A strain of Paracoccus denitrificans, NRRL B-15710, is an overproducer of a fumarase which is readily purified and relatively thermostable. The fumarase so produced converts fumaric acid to L-malic acid without formation of any significant amounts of a coproduct.

BACKGROUND OF THE INVENTION 
Although acidulants have various functions in food processing and products, 
their principal role is to adjust the pH of foods, primarily to enhance 
and to modify flavor, but also to preserve foods. Acidulants are used to 
add sourness and a desired tartness to many food products. Because of the 
low pH created when used, food acids can also prevent growth of 
microorganisms that might cause food to spoil or cause food poisoning or 
diseases. Through chelation of trace metal ions and/or low pH, acidulants 
also prevent rancidity and discoloration of foods by functioning as 
synergists to antioxidants such as BHA, BHT, and ascorbic acid. They also 
are used as buffers during various stages of food processing as well as in 
finished products. Food acidulants may also be used for purposes other 
than imparting flavor or aiding in preservation. They function as melting 
modifiers for cheese spreads and mixtures used in making hard candy, as 
gelling agents, and as viscosity modifiers for doughs. The versatility of 
acidulants will certainly be a factor in their general increased use as 
food additives in the future. 
U.S. markets for food acidulants include carbonated beverages, powdered 
soft-drink mixes, noncarbonated beverages (e.g., fruit juice drinks), 
flavored gelatin desserts, jams and jellies, candies, canned fruits and 
vegetables, pie fillings, yogurts, starch puddings, and wines. 
Beverages--including liquids and powders--are the largest end use for 
acids in food products. Acids are used in soft drinks to provide tartness 
and to modify sweetness, and they are used in canning tomatoes to alter pH 
in order to optimize heat processing. Properties of the various acids are 
important to consider for the different applications; in some uses, high 
solubility is important (e.g., for flavor concentrates), whereas in other 
uses sourness and speed of solution may be the critical factors (e.g., 
powdered beverage mixes). 
Of all acids used in foods, citric acid is the most widely used. Malic acid 
generally is recognized as being versatile and a potential competitor with 
citric acid. Used as a food acidulant in the United States for only about 
fifteen years, malic acid has made its greatest inroads when new products 
are developed and when old products need improvement. It has been 
successful in replacing citric acid, to some degree, in some major uses, 
such as dry powder mixes and candies. In new products, it is sometimes 
used in combination with citric acid. 
Beverages based on malic acid use 8 to 12% less acidulant by weight, on the 
average, than beverages using citric acid, with extremes of 5 to 22% less 
acidulant having been reported. Although the level of acid required for 
replacement varies with the type of flavor, level of sweetness, and level 
of carbonation used, there remains a clear economic incentive for 
replacing citric acid by malic acid. 
Malic acid has penetrated some of citric acid's uses in the nectar and 
diet-drink market; some sugar-free and low-calorie soft-drink producers 
have switched to malic from citric acid because malic acid masks the 
off-taste produced by sugar substitutes (e.g., saccharin). Malic acid is 
used to enhance the flavor in fruit-flavored (especially the berry 
flavors) carbonated beverages and cream sodas. It is used in beverages by 
itself and, in some instances, in combination with citric acid. Newly 
marketed food and beverage products have been (and are expected to 
continue to be) malic acid's major acidulant growth market. Other 
carbonated and still beverages, candies, dessert powders, instant tea, 
syrups, and preserves are targets for growth. It is believed that malic 
acid's use in fruit drinks, particularly apple and berry flavors, will 
increase because of the apparent change in consumer preference for 
beverage flavors; a faster growth rate in consumption of non-cola-flavored 
beverages compared to cola beverages is foreseen. 
Domestic demand for malic acid as a food and beverage acidulant has 
increased from about 4 million pounds in 1967 to about 11 million pounds 
in 1979. Domestic production in 1979 was over 15 million pounds, with the 
difference between production and demand being exported. The average 
annual growth rate for malic acid has been estimated between 3 and 8% per 
year. 
Malic acid is naturally found in many fruits, such as apples, and is there 
produced not as a racemate but as L-malic acid. Regulations in most of 
Western Europe, with the exception of the United Kingdom, Norway, and 
Denmark, do not permit additives of a synthetic origin in food and 
beverages. Therefore, racemic malic acid is not allowed in food and 
beverages except in those countries mentioned. Consequently, there is a 
great economic incentive for a method of producing L-malic acid relatively 
inexpensively. 
