GB2192890A - Improvements in or relating to pullulanase production - Google Patents

Abstract

In a fed batch method of producing pullulanase under aerobic conditions, a pullulanase-producing micro-organism, such as klebsiella aerogenes, is incubated in a simple culture medium comprising any of sucrose, dextrose and fructose as a carbohydrate source. The medium is free of starch, proteins, yeast extract or other complex nutrients, and the carbohydrate source is fed to the culture medium at a controlled rate sufficient to maintain the energy requirements for pullulanase production but not so great as to repress the pullulanase production.

Description

SPECIFICATION Improvements in or relating to pullulanase production This invention is concerned with improvements in or relating to pullulanase production.

Pullulanase (amylo-1,6-glucosidase) is an enzyme which is specific to the hydrolysis of alpha1,6-glucosidic starch linkages, and is an important enzyme in the production of high dextrose syrups. A process for the production of pullulanase is described for example in U.K. Patent Specification No. GB-1,499,340-A of A.E. Staley Manufacturing Company.

Generally the biosynthesis of enzymes is controlled at the genetic level.

In an inductive system, for example, repressor molecules are produced which block the formation of the necessary mRNA. In such cases an inducer molecule is used to neturalise this effect of the repressor molecules.

On the other hand, in a constitutive system this particular type of repression apparently does not occur and it is not necessary to use an inducer molecule. However if the rate of carbon source metabolism is too great, repression ("metabolic repression") takes place by mechanisms not fully understood: but the rate of metabolism must be sufficient to maintain the energy requirements for the enzyme production.

It has previously been considered that pullulanase production was essentially an inductive system and that carbohydrate inducers other than dextrose were required: see again GB 1 ,499,340A. - We now believe however (without being bound by theory) that surprisingly pullulanase production can in fact be a constitutive system; and any of sucrose, dextrose and fructose can be used as the sole source of carbohydrate. It will be realized that sucrose is hydrolyzed to dextrose and fructose.

Complex media made up from various natural plant and animal sources have traditionally been used to grow bacteria and produce enzymes, with the optimum composition of the initial medium being determined empirically. Complex media components are variable in character and productivity varies from batch to batch. In order to overcome these difficulties it has been previously proposed to use simple media free of proteins, yeast extract, starch or other complex nutrients, but in an inductive system it is necessary to specifically include an inducer molecule in the medium: in the case of a complex medium for inductive systems the inducer molecule is normally present naturally.

We have found that pullulanase can be produced using for example a dextrose-containing simple medium, but too high an initial dextrose concentration in the medium will result in metabolic repression.

In a fed batch method of producing an enzyme, the micro-organism capable of producing the enzyme is incubated in a culture medium in a batch mode, and nutrients are fed to the batch medium on a continuous or intermittent basis.

It is an object of the present invention to provide an improved method of producing pullulanase.

The present invention provides, in one of its aspects, a fed batch method of producing pullulanase under aerobic conditions wherein a pullulanase-producing micro-oganism is incubated in a simple culture medium comprising any of sucrose, dextrose and fructose as a carbohydrate source and which is free of starch, proteins, yeast extract or other complex nutrients, and the carbohydrate source is fed to the culture medium at a controlled rate sufficient to maintain the energy requirements for pullulanase production but not so great as to repress the pullulanase production.

A method according to the invention is for example carried out at a temperature of 25-35"C and the dissolved oxygen concentration in the culture is maintained at least 10% of saturation, for example at least 30%. The range of oxygen concentration employed is for example 10-60% of saturation preferably 30-40%.

Examples of puliulanase-producing bacteria include Klebsiella aerogenes ("Klebsiella pneumoniae" in GB-1,499,390-A), ATCC-15050 and ATCC 8724; NRRL-B-5780, NRRL-B-5783 and NRRL-B-5784; Bacillus acidopullulyticus as described in U.K. Patent Specification No. GB2,097,405-A of Novo Industri A/S. Mutant and genetically engineered variants of any of these bacteria are encompassed.

There is also a nutritional nitrogen requirement for a simple medium and this can be provided ammoniacally but too high an initial ammonia concentration in the culture medium can inhibit growth of the micro-organism. Again the natural characteristics of a complex medium usually prevent this problem arising.

