activity of the enzyme preparation for immobilization was obtained by purifying the enzyme by selective adsorption on Na-Ca-silicate.     

D-amino acid oxidase fromTrigonopsis variabilis: Comparison of enzyme specificity “in vivo” and “in vitro”

Summary A method is described for permeabilization of intact cells of the yeastTrigonopsis variabilis with respect toin vivo measuring D-amino acid oxidase activity. The kinetic results so obtained differ from those obtained with the purified enzyme, pointing to the advantage of using the purified enzyme or the permeabilized cells in the oxidative deamination of different D-amino acids.     

The continuous production of glucose isomerase

Summary It is shown that the enzyme glucose isomerase may be produced effectively by suitable continuous culture techniques using species of Arthrobacter and Mycobacterium. Carbon-limited growth conditions gave better carbon conversion efficiencies and higher specific enzyme activities than batch or nitrogen-limited conditions.This work was completed whilst the author was a member of the staff of I.C.I. Agricultural Division, Billingham, Teesside. Its contents are the subject of British Patent 1 492 258.     

Adsorption of aspartase onto hydrophobic cloth for conversion of ammonium fumarate

Summary A simple aspartase assay was developed. Aspartase fromEscherichia coli Crooks strain was adsorbed to -naphthyl cotton cloth by hydrophobic interaction. The adsorbed enzyme did not desorb in 1 M ammonium fumarate. The adsorbed enzyme exhibited the same pH vs. activity curve as free enzyme and had a half life of approx. 40 weeks. A column packed with the adsorbed aspartase showed 100% conversion of 1 M ammonium fumarate at a space velocity of approx. 2.     

Fluorocitrate inhibition of aconitase. Reversibility of the inactivation

Fluoride ion is released nearly stoichiometrically when (?)-erythro-fluorocitrate is incubated with aconitase. The release of F^? parallels the loss in activity and could arise from direct displacement of F^? by a base on the enzyme or from dehydration to fluoro-cis-aconitate and attack of an enzymic base to release F^?. Aconitase inactivated by ¹⁴C-fluorocitrate does not retain radioactivity when passed through G-50 Sephadex or precipitated by ammonium sulfate. Fullenzymicactivity can be regained after either of these treatments by activation by cysteine and ferrous salts. These data are consistent with the report of fluorocitrate being a competitive (and non-competitive) inhibitor of aconitase (Villafranca, J.J. (1972) Intra-Science Chem. Rept. 6 (4), 1–11) which rapidly inactivates the enzyme. This inactivated enzyme may be a very labile covalent complex, a very tight complex between enzyme and fluoro-cis-aconitate or a tight complex between a defluorinated deravitive of fluorocitrate.In the course of Peters (1957) extensive work on the toxic effects of fluoroacetate, he determined that fluoroacetate was metabolically converted to fluorocitrate. This finding and the fact that citrate levels rise soon after ingestion of fluoroacetate led to the suggestion that fluorocitrate inactivates aconitase (E.C. 4.2.1.2).Recently, conflicting reports concerning the site of inactivation in mitochondria by the inhibiting isomer of fluorocitrate, (?)-erythro-fluorocitrate (1R:2R, 1-fluoro-2-hydroxy-1,2,3-propanetricarboxylate) have appeared (Eanes et al. 1972; Brand et al, 1973). Eanes et al. (1972) contends that the tricarboxylate carrier is the site of inhibition, while Brand et al. (1973) has compelling evidence that aconitase is the site of inhibition. This controversy is a matter of intrepretation of the results and a greater knowledge of the inactivation of aconitase by fluorocitrate may be useful in these interpretations. The results reported herein are concerned with the mechanism of inactivation of purified mitochondrial aconitase by fluorocitrate and demonstrate that this reaction is readily reversible.     

Presence of two subunit types in ribulose-1,5-bisphosphate carboxylase from blue-green algae.

Ribulose-1,5-bisphosphate car?ylase (E.C. 4.1.1.39) from 2 blue-green algae, Plectonema boryanum and Anabaena variabilis, was isolated by sucrose density gradient centrifugation. Both enzymes had a sedimentation value of about 18s, similar to that of Chromatium enzyme. The presence of two subunits (A, B) in the algal enzyme was demonstrated by Nadodecyl sulfate polyacrylamide gel electrophoresis. The molecular weight of the two subunits was determined: for Plectonema A, 5.4 × 10⁴ and B, 1.3 × 10⁴ and Anabaena A, 5.2 × 10⁴ and B, 1.3 × 10⁴, respectively. The car?ylase reaction catalysed by the algal enzyme was similar to the higher plant enzyme in exhibiting the Mg²⁺-effect, the optimal pH shifting from alkaline to neutral by elevating the concentration of Mg²⁺ in the assay mixture. The rabbit antisera developed against the spinach ribulose-1,5-bisphosphate car?ylase and its catalytic oligomer exhibited significant inhibitory effects on the car?ylation reaction catalysed by the algal enzyme.     

Novel biotechnology process for the production of (+)-2-aminobutyrate

Summary The production of (+)-2-aminobutyrate was catalyzed by a transaminating enzyme with 2-ketobutyrate and alanine as the reactants. The required enzyme was obtained from a plasmid-bearingE. coli strain.     

Screening of yeast strains capable of hyperproducing amylolytic enzymes

Summary Fifteen strains of yeast, which produced an extracellular amylolytic enzymes, were isolated from nature. One of them produced more than 100 times the enzyme activity in comparison with the 14 strains and the extremely hyperproducing strain of yeast was identified asCandida sp. 347. Paper chromatograms of the amylolytic enzyme demonstrated activity of amyloglucosidase. The optimum pH for activity of the enzyme was 5.5–6.0 and optimum temperature was 60°C.