EP0179789A1 - Method of production of quinoproteins - Google Patents

Abstract

Process for the production of a quinoprotein dehydrogenase from an adequate cell culture, in which the production of the quinoprotein is stimulated by the treatment of said culture with a stimulator, consisting of an alkane, and the culture is cultivated in the presence of a nonionic detergent. Also described is a process for producing a quinoprotein from an adequate cell culture of gram-negative bacteria, this culture being cultured in the presence of a nonionic detergent. The processes described here allow the production of these enzymes in gram quantities and in a relatively pure form.

Description

METHOD OF PRODUCTION OF OUINOPROTEINS

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.

Development of 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.

Although many cell export mechanisms are known for the products of gram-positive bacte.ria, a transport mechanism is not known to exist for the transportation of many of these gram negative source enzymes across the outer of the two membranes and thus production has required the harvesting of cells and subsequent lytic extraction of the desired product. Other suggested methods for the release of these proteins have included the use of osmotic shock techniques.

The production of, for example, methanol dehydrogenase (MDH) by traditional biochemical methods has been based o on the purification procedure of Anthony and Zatman (Biochem J. 104; 953-959), involving salt precipitation or heat-treatment of cell-free extracts followed by chromatographic resolution using molecular exclusion, ion exchange or affinity chromatography.

Homogeneous preparations of methanol dehydrogenase have been derived from a wide variety of methylotrophic sources using these procedures, but unfortunately, these procedures are time-consuming, require considerable centrifugation and are difficult to scale up with ease.

o A ten-fold purification of methanol dehydrogenase from the cell-free extract of Methylophilus methylotrophus produced a homogenous enzyme preparation has previously been described by Ghosh et al. (Biochem J. 199 245-250).

A large-scale method for the purification of non-quinoproteins has been described by Kroner et al. ((Scale-up of formate dehydrogenase by partition) J. Chem Technol. Biotechnol. .32. 130-137). More particularly the NAD-linked dehydrogenases, such as 5 formaldehyde, formate and isopropanol dehydrogenase have been extracted from Candida Boidinii.

Detergents have been reported to be active in stimulating production of extracellular enzymes by gram-positive bacteria (Appl.Microbiol. r7 (1969) 242

IO and J. Ferm. Technol 58, (1980) 1), but not of the periplasmic, quinoprotein dehydrogenases.

Alkanes have been noted to stimulate growth on alkanes of Pseudomonas aeruαinosa (long known as Bacillus pyocyaneus). a plasmid carrying, gram-negative soil i 5 bacteria.

According to a first aspect of the present invention, there is provided 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.

By performing the method of the present invention it is possible to produce quinoprotein dehydrogenases in gram quantities. It should be understood the 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.

Typically, 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.

It has been determined that this method of separation is particularly useful when used in combination with the method of the present invention. It is preffered that the polyalkylene glygol is polyethylene glycol, and that the soluble salt is soluble to at least 30% w/v and is an alkali metal phosphate salt, such as potassium phosphate. However, although 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.

According to a second aspect of the present invention there is provided 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.

It is believed that the presence of the detergent stimulates export of the enzyme, either by partial cell breakage, stimulation of some transport mechanism or •5 disruption of the membrane structure, although no firm mechanism has been identified.

Conveniently, 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.

It is believed that the presence of the detergent stimulates growth on alkanes, possibly by the emusification of the alkane into droplets, as detergents which do not stimulate growth generally do not emulsify the particular alkane.

A concentration of 0.05% Triton X-100 is known to give particularly good yield.

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.

s in a particular embodiment of the present invention the alkane is selected from the group of C_b-C_ £._Δ alkanes, and is more preferably selected from the group comprising C₅. Cg, ^C ₁₂ ^~C18 ^{and C}22"

Particular utility has be found with C.,. C,_ and i ct C,_a especially in the case of C_,_.

More preferably the quinoprotein is selected from the group comprising; methanol dehydrogenase and glucose dehydrogenase.

Enzyme stability is enhanced in the above system, ι5 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.

The organism of preference is 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

15 conditions. Furthermore, numerous thin fimbriae are produced at the cell surface, leading to the speculation that such are responsible for the adherence of the organisms to hydrocarbon droplets.

In order that the invention may be better understood it

-2.0 will be described hereafter by way of the following examples and with reference to the accompanying figures wherein;

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.

General Technique;

All reagents were obtained from Baker except hexadecane and heptadecane obtained from Janssen and Fluka, reapectively. The non-ionic detergents were obtained ιo from Sigma.

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.

All measurements were performed at room temperature unless otherwise stated.

EXAMPLE (A) Purification of methanol dehydrogenase from Methylophilus methylotrophus

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. Using a PEG 400/phosphate system, the enzyme was located entirely in the upper, polyethylene-glycol-rich phase.

Use of 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).

Methanol addition ensured the long term stability of the enzyme. The reaction mixture was stirred briefly (5 min, 300 rpm) to attain equilibrium. The phases could then be separated either under gravity (24 h) or by a 2.0 short centrifugation run (5 min. 2,500g).

Under gravity, the cell debris was distributed throughout the phosphate-rich bottom phase.

After centrifugation, it accumulated as a tight pellet at the interface and the clear, pale yellow. enzyme-containing phosphate-rich bottom layer was then carefully removed.

Phosphates and low molecular weight contaminants were removed by diafiltration against phosphate buffer (50mM,

5 pH 7.0, containing lOmM methanol) and the solution concentrated (to approximately 200ml) using a tangential flow ultrafiltration system (Millipore Pellican Cassette System. membrane molecular weight cut-off, 10,000). This purification scheme and the characteristics of the

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

15 by two phase aqueous partition was judged to be at least 95% pure. However, the preparation did contain large quantities of nucleic acids (Fig. 7), which may be removed by protamine sulphate precipitation. In addition, the yield of enzyme was also far greater than

_2.o that obtained by conventional methods. The methods of the prior art, such as described by Ghosh et al. reported a yield of only 13%. whilst most other preparations yield 30-50%. The specific activity of enzyme preparations using this method is also

2.S considerably higher than previously reported (5.6-12.2 umole (mg protein min)- ). The method disclosed herein may easily be scaled up to produce several grams of enzyme, of reasonable purity, that may be further resolved by HPLC or affinity chromatography.

This procedure has been developed to effect the one step resolution of methanol dehydrogenase from disrupted cell mass.

EXAMPLE (B) Purification of Glucose dehydrogenase from Acinetobacter calcoaceticus. NCTC 7844

Acinetobacter calcoaceticus. NCTC 7844 was grown in mineral salt medium (FAM 2) with acetate (0.1% w/v) as the sole carbon source. In order to establish that the method of the present invention could be employed with a large biomass, 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.

Similar behaviour in the PEG 1000/phosphate system was observed as in the example given above, that is GDH partitions into the lower phase of the mixture, thus producing a similar degree of purification as the MDH (approximately 60 fold purification from disrupted 5 organisms; 7-9 fold purification from cell-free extracts).

EXAMPLE (C) Purification of Glucose dehydrogenase from Acinetobacter calcoaceticus. LMD 79.41. with induction by an alkane.

ιo Acinetobacter calcoaceticus LMD 79.41 (obtained from Prof. J. Hauge; Hauge J. G. Biochem Biophys Acta (1960), 45, 263.) was grown on 0.5% hexadecane plus 0.05% Triton-X 100 in a mineral salts medium, buffered with 0.1 Molar potassium phosphate, at pH7. and at 30°C in

15 shake flasks.

As can be seen from figure 1, substantial amounts of GDH appeared in the culture fluid after a relatively short incubation time. 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.

A substantially similar procedure to that employed in example (A) was then followed, and it was noted that the GDH derived from the strain Acinetobacter calcoaceticus LMD 79.41 does not partition into the lower phase of the mixture, as does the GDH derived from the strain Acinetobacter calcoaceticus. NCTC 7844 possibly indicating that these two GDH's are distinct.

EXAMPLE (C(a)) INDUCTION BY ALKANES

The following alkanes were investigated in some detail; Cb_» CO-» C-_._-_-C.10- and C t,Δ_. It was found that

C -. C₁₇ and C.- gave the best yield. and that C.- gave the best stimulation. It appears that the amount of stimulation bears a linear relation to the quantity of alkane present in the growth medium.

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.

EXAMPLE fCfb)) EFFECT OF DETERGENTS.

Fig.3 shows the enzyme activity with heptadecane and 5 varying concentrations of detergent,

A) activity of glucose dehydrogenase;

B) activity of malate dehydrogenase; and.

C) activity of alkaline phosphatase, in Triton X-100 concentrations of; o a) 0%, b) 0.005%, and, c) 0.04%.

It is noted that there is production of alkaline phosphatase at low concentrations of Triton. Alkaline 5 phosphatase is a periplasmic enzyme. It is further noted that the production of 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.

Electron microscope evidence shows that at high detergent concentrations many of the cells are empty. 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.

These figures show that the process of lysis is not abrupt, since the enzyme production follows the growth curve, indicated by the absorbance at 660nm.

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.

As is illustrated in figure 5. a concentration of 0.05% 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 bioelectroniσ 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.