For example, in our case of pullulanase production using a simple medium we can employ a low initial concentration of ammonium sulphate, ammonium chloride or mixtures thereof as the sole source of nitrogen in the initial medium. As the microbiological reaction proceeds ammonium ion is depleted and the pH of the medium tends to fall: ammonium hydroxide, for example, is then fed (e.g. by a pump) at a controlled rate to control the pH and provides additional source of nitrogen at the rate demanded by the microbiological reaction; alternatively, for example, the pH is controlled and additional nitrogen provided by sparging in ammonia gas.

The description which follows is given with reference to dextrose but it will be realized that it is applicable as appropriate also to sucrose or fructose.

As indicated above a solution of for example dextrose is fed (e.g. by a pump) to the culture medium at a controlled rate sufficient to maintain they energy requirements for pullulanase production but not so great as to repress the pullulanase production.

If for example, contrary to our procedure, the feed rate of dextrose is maintained constant, then when the cell population in the medium is low the metabolic rate may be great enough to cause some repression. On the other hand as the cell population increases the dextrose available to each cell falls until repression no longer takes place. However, eventually the population increases to a level such that the cells do not have sufficient energy to express pullulanase and production is seriously retarted. Only a short period will occur in such a scheme in which the specific rate of dextrose uptake is in the range which would be economical for the production of pullulanase.

Ideally, the dextrose feed rate is slowly increased according to the increase in cell population so that the optimum specific rate of dextrose uptake Go (weight of dextrose/weight of cells.

hour) is maintained; and enzyme production will occur at the optimum rate at all times being neither repressed nor short of energy: Go can be determined empirically and may be dependent on various factors such as the nature of the micro-organism, pH and temperature.

The dextrose feed rate F (grams dextrose/litre of culture. hour) required can be determined at any particular time t by measuring the cell concentration x (grams cells/litre culture); and: F=Gox (1) As x increases in the culture medium F can be increased according to equation (1).

It is possible to predict x2 the cell concentration at a time t2 knowing the value of the cell concentration x1 at an earlier time t1.

The equation is: x2=x1 e OYet2- 1) (2) where Y is the yield of cells on dextrose fed to the culture (weight of cells/weight of dextrose).

Conveniently, x, may be the initial cell concentration xj at the moment in time tj when the dextrose feed is commenced. Then at any time t the cell concentration x is given by: x=xi e 0,Y(t-tj (3) The feed rate F can then be determined at any time t from the initial values x, at t by combining equations (1) and (3): F=Go xi e G,y(tt (4) or F=Fj e GoY(t G,y (5) If t2-t1 in equation (2) is very short and passes from t to (t+At), with a constant value of At, the feed rate F at time (t+At) becomes from equation (5): : Ft=F e G,y (6) As indicated hereinbefore in equation (1) x is expressed in grams cells/litre culture. However x is proportional to the optical density of the culture and so conveniently equations (1)-(6) can be re-expressed in these terms; for example: F=Go0D X OD (7) xoD=xoD e G0 Y 00(t-t (8) F=F e C,0 y OD(tt,) (9) F,=F e Gs" Y ODa (10) where:: xOD is the optical density at 510 nm of a sample taken at time t and diluted to 1/50th GO D is now expressed in terms of grams dextrose/litres. optical density unit. h.

YOD is now expressed in terms of optical density units. litre/grams dextrose.

We have found that Go D and YOD are generally constant for a given micro-organism, pH and temperature.

For Klebsiella aerogenes or variant thereof at 28"C and a pH of 7.5 , Go0D is apparently in the range of 6-8.5 e.g. About 7.0 and YOD in the range 0.02-0.04 e.g. about 0.03. Go D YOD is then in the range 0.12-0.34 e.g. about 0.165.

In embodiments of the invention, as well as sources of carbon and nitrogen the medium comprises sources of magnesium, phosphate, and potassium: citric acid and trace salts e.g.

ferrous sulphate, calcium chloride, manganese sulphate and zinc sulphate. The citric acid carries out mild sequestration of trace metal ions to prevent their precipitation.

For example the initial culture medium comprises the following ranges of components in aqueous solution (grams per litre except where otherwise stated).

Dextrose 4-6 Citric acid 0.5-1 Magnesium sulphate 0.5-1 Ammonium sulphate 5-9 Potassium sulphate 0-1.5 Potassium chloride 0-1.5 Potassium nitrate 0-7 Monosodium phosphate 6-20 Trace salts 0.02-0.05 Polypropylene glycol (PPG) 1-1.5 ml PPG is an anti-foaming agent.

The initial pH is adjusted preferably to a figure above 7.0 but not exceeding 8 (e.g. 7.3-7.6) using sodium hydroxide.

In carrying out a fed batch method embodying the invention an inoculum of a pullulanase bacterium e.g. Klebsiella aerogenes or a variant thereof is prepared and added to the initial medium in a fermenter under aerobic conditions which are maintained throughout the method.

After a time lag of say about 6 hours the pH begins to drop and the controlled addition of ammonium hydroxide solution is commenced so that the pH is maintained in the range above 7.0 but not exceeding 8 (e.g. 7.3-7.6) preferably about 7.5: the ammonium hydroxide concentration in the solution to be added is for example in the range 30-40% by weight preferably about 35%.After approximately another one or two hours the dextrose in the medium has been substantially consumed as indicated by an optical density measurement of the cell suspension (510 nm 1/50 dilution) of 0.15-0.2: other indications are a rise in dissolved oxygen, rise in outlet gaseous oxygen and reduction in outlet carbon dioxide: at this point x,OD is determined and (knowing G,OD) the controlled addition of dextrose is commenced substantially according to equation (7): F=GoOD x OD (7) The dextrose concentration in the solution fed to the batch culture is for example in the range 300-400 grams/litre, e.g. about 350 grams/litre.

A simple way of operating substantially in accordance with equation (7) is to take frequent samples, determine the xOD for the sample and calculate F from equation (7); it will be appreciated that xOD would be increasing between samples while F remains constant, but we have found there is a measure of self compensation.

Preferably a pump adapted for continuously variable feed rate is used for the dextrose feed but an alternative is to use a simple pump, switching on and off at appropriate intervals, but again it will be appreciated this may involve further departures from the ideal of equation (7).

Alternatively a table or graph can be prepared from equation (7) to give the appropriate value of F at any given optical density x D. The procedure being say at frequent intervals of not more than one hour to calculate xoD from equation (8), read F from the table or graph and adjust the feed rate of dextrose accordingly. A check can be made periodically by taking a sample and measuring the actual xoD.

In any event, typically the method is terminated after say about 20 hours from the original inoculation, when an enzyme activity of at least 35 units preferably of at least 40 or 100 units has been achieved.

A unit of pullulanase activity is defined as that activity which will produce reducing sugars equivalent to 1 mg. of anhydrous maltose in one minute under the conditions of an assay described in "Food and Nutrition Paper" No. 19 published by the FAO of the United Nations, Rome, Italy 1981.

Still higher yields can be obtained by continuing the method as long as possible, i.e. until the oxygen supply relative to the cell population of the culture becomes limiting, and this period can be extended by sparging with pure oxygen.

In the accompanying drawings: Figures 1 and 2 show graphs which are illustrative of aspects of the invention by way of example thereof and not by way of limitation; and Figure 3 illustrates the results of a comparative test.

Fig. 1 is a graph relating xOD in terms of F for Go D=8.0.

Rather then carrying out the adjustment to the dextrose feed rate at relatively long intervals of time, superior yields of pullulanase may be obtained by varying the feed rate substantially continuously in accordance with equation (10) Ft=F e Gg YODAX (10) with a very small time interval At between feed rate adjustments for example less than one minute, e.g. about 30 seconds. A computer can be used to generate an output signal corresponding to the required value of Ft from the value of F at an earlier time and this output signal can be utilized to control the dextrose solution feed pump.

The ammonium hydroxide feed rate can also be controlled directly by a computer in which case an input signal is provided corresponding to the pH and an output signal corresponding to the ammonium hydroxide feed rate which signal is utilized to control the ammonia solution feed pump.

