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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/001—Enzyme electrodes
  • C12Q1/004—Enzyme electrodes mediator-assisted

Definitions

• the present invention is concerned with the production of quinoproteins. particularly the quinoproteins ethanol dehydrogenase and glucose dehydrogenase. and relates to the technical field of enzyme production and •5 isolation.
• Quinoproteins are PQQ-containing enzymes which are found in many gram-negative bacteria.
• One particular enzyme, glucose dehyrogenase (GDH) is known to occur is many gram-negative bacteria and is believed to occur in the pe iplasm, that is the space between the inner and outer membranes of the gram-negative cell. This enzyme has particular utillity in the estimation of glucose in biosensors and has been in this particular aspect the subject of many patent applications.
• glucose dehyrogenase and other quinoprotein based enzyme sensor electrodes and fuel cells necessitates the production of gram quantities of these enzymes. Furthermore, a growing number of industrially used enzymes are dehydrogenases.
• MDH methanol dehydrogenase
• Alkanes have been noted to stimulate growth on alkanes of Pseudomonas aeru ⁇ inosa (long known as Bacillus pyocyaneus). a plasmid carrying, gram-negative soil i 5 bacteria.
• a method of production of a quinoprotein dehydrogenase from a suitable cell culture CHARACTERISED IN THAT, the production of the said o quinoprotein is stimulated by the treatment of the said culture with an stimulator, and wherein the stimulator is an alkane. and in that said culture is grown in the presence of a non-ionic detergent.
• quinoprotein dehydrogenases By performing the method of the present invention it is possible to produce quinoprotein dehydrogenases in gram quantities. It should be understood the terms “enzyme” and “dehyrigenase” are intended not only to mean the wild-type enzyme as found in the majority of strains but to extend to apoenyme mutants and other variants, such as that given by way of example below.
• said quinoprotein is separated from a cell extract containing the said quinoprotein in an aqueous two-phase partition system consisting of at least one polyalkylene glycol and at least one soluble salt.
• the polyalkylene glygol is polyethylene glycol
• the soluble salt is soluble to at least 30% w/v and is an alkali metal phosphate salt, such as potassium phosphate.
• potassium phosphate is a preffered reagent it may be replaced by any other suitably soluble salt, such as magnesium sulphate or ammonium chloride. More preferably the method of purification is carried out at or near room temperature.
• a method for the production of a protein from a gram-negative bacterial culture CHARACTERISED IN THAT the bacterial culture is grown in the presence of a non-ionic detergent.
• the cell culture is grown in the presence of a detergent capable of emulsifying the alkane, preferably one selected from the group comprising; Triton X-100, Triton X-35. Triton X-45, Triton X-102, Triton N-57 and Triton N-101.
• a detergent capable of emulsifying the alkane preferably one selected from the group comprising; Triton X-100, Triton X-35. Triton X-45, Triton X-102, Triton N-57 and Triton N-101.
• Triton X-100 A concentration of 0.05% Triton X-100 is known to give particularly good yield.
• Triton X-100 On addition of low concentrations of Triton X-100 to a o mineral culture medium suplemented with an alkane, a marked stimulation of growth rate is observed and appreciable amounts of quinoprotein dehydrogenase are found in the culture medium. At higher concentrations of Triton X-100 the enzyme production increases still further but cytoplasmic enzyme activities and a substantial amount of protein are found in the medium, indicating that some lysis is occu ing.
• the alkane is selected from the group of C_b-C_ £._ ⁇ alkanes, and is more preferably selected from the group comprising C 5 . Cg, C 12 ⁇ C 18 and C 22"
• the quinoprotein is selected from the group comprising; methanol dehydrogenase and glucose dehydrogenase.
• Enzyme stability is enhanced in the above system, ⁇ 5 allowing purification procedures to be carried out at room temperature. It believed that the reason for this is that the cells are not broken during the extraction process and therefore enzymes which may themselves degrade the desired product are not released into the 2.0 growth medium.
• the above method has been found to have a particular utillity in the production of; a) methanol dehydrogenase from Methylophilus methylotrophus. and, b) methanol dehydrogenase from Methylosinus trichosporium strain OB3B. c) glucose dehydrogenase from Acinetobacter s calcoacetius. strains LMD 79.41. ATCC 23055 (type strain). ATCC 23220. ATCC 23236 (strain HOI) . and, NTCC 7844.
• Acinetobacter calcoacetius which is known to be a versatile organism capable of ic growing on a variety of carbon sources. During growth on alkanes a number of distinct morphological changes become apparent, and extracellular membrane particles having a composition similar to that of the cell membrane have been noted in culture fluids under these
• Fig.l shows the results of growth on heptadecane with or without Triton X-100, 2.5 a) without Triton X-100. precultured on heptadecane; b) without Triton X-100, precultured on acetate; c) with 0.04% Triton X-100, precultured on heptadecane; d) with 0.04% Triton X-100, precultured on acetate.
• Fig.2 shows the glucose dehydrogenase activity in the -5 medium after growth on different straight chain alkanes (0.5%) plus 0.04% Triton X-100.
• Fig.3 shows the enzyme activity with heptadecane and varying concentrations of detergent, A) activity of glucose dehydrogenase; o B) activity of malate dehydrogenase; and, C) activity of alkaline phosphatase, in Triton X-100 concentrations of; a) 0%, b) 0.005%, and, c) 0.04%.
• Fig.4 shows the compared malate dehydrogenase activity and glucose dehydrogenase activity, both during growth on heptadecane (0.5%) and Triton X-100 (0.04%) a) absorbance at 660nm. 0 b) glucose dehydrogenase activity, and, c) malate dehydrogenase activity.
• Fig.5 shows a table of the effects of a range of detergents on growth and glucose dehydrogenase production.
• Fig.6 shows a table of the effects of a range of growth conditions on the specific activity and amount of glucose dehydrogenase produced in a culture, and.
• Fig.7 shows the purification scheme in the two-phase S partition system.
• Glucose dehydrogenase activity was estimated as described by Duine and Frank [FEBS letters (1979) 108, 443], measuring the rate of reduction of Wurster's Blue in Tris/HCl buffer at (0.1M). pH 7.0.
• Malate Dehydrogenase activity (EC 1.1.1.37) was estimated by following the rate of oxidation of NADH (0.2mM) with oxaloacetic acid (0.5mM) in potassium phosphate buffer (0.1M) at pH 7.5.
• Alkaline Phosphatase activity (EC 3.1.3.1) were o estimated by following the production of p-nitrophenol at 420nm in p-nitrophenylphosphate solution (440uM) in Tris/HCl (0.1M) buffer at pH 7.
• the exemplary method performed herein employed an aqueous two-phase partition system consisting of polyethylene glycol and potassium phosphate.
• the behaviour of methanol dehydrogenase in the said aqueous two-phase system was determined by preliminary investigation on a small scale.
• the partition coefficient ([Enzyme] upper phase : [Enzyme] lower phase) was profoundly affected by the molecular weight of the polyethylene glycol.
• the enzyme was located entirely in the upper, polyethylene-glycol-rich phase.
• PEG 1000 caused a rapid reversal of partition of this enzyme, although the majority of the total protein remained in the upper phase. It should be noted that polyethylene glycols do act as substrates for methanol dehydrogenase in the presence of appropriate electron acceptors and enzyme activators. The reversal of partition of the enzyme in the PEG 1000 system appeared to be total. The method was then scaled up fity fold to attempt the medium scale isolation of the enzyme.
• Thawed cell paste (200g) previously prepared from Methylophilus methylotrophus. was resuspended in phosphate buffer (50 mM, pH 7.0; 600 ml) and disrupted 5 by a single passage through a continuous flow cell-disrupter (Stansted cell disrupter, Stansted Fluid Power Ltd. Stansted, Essex, U.K. , operating pressure, 30.00 psi). The disrupted cell mass was immediately cooled on ice. An aqueous two phase system was io constructed in a small stirred fermenter vessel from the following constituents: a) Disrupted cell suspension (approx. 600 ml), b) Aqueous PEG 1000 (50% v/v. 1400 ml), c) Potassium phosphate (50% w/v, pH7.0; 1050ml), and, 15 d) Aqueous methanol solution (100 mM. 350 ml).
• phosphate buffer 50 mM, pH 7.0; 600
• IO preparation are shown in fig.7.
• the methanol dehydrogenase was compared with the extract of Methylophilus ethylotrophus described by Ghosh et al. -Using this criterion, and protein analysis by polyacrylamide gel electrophoresis, the enzyme prepared
• NCTC 7844 was grown in mineral salt medium (FAM 2) with acetate (0.1% w/v) as the sole carbon source.