Presently, malic acid is made by hydration of maleic anhydride to afford 
racemic malic acid. Resolution of the racemate to obtain L-malic acid is 
expensive, hence its supply remains limited. The demand for L-malic acid 
remains high, and for the aforementioned reasons the use of malic acid as 
an acidulant, especially in Europe, undoubtedly would increase were 
L-malic acid economically competitive with citric acid. 
Fumarase is an enzyme which catalyzes the interconversion of fumaric and 
L-malic acids under mild conditions typical of enzymatic reactions. The 
invention herein is based on our discovering a bacterial strain which 
produces a fumarase which can be easily purified, which has substantial 
thermal stability even at 50.degree. C., which converts fumaric acid to 
L-malic acid without any other detectable coproducts, and which can be 
efficiently immobilized. Our discovery thus makes possible a commercially 
feasible process of making L-malic acid. 
In one aspect our invention is a biologically pure culture of a strain of 
Paracoccus denitrificans, FUM-14, NRRL B-15710. In another aspect our 
invention is a method of producing a fumarase by growing FUM-14 
aerobically in a medium containing an assimilable source of carbon, 
nitrogen, and mineral nutrients, at a temperature from about 20.degree. to 
about 45.degree. C., and recovering the fumarase produced thereby. In 
still another aspect our invention is the fumarase produced by Paracoccus 
denitrificans when grown under the aforesaid conditions. 
DESCRIPTION OF THE INVENTION 
Fumarase is an enzyme found widely in nature which catalyzes the reaction, 
##STR1## 
Just as all microorganisms are not equivalent in their ability to produce 
the enzyme, so are all fumarases not equivalent in their physical-chemical 
characteristics. 
We have discovered a microorganism, isolated from a soil sample, which 
produces fumarase in greater than usual amounts. Additionally, and equally 
important from the process standpoint, the fumarase so produced is readily 
purified, evidences good thermal stability, can be efficiently 
immobilized, and as an immobilized enzyme converts fumaric to L-malic acid 
with the equilibrium mixture containing about 80% of the latter, without 
any substantial amount of byproduct being coproduced. Our discovery makes 
possible a method of making L-malic acid from fumaric acid using the 
immobilized fumarase from FUM-14. 
The microorganism FUM-14, NRRL B-15710, isolated by the enrichment culture 
technique from a soil sample, is an overproducer of fumarase. Briefly, a 
soil sample was grown in a medium containing d, 1-malic acid as the sole 
carbon source. Successive transfers were made to a similar medium, and 
after six such transfers the mixed culture was streaked out to isolate 
single colonies. Colonies were screened for fumarase activity, and FUM-14 
was among those exhibiting particularly high fumarase activity. 
The enzyme is produced intracellularly and is released into solution by 
rupture of the cell walls. Such rupture can be performed by mechanical 
means, such as by homogenization, or by methods such as sonication or 
digestion with lysing enzymes, such as lysozyme. 
After rupture of the cells debris is removed by any suitable means, such as 
centrifugation; the solution which results is the crude extract of 
fumarase. It has been found that although the fumarase from this crude 
extract can be immobilized, other materials in the crude extract lead to 
decreased activity of the immobilized fumarase. More specifically, 
although the activity of immobilized fumarase initially increases with 
increased offering of crude extract, the observed fumarase activity 
subsequently declines as more crude extract is offered in an attempt to 
obtain an immobilized fumarase system with greater activity. Thus, it 
appears necessary to purify, at least partially, the fumarase before its 
immobilization. 
To effect an approximately 30-fold purification of fumarase, the crude 
extract is first diluted with polyethylene glycol and treated with 
potassium chloride at a temperature under about 10.degree. C., causing 
precipitation of unwanted proteins, dialyzing the supernatant from the 
prior described treatment to remove potassium chloride and other salts, 
chromatographing the dialyzed solution on a diethylaminoethyl cellulose 
column, precipitating additional fumarase-inactive protein with a 
concentrated salt solution at a temperature under 10.degree. C., 
increasing the salt concentration in the supernatant to precipitate 
fumarase, and collecting the precipitate formed thereby. The sequence of 
steps is important; the degree of purification using a different sequence 
of the above steps will be less than that attained in the given sequence. 
Partial purification may be effected using chromatographic separation 
alone. 