Example I Culture Medium (Grams/litre unless otherwise stated) Dextrose 5.14 Citric acid 0.71 Magnesium sulphate 0.71 Ammonium sulphate 7.29 Potassium sulphate 1.43 Monosodium phosphate 17.14 Trace salts:

Ferrous sulphate 0.029 Calcium chloride 0.013 Manganese sulphate 0.0023 t 0.0466 Zinc sulphate 0.0023 PPG 1.43 ml Water to 1 litre pH adjusted to 7.5 using sodium hydroxide Ammonium Hydroxide Feed 35% by weight aqeuous ammonium hydroxide solution Dextrose Feed 336 grams/litre aqueous solution.

Inoculum The inoculum was prepared from a freeze dried culture of a variant of Klebsiella aerogenes derived from NRRL-B5780 by strain selection and stored in ampoules in the dark at 5"C.

An ampoule was opened and 1 ml. of sterile, deionized water added. The suspension was shaken, streaked onto an agar plate and incubated at 28"C for 18 hours.

Single colonies were picked off aspetically and transferred to a bottle containing 150 ml. of sterile cooked meat medium (Difco B 267, Difco Laboratories, Detroit, Michigan, U.S.A.), incubated at 28"C for 18 hours and stored at 5"C until required.

5 ml of medium from this bottle was introduced into 200 ml of a broth containing bactopeptone (Difco B 118), starch and salts, and incubated for 18 hrs. at 28"C.

Procedure A 7 litre batch of the culture medium was introduced into a well stirred fermenter and inoculated with the inoculum; initially there was no feed of either ammonium hydroxide or dextrose. Aerobic conditions were maintained throughout with dissolved oxygen maintained at least 30% of saturation using an aeration device and the pH continuously monitored.

At 6 hours from inoculation the pH commenced falling and when it reached 7*3 which was between 6 and 7 hours, the ammonium hydroxide feed was commenced to maintain the pH at about 7.5 for the remainder of the run.

After 8 hours the optical density (510 nm 1/50 dilution) was 0.15-0.2, dissolved oxygen was rising, and outlet carbon dioxide was falling. At this point the dextrose feed was commenced and maintained approximately according to equation (7), with Go0D selected at 8.0. For this purpose calculations of xOD were made at hourly intervals using equation (8) and corresponding adjustments made manually to the dextrose feed rate in accordance with the graph of Fig. 1.

After about 20 hours from inoculation the run was terminated, and an enzyme activity of about 40 units of pullulanase had been achieved.

Total consumption of the fed ammonium solution was about 28 ml per litre of initial culture and of the fed dextrose solution about 145 ml per litre of initial culture.

The-temperature was maintained at 28"C throughout.

EXAMPLE II Culture Medium (Grams/litre unless otherwise stated) Dextrose 4.5 Citric acid 0.625 Magnesium sulphate 0.625 Ammonium sulphate 6.375 Potassium chloride T.25 Monosodium phosphate 15.0 Trace salts:

Ferrous sulphate 0.025 Calcium chloride 0.011 Manganese sulphate 0.002 0.04 Zinc sulphate 0.002 PPG 1.25 ml Water to 1 litre.

pH adjusted to 7.5 using sodium hydroxide The ammonium hydroxide and dextrose feed solutions, and the inoculum were as in Example I.

Also the procedure of Example I was substantially followed and the final enzyme activity achieved was again about 40 units of pullulanase.

In this case the enzyme activity was monitored at regular intervals and Fig. 2 is a graph of enzyme activity in units of pullulanase against time. The continuously rising nature of the curve of Fig. 2 will be noted.

Comparative Test The procedure of Example II was substantially followed but with a constant dextrose feed rate of 3.82 grams/litre of original culture hr. Fig. 3 is a graph of enzyme activity against time, and the falling off in activity well before completion of fermentation will be noted.

EXAMPLE 111 The culture medium, ammonium hydroxide and dextrose feed solutions, and the inoculum were as in Example II.

Feed pumps for the ammonium hydroxide and dextrose solutions were controlled by a com puter: the ammonium hydroxide feed pump in response to a pH signal to maintain the pH of the culture medium at about 7.5: and the dextrose feed pump substantially in accordance with equation (10) with GO D=7.0 YOD=0.02 and At=32 seconds. The procedure was otherwise substantially as in Example II and the final enzyme activity achieved was 41.7 units of pullula nase with a profile of enzyme activity against time similar to Fig. 2.