• FAM 2 mineral salt medium
• the organism was grown in batch in an 80 litre fermenter, harvesting in late log phase to avoid cell lysis and foaming.
• the purification of the glucose dehydrogenase was carried out both according to the method of the present invention and by conventional methods of ion exchange and affinity chromatography. to provide a comparison between these two protocols.
• ⁇ o Acinetobacter calcoaceticus LMD 79.41 obtained from Prof. J. Hauge; Hauge J. G. Biochem Biophys Acta (1960), 45, 263.
• Fig.l shows the results of growth on heptadecane with or without Triton X-100 as follows; 2.o a) without Triton X-100. precultured on heptadecane; b) without Triton X-100. precultured on acetate; c) with 0.04% Triton X-100, precultured on heptadecane; d) with 0.04% Triton X-100. precultured on acetate.
• the level of GDH increased with further incubation. until a concentration of GDH is reached which was about five times higher than can be extracted from a cell pellet grown on ethanol or acetate in a comparable culture volume. After centrifugation, the culture fluid is diluted five times with distilled water, CM-Sepharose is added and the enzyme adsorbed.
• Figure 2 shows the effect on the glucose dehydrogenase activity in the medium after growth on different straight chain alkanes (0.5%) plus 0.04% Triton X-100. It can be seen that the best growth was obtained with the C-_ alkane heptadecane.
• Fig.3 shows the enzyme activity with heptadecane and 5 varying concentrations of detergent
• alkaline phosphatase there is production of alkaline phosphatase at low concentrations of Triton.
• Alkaline 5 phosphatase is a periplasmic enzyme.
• malate dehydrogenase a cytoplasmic enzyme occurs at higher concentrations of the detergent, indicating that some cell lysis has occurred.
• the occurence of lysis at high concentrations o of the detergent is further supported by the protein content found at high concentrations and given in figure 6.
• Fig. shows the compared malate dehydrogenase activity and glucose dehydrogenase activity, both during growth on heptadecane (0.5%) and Triton X-100 (0.04%) a) absorbance at 660nm, b) glucose dehydrogenase activity, and, c) malate dehydrogenase activity.
• Fig.5 shows a table of the effects of a range of detergents on growth and glucose dehydrogenase production.
• Figure 5 shows that other non-ionic detergents are active in stimulating growth. Buffering of the medium is important as no production was found outside of the pH range 6.3 to 7.5.
• Triton X 100 gives particularly good growth.
• the enzyme in the culture medium was found to be stable over a few days, as incubation at 30°C during several days retained the specific activity. This also applied to a mutant PQQ-apoenzyme form in contrast to a similar apoenzyme obtained from a cell free extract.
• Figure 6 shows that the enzyme has a high specific activity relative to that produced from a cell extract. This is a particularly attractive feature if the enzyme is to be employed in bioelectroni ⁇ sensors.
• Figure 6 also shows the effect of changes in the particular carbon source employed, compared with the growth conditions of the present invention. From fig. 6 it is seen that the highest production was obtained with heptadecane, while other carbon sources were less effective at stimulating production of the enzyme.
• GDH is a constitutive enzyme in this organism. Either there is a continuous synthesis to replace the detached enzyme or there is lysis of cells and constant production of new cells. The second possibility cannot be excluded because significant levels of NAD-dependent 5 malate dehydrogenase have been found in the culture fluid.
• the present invention may be applied to other quinoproteins, such as the general aldehyde dehydrogenase of methylotrophic bacteria.
• the present invention can be employed in the resolution of glucose and/or methanol dehydrogenases from Methylosinus trichosporium QB3b. Pseudomonas aeruqinosa and Pseudomonas extorquens.
• (a) electrodes having on or at a surface thereof, the combination of an enzyme as disclosed herein and a mediator compound capable of transfering charge from the enzyme to the electrode when the enzyme is catalytically active.
• mediator-enzyme-antibody combination a chemically-linked mediator-enzyme combination, mediator-enzyme-antibody combination, or, mediator-enzyme-antigen combination, where the enzyme is prepared according to the method disclosed hereinbefore.

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• 1984
  • 1984-04-10 GB GB8409208A patent/GB8409208D0/en active Pending
• 1985
  • 1985-04-10 WO PCT/GB1985/000156 patent/WO1985004671A1/en not_active Application Discontinuation
  • 1985-04-10 EP EP19850901526 patent/EP0179789A1/de not_active Withdrawn

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