To the crude extract is added enough polyethylene glycol to form from about 
a 10 to about a 15% V/V solution, followed by sufficient salt to give from 
about a 1 to about a 5% W/V solution at a temperature less than about 
10.degree. C., preferably less than about 5.degree. C., and most desirably 
from about 0.degree. to about 2.degree. C. Polyethylene glycol of a 
molecular weight in the range from about 1000 to about 10,000 can be used 
satisfactorily, although polyethylene glycol of different molecular weight 
also may be used but not necessarily with equivalent results. The salt 
used is typically an alkali metal halide chosen for convenience only, with 
a concentration of about 3% for potassium chloride being preferred. 
Addition of both materials is done with stirring and solid is removed by 
suitable means, such as centrifugation. 
The supernatant from the centrifuged material is dialyzed at a pH of about 
5 in order to remove the salt used in the aforedescribed precipitation and 
to adjust the pH before the subsequent treatment. Any solids formed during 
dialysis are removed and discarded. 
The dialysate then is applied to a diethylaminoethyl cellulose column which 
has been pre-equilibrated with a buffer at a pH of about 5. Elution is 
performed with a gradient of an alkali halide salt solution. Using 
potassium chloride as an example a gradient between 0 and 0.4 molar 
potassium chloride in a suitable buffer at a pH of about 5 may be used. 
Fractions are assayed for fumarase activity and those containing the 
enzyme are pooled prior to subsequent salt fractionation. 
The enzyme in the pooled fractions is now further purified by salt 
fractionation. A salt is added to the cooled solution in an amount 
corresponding to about 25 to about 35% of its saturation point (i.e., the 
total amount of salt which can be dissolved in the solution). In a 
preferred embodiment the solution is cooled to a temperature between about 
0.degree. and about 10.degree. C., more preferably between about 0.degree. 
and about 5.degree. C. To be useful the salt must have a solubility such 
that about a 4 molar solution at 0.degree. C. can be prepared, but is 
otherwise without limitation. Examples of such salts include ammonium 
sulfate, ammonium acid sulfate, sodium chloride, potassium acetate, 
potassium carbonate, and potassium chloride. In another preferred 
embodiment the salt is an alkali metal or alkaline earth metal sulfate, 
such as lithium sulfate, sodium sulfate, potassium sulfate, rubidium 
sulfate, cesium sulfate, and magnesium sulfate. After allowing the solids 
to precipitate they are removed by suitable means, as by centrifugation, 
and discarded. At this point an additional amount of salt is dissolved 
such that the total amount in solution corresponds from about 50 to about 
65% of its saturation point, thereby causing precipitation of fumarase. 
After precipitation is complete the solid containing purified fumarase is 
collected by suitable means, such as centrifugation. 
Salts can be removed from the purified fumarase by any means, such as, for 
example, by dialysis or gel permeation chromatography. The solid which is 
collected as described above is redissolved in a buffer at a pH from about 
6 to about 8. This enzyme solution then is dialyzed overnight against the 
same buffer. Purification practiced as described above can lead to 
enrichment, in terms of units of activity per milligram protein, of over 
30-fold with total fumarase recovery in excess of 50%. 
Native fumarase from NRRL B-15710 is relatively thermally stable. Although 
at 55.degree. C. it loses about 80% of its activity in about 30 minutes, 
it appears to be indefinitely stable at about 40.degree. C. Fumarase 
activity peaked at a pH of about 7.8, showed half that activity at a pH of 
about 7.3, and was virtually inactive at a pH of about 6.0. At a pH of 
about 7.0 the activity of fumarase doubled in going from about 30.degree. 
C. to about 50.degree. C. 
An exceedingly important characteristic of the fumarase of this invention 
is that it produces L-malic acid from fumaric acid without the formation 
of any detectable byproducts. That is to say, when the reaction mixture 
was assayed for product L-malic acid and reactant fumaric acid 100% 
product balance was obtained.

The examples below are merely illustrative of this invention and do not 
limit it in any way. 
Fumarase activity was assayed spectrophotometrically using a substrate 
containing 0.05 molar L-malate in 0.1 molar potassium phosphate buffer, pH 
7.8. One milliliter of the substrate was preincubated in 
spectrophotometric cells for 5-10 minutes before addition of enzyme. After 
enzyme was added the reaction was allowed to proceed for 3 minutes at 
40.degree. C. The change in absorbance was determined at 240 nm at 60 
second intervals, using an extinction coefficient for fumarate of 2.44 
cm.sup.2 /umole. Enzyme activity was expressed as micromoles of L-malate 
converted into fumarate per minute. 
EXAMPLE 1 
A soil sample from the Chicago, Ill. area was incubated in a medium of 
bacto yeast nitrogen base, 0.5% D-malic acid, 0.5% (NH.sub.4).sub.2 
SO.sub.4, and 0.01 M potassium phosphate buffer at pH 7.0 at a temperature 
of 30.degree. C. for about 16 hours. After that time an inoculum was 
transferred to a similar medium which was incubated at the same 
temperature for about the same time. After five transfers the culture was 
streaked out on Petri plates containing the same medium plus agar. 
Isolated colonies were screened for fumarase activity, and among those 
exhibiting high fumarase activity was one originating from FUM-14. 
The strain FUM-14 was identified as Paracoccus denitrificans based on the 
carbon utilization data. This strain was composed of gram negative 
coccal-bacillary cells, arranged singly, in pairs, aggregates or short 
chains. On nutrient agar after 48 hours this isolate produced two colony 
types. While both were circular, entire, glistening, flat and creme color, 
one was opaque and the other translucent. The opaque colony produced the 
two colony types, hence the culture was considered to be pure, with 
colonial variation. 
The physiology and biochemistry of FUM-14 is summarized below. 
______________________________________ 
Motility - Urease - 
4.degree. C. growth 
- Nitrate to nitrite 
+ 
25.degree. C. growth 
+ Nitrite reduction + 
30.degree. C. growth 
+ Nitrite to N.sub.2 
+ 
37.degree. C. growth 
+ Hydrogen sulfide (TSI) 
- 
41.degree. C. growth 
- Lysine decarboxylase 
- 
Fluorescein produced 
- Arginine (Mollers) 
- 
Pyocyanine produced 
- Ornithine decarboxylase 
- 
Pigment produced 
- DL-arginine deamination 
- 
pH 6.0 growth + Phenylalanine - 
3% NaCl growth + Lecithinase - 
6.5% NaCl growth 
- Phosphatase - 
MacConkey agar growth 
+ Catalase + 
Skim milk agar growth 
+ Oxidase + 
Aesculin hydrolysis 
- Gluconate oxidation 
- 
Casein hydrolysis 
- Tyrosine degradation 
+ 
Starch hydrolysis 
- dl-hydroxybutyrate growth 
+ 
Gelatinase - Poly-B--Hydroxybutyrate 
+ 
accumulation 
Tween 20 hydrolysis 
- Deoxyribonuclase + 
Tween 70 hydrolysis 
- Growth on 0.05% cetrimide 
- 
Indole - Growth on acetate as 
+ 
sole carbon source 
Simmons citrate growth 
+ Testosterone degradation 
- 
______________________________________ 
Fermentation of FUM-14 after 7 days in the presence of various sugars 
afforded the following results. 
______________________________________ 
Acid from L-arabinose 
+ Acid from maltose 
+ 
Acid from cellobiose 
K Acid from D-mannitol 
+ 
Acid from ethanol + Acid from D-mannose 
K 
Acid from D-fructose 
+ Acid from L-rhamnose 
K 
Acid from D-glucose aerobically 
+ Acid from D-ribose 
+ 
Acid from D-glucose 
- Acid from sucrose 
+ 
anaerobically 
Acid from glycerol 
+ Acid from trehalose 
+ 
Acid from i-inositol 
+ Acid from D-xylose 
K 
Acid from lactose K Control K 
______________________________________ 
+ = acid produced 
K = alkaline produced 
- = no change 
Utilization by FUM-14 of carbohydrates as the sole carbon source after 12 
days incubation gave results which are summarized below. 
______________________________________ 
L-arabinose 
+ M--hydroxybenzoate 
+ 
cellobiose - 2-ketogluconate 
+ 
D-fructose + DL-lactate - 
D-glucose + L-malate + 
lactose - pelargonate - 
maltose + propionate + 
D-mannitol + quinate - 
L-rhamnose - succinate + 
D-ribose + L-+-tartrate - 
D-sorbitol + valerate - 
sucrose + B--alanine + 
trehalose + D-A--alanine + 
D-xylose - betaine + 
adonitol + glycine + 
erythritol - L-histidine + 
glycerol + DL-norleucine + 
ethanol + L-proline + 
geraniol - D-tryptophan - 
i-inositol + L-valine - 
sebacic acid 
- DL-arginine - 
acetamide - benzylamine + 
adipate - butylamine + 
benzoate - putrescine - 
butyrate - mesoconate - 
citraconate 
- DL-glycerate + 
D-gluconate 
+ L-tryptophan - 
Poly-OH butyrate 
+ 
______________________________________ 
EXAMPLE 2 
A cell pellet of FUM-14, NRRL B-15710 was resuspended in a small amount of 
0.02 M potassium phosphate buffer at a pH of 7.0. To this was added 
lysozyme and 10.sup.-3 M ethylenediamine tetracetic acid and the mixture 
was incubated at 37.degree. C. with agitation until there was visual 
evidence of cell lysing (ca. 30-60 minutes). After this mixture was 
chilled in an ice bath cells were further ruptured by sonification using a 
Branson sonicator, debris was removed by centrifugation at 12,000 rpm for 
30 minutes and supernatant was collected by decantation to afford a crude 
extract of fumarase. 
EXAMPLE 3 
To a crude extract was added sufficient polyethylene glycol, molecular 
weight about 6000, to afford an 11.5% V/V solution, to which was further 
added sufficient potassium chloride to afford a 3.3% W/V solution, the 
mixture being continually immersed in an ice bath with stirring. When 
precipitation of solids was complete the mixture was centrifuged at 12,000 
rpm for about 20 minutes and the supernatant was collected. This 
supernatant was then dialyzed overnight against 0.02 M sodium citrate, pH 
5.2, to remove potassium chloride and to adjust the pH before 
chromatography. Any solids which form are removed by centrifugation before 
chromatography. 
A column of diethylaminoethyl cellulose was equilibrated with sodium 
citrate buffer, 0.02 molar, pH 5.2. Dialysate was applied and the column 
was washed with the same buffer. Enzyme was then eluted with a gradient of 
0 to 0.4 M potassium chloride in the same buffer. Individual tubes were 
assayed to determine the location of the fumarase activity and the enzyme 
fractions were pooled. 
The pooled fractions were chilled in an ice bath to a temperature less than 
5.degree. C. Ammonium sulfate was then added in an amount sufficient to 
give a 30% saturated solution. When precipitation was complete solids were 
removed by centrifugation at 18,000 rpm for 15 minutes. The supernatant 
was decanted, immersed in an ice bath, and additional ammonium sulfate was 
added thereto in an amount sufficient to afford a 60% saturated solution. 
Fumarase was precipitated and collected by centrifugation. This solid was 
redissolved in 0.02 M potassium phosphate buffer, pH 7.0, and dialyzed 
against the same buffer to remove ammonium sulfate. 
The course of purification is summarized in the accompanying table. 
TABLE 1 
__________________________________________________________________________ 
Purification of Fumarase 
Specific 
Volume 
Activity 
Total 
Protein 
Activity 
Yield 
Procedure 
(mL) (Units/mL) 
Units 
(mg/mL) 
(Units/mg) 
(%) 
__________________________________________________________________________ 
Crude Extract 
645 26.3 16,960 
18.6 1.41 (100) 
PEG-KCl 860 17.4 14,960 
21.0 0.83 88.2 
After Dialysis 
1,700 
8.9 15,130 
4.9 1.80 89.2 
pH 5.2 
Chromatography 
59 177.0 10,440 
-- -- 61.6 
30-60% Sat. 
10 916.0 9,160 
29.1 31.5 53.4 
(NH.sub.4).sub.2 SO.sub.4 
__________________________________________________________________________ 
As can be seen, a nearly 32-fold purification was achieved with overall 
recovery of over 53% fumarase. 
EXAMPLE 4 
The activity of native fumarase was determined by assaying the crude 
extract for fumarase activity as a function of pH. Results, which are 
reported as relative activity, are summarized in the accompanying table. 
TABLE 2 
______________________________________ 
pH Profile of Fumarase as Free Enzyme 
Relative 
pH Activity (%) 
______________________________________ 
6.0 5 
6.3 8 
6.6 15 
6.9 27 
7.2 42 
7.5 64 
7.8 100 
8.1 97 
______________________________________ 
EXAMPLE 5 
A solution of fumarase, partially purified by chromatography on 
diethylamino ethyl cellulose, was assayed at various temperatures to 
determine the temperature-activity profile. Results are summarized in the 
accompanying table. 
TABLE 3 
______________________________________ 
Effect of Reaction Temperature on Free Fumarase Activity 
Activity 
Temperature, .degree.C. 
(Units/mL) 
______________________________________ 
22 100 
27 104 
32 108 
37 137 
42 162 
48 207 
55 211 
______________________________________