As stated above, and without wishing to impose any limitation to any particular theoretical basis of the present invention, it is believed that the following mechanism is most probable: growth on alkanes induces alterations in the outer cell-membrane, degrading it in some way so that small particles of this membrane are found in the culture fluid (J.Bact. 140 (1979) 707). This may result in a passage for periplasmic GDH to escape. As GDH tends to stick to the cytoplastmic membrane, a non-ionic detergent (in this case best exemplified by Triton X-100) is added to the culture medium to ensure that the yield is maintained.

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.

It should be stressed that only the combination of alkane and a non-ionic detergent such as Triton X 100 is able to induce enzyme production. Combinations of Triton ιo X-100 with other carbon sources were ineffective.

Various modifications can be made within the scope of the present invention. for example, to include a preliminary partition step in which cell debris and nucleic acid are removed, whilst retaining the enzyme in is a low molecular weight PEG (400)-rich phase.

Furthermore, it is envisaged that the present invention may be applied to other quinoproteins, such as the general aldehyde dehydrogenase of methylotrophic bacteria. As a further example of this general J2.o utillity, it is envisaged that the present invention can be employed in the resolution of glucose and/or methanol dehydrogenases from Methylosinus trichosporium QB3b. Pseudomonas aeruqinosa and Pseudomonas extorquens.

Although the present invention has been described in terms of a method it should be understood that is further extends to;

(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.

(b) cells defining a liquid chamber and incorporating such an electrode and a reference electrode,

(c) equipment including such cells,

(d) assay methods including the use of any of (a, b or c) as defined above, and

(e) 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.

Claims

1) A method of production of a quinoprotein dehydrogenase from a suitable cell culture, CHARACTERISED IN THAT. the production of the said quinoprotein is stimulated by the treatment of the said

5 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.

2) A method for the production of a quinoprotein from a suitable gram-negative bacterial cell culture, iθ CHARACTERISED IN THAT said culture is grown in the presence of a non-ionic detergent.

3) A method as claimed in claim 1 or 2 wherein, the detergent is capable of emulsifying the alkane.

4) A method as claimed in claim 1 or 2 wherein, the 15 detergent is at least 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.

5) A method as claimed in claim 4 wherein, the detergent is Triton X-100.

2.0 6) A method as claimed in claim 5 wherein, the detergent is at a concentration of 0.05%. 7) A method as claimed in claim 1 wherein, the alkane is selected from the group of C_ς-C₂₂ alkanes.

8) A method as claimed in claim 7 wherein, the alkane is selected from the group comprising C_b, Co._r

^C12-^C18 ^{aild C}22 ^alkanes-

9) A method as claimed in claim 8 wherein, the alkane is a C-- alkane.

10) A method as claimed in either of claims 1 or 2 wherein the quinoprotein is selected from the group o comprising; methanol dehydrogenase and glucose dehydrogenase.

11) A method as claimed in either of claims 1 or 2 wherein said culture is a culture of bacterial selected from the group comprising; Methylophilus methylotrophus. Methylosinus trichosporium strain OB3B. Acinetobacter calcoacetius. strains LMD 79.41. ATCC 23055 (type strain). ATCC 23220. ATCC 23236 (strain HOI), and. NTCC 7844.

12) A method of claim 11 wherein said culture is a o culture of Acinetobacter calcoacetius

13) A method according to either of claims 1 or 2 wherein, the quinoprotein is methanol dehydrogenase from Methylophilus methylotrophus.

14) A method according to either of claims 1 or 2 wherein, the quinoprotein is glucose dehydrogenase from

-5 Acinetobacter calcoacetius. strain LMD 79.41

15) A method according to either of claims 1 or 2 wherein, the quinoprotein is glucose dehydrogenase from Acinetobacter calcoacetius. strain NTCC 7844.

16) A method according to either of claims 1 or 2 \ o wherein, the quinoprotein is glucose dehydrogenase from

Acinetobacter calcoacetius. strain ATCC 23055.

17) A method according to either of claims 1 or 2 wherein, the quinoprotein is glucose dehydrogenase from Acinetobacter calcoacetius. strain ATCC 23220.

18) A method according to either of claims 1 or 2 wherein, the quinoprotein is glucose dehydrogenase from Acinetobacter calcoacetius. strain ATCC 23236.

19) A method according to either of claims 1 or 2 wherein, the quinoprotein is methanol dehydrogenase from Methylosinus trichosporium. strain OB3B.

20) A method according to either of claims 1 or 2 wherein, the quinoprotein is a dehydrogenase from a Pseudomonad

21) An electrode having on or near a surface thereof a quinoprotein produced by the method of claim 1 or 2.

S . 22) A method as claimed in claim 1 or 2 in which the quinoprotein is an aldehyde dehydrogenase of a methylotrophic bacteria.