EXAMPLE IV The procedure of Example Ill was substantially followed but with Go D=6.5 and the final enzyme activity achieved was 48.08 units of pullulanase.

EXAMPLE V The procedure of Example lli was substantially followed but with Go D=6.0 and the final enzyme activity achieved was 31.6 units of pullulanase.

EXAMPLE VI The procedure of Example III was substantially repeated but with Go D=6.5 and YOD=0.03. The final enzyme activity achieved was 46.8 units of pullulanase.

Claims (20)

1. A fed batch method of producing pullulanase under aerobic conditions wherein a pullulanase-producing micro-organism is incubated in a simple culture medium comprising any of sucrose, dextrose and fructose as a carbohydrate source and which is free of starch, proteins, yeast extract or other complex nutrients, and the carbohydrate source is fed to the culture medium at a controlled rate sufficient to maintain the energy requirements for pullulanase production but not so great as to repress the pullulanase production.

2. A method according to claim 1, wherein the micro-organism is a bacterium.

3. A method according to claim --1, wherein the micro-organism is Klebsiella aerogenes, or a variant thereof.

4. A method according to any one of claims 1, 2 and 3, wherein the medium comprises sources of nitrogen, magnesium, phosphate, and potassium; citric acid and trace salts.

5. A method according to claim 4, wherein the initial medium comprises in aqueous solution (grams/litre): Dextrose 4-6 Citric acid 0.5-1 Magnesium sulphate 0.5-1 Ammonium sulphate 5-9 Potassium sulphate 0-1.5 Potassium chloride 0-1.5 Potassium nitrate 0-7 Monosodium phosphate 6-20 Trace salts 0.02-0.05.

6. A method according to any one of claims 1 to 4, wherein dextrose is the sole carbohydrate source.

7. A method according to any one of the preceding claims, wherein the feed rate of sucrose, dextrose or fructose is controlled substantially according to the equation: F=Go xj e G,Y(ttj where: F is the required feed rate after a time interval from time t to time t Go approximates to the optimum specific rate of uptake of sucrose, dextrose or fructose xj is the initial cell concentration of the culture medium at the commencement of the feed Y is the yield on sucrose, dextrose or fructose.

8. A method according to claim 7, wherein the feed rate is adjusted at intervals not exceeding one hour.

9. A method according to any one of claims 1 to 6, wherein the feed rate of sucrose, dextrose or fructose is controlled substantially according to the equation: Ft=F e G,yAt where: Ft is the required feed rate at time t+At F is the required feed rate at time t Go approximates to the optimum specific rate of uptake of sucrose, dextrose or fructose Y is the yield on sucrose, dextrose or fructose and the feed rate is adjusted substantially continuously.

10. A method according to any one of claims 7, 8 and 9, wherein G,, xi and Y are defined in terms of optical density at 510 nm and 1/50th dilution, and for Klebsiella aerogenes or a variant thereof at 28"C and a pH of 7.5 and G,OD is the range 6-8.5 grams/litres original culture. OD.h.

11. A method according to any one of claims 7 to 10, wherein YOD is in the range 0.02-0.04 OD. litres/grams.

12. A method according to any one of claims 7 to 11, wherein GO D YOD is in the range 0.12-0.34.

13. A method according to any one of the preceding claims, wherein an aqueous solution of ammonium hydroxide or ammonia gas is fed at a controlled rate to the culture medium to maintain nutritional nitrogen and maintain the pH of the culture within a required range.

14. A method according to claim 13, wherein said pH range is above'7 and not exceeding 8.

15. A method according to any one of the preceding claims carried out at a temperature of 25-35"C.

16. A method according to any one of the preceding claims, wherein the dissolved oxygem concentration in the culture is maintained at least 10% of saturation.

17. A method according to any one of claims 1 to 15, wherein the dissolved oxygen concentration is maintained in the range of 10-60% of saturation.

18. A method according to any one of claims, 1 to 15, wherein the dissolved oxygen is maintained at least 30% of saturation.

19. A method of producing pullulanase substantially as hereinbefore described with reference to any one of the Examples.

20. Pullulanase when produced by a method according to any one of the preceding claims.