TECHNICAL FIELD

The present invention relates to novel cholesterol esterases. More specifically, the invention relates to novel cholesterol esterases derived from strains 5 of Pseudomonas cepacia. In a more specific embodiment, the invention relates to a cholesterol esterase acting lipase.

BACKGROUND ART

It is well known that some of the members belonging to the genus Pseudomonas possess the ability to produce cholesterol esterases, other members 0 possess the ability to produce lipases, and that furthermore some members possess the ability to produce both types of enzymes.

In a study by Smirnovet. al. [Smirnov, V.V.; Komyushenko, O.N.; Bioko, O.I.; Kolesova, E.A.; Govseeva, N.N.; Endt, V.P.; and Kiprianova, E.A.; Mikrobiologicheskii Zhurnal (Kiev); 42 (5), 1980, 566-570], a total of 591 strains of 25 s species of bacteria belonging to the genus Pseudomonas were studied for their ability to synthesize extracellular cholesterol esterase. Only individual strains of P. aeruginosa, P. pseudoacaligenes, P. fluorescens, P. putida, and P. maltophilia (3.4% of the studied cultures) had cholesterol esterase activity. Lipolytic activity was found in P. aureofaciens (in 100% of the studied strains), P. cepacia (in 75%), P. o maltophilia (in 60%), P. fluorescens (in 13.3%), and P. aeruginosa (in 60%).

From this study it is noticed that none of the P. cepacia cultures examined were able to produce cholesterol esterases.

SUMMARY OF THE INVENTION

According to the present invention it has now been found that at least 5 one strain of Pseudomonas cepacia is able to produce extracellular cholesterol

esterase. Moreover, as regards this strain, it has surprisingly been found that the component responsible for the cholesterol esterase activity is also the component responsible for the lipase activity.

Accordingly, the invention provides cholesterol esterases obtainable from a strain of Pseudomonas cepacia.

In another aspect, the invention provides a process for the preparation of cholesterol esterases of the invention, which process comprises cultivation of a cholesterol esterase producing strain of Pseudomonas cepacia in a suitable nutrient medium, containing carbon and nitrogen sources and inorganic salts, followed by recovery of the desired enzyme.

In a further aspect, the invention relates to the use of a lipase obtainable from the strain P. cepacia DSM 3401 as a cholesterol esterase.

BRIEF DESCRIPTION OF DRAWINGS

The present invention is further illustrated by reference to the accompanying drawings, in which:

Fig. 1 shows the kinetic curves (absorbance versus time) as obtained from the spectrophotometer (1) Cholesterol esterase, Sigma ^® , OD _2B0 =0.062; 2) P. cepacia 7510A lipase, 00 ₂₈₀ =0.061; 3) Mucor lipase, OD- ₃ ^0.063; 4) Candida B lipase, 00 ₂₈₀ =0.060; 5) Upper to lower curve: Humicola lipase, 00 ₂₈₀ =0.062; Guinea pig lipase LGF 1000, OD-,^0.062; Candida A lipase, 00 ₂₈₀ =0.054; and Candida cylindracia lipase,

DETAILED DISCLOSURE OF THE INVENTION

It has now been demonstrated that at least one strain of P. cepacia possesses the ability to produce an enzyme having cholesterol esterase activity. Accordingly, the present invention provides a cholesterol esterase obtainable from a strain of Pseudomonas cepacia.

Moreover, it has surprisingly been found that in this case the enzyme responsible for the cholesterol esterase activity is identical to the enzyme responsible for the lipase activity, i.e. the very same protein possesses lipase activity as well as cholesterol esterase activity. Accordingly, the invention provides an enzyme having 5 cholesterol esterase activity as well as lipase activity, i.e. being a cholesterol esterase acting lipase or vice versa.

The lipase activity of P. cepacia DSM 3401 and its applicability in detergent compositions have been described in EP Patent Specification No. 214,761 . However, in this publication there is no indication regarding the lipase having any ιo cholesterol esterase activity. The strain, P. cepacia DSM 3401 , was deposited on 22 July 1985 at Deutsche Sammlung fur Mikroorganismen under the terms and conditions of the Budapest Treaty, and is now available according to the above EP Patent Specification No. 214,761 .

Accordingly, the invention provides novel cholesterol esterases is obtainable from a strain of P. cepacia, preferably the strain P. cepacia DSM 3401 , or a mutant or a variant thereof.

The cholesterol esterase of the invention can be further described by the following physical-chemical characteristics. The enzyme possesses cholesterol esterase activity at pH values of from below pH 4 to above pH 10, determined at

20 37°C with cholesterol oleate as substrate. The enzyme has a pH optimum in the range of from pH 5 to pH 7, more specifically around pH 6. The enzyme has a molecular weight of approximately 30 kD, as determined by SDS-PAGE, and a pi of approximately 4.0. Maximum activity, among the substrates tested, on cholesterol- palmitate.

25 In a more specific aspect, the cholesterol esterase of the invention has the amino acid sequence identified by the sequence listing attached to this specification.

The cholesterol esterase of the invention can be obtained by cultivation of a cholesterol esterase producing strain of P. cepacia, preferably the strain DSM

30 3401 , in a suitable nutrient medium, containing carbon and nitrogen sources and inorganic salts, followed by recovery of the desired enzyme.

A highly purified lipase preparation, designated Pseudomonas cepacia 751 OA lipase, was obtained from the strain DSM 3401 by the methods described in Examples 1-2 of this specification. By amino acid degradation, the amino acid sequence of the enzyme was found to be the one identified in the sequence listing 5 attached to this specification. By the method described in EΞxample 3 of this specification it was demonstrated that this enzyme also possesses cholesterol esterase activity.

The cholesterol esterase of the invention may also be obtained by recombinant DNA-technology. By expression of only one protein it is possible to ιo achieve two enzymatic activities, lipase activity and cholesterol esterase activity.

Both cholesterol esterases and lipases have widespread industrial applications. By applying one and the same enzyme possessing both activities especial advantages may be obtained. Accordingly, the invention also relates to the use of a cholesterol esterase acting lipase for hydrolysis of eggs, is In a further aspect, the invention relates to use of a cholesterol esterase of the invention in processes for hydrolysis of resin in pulp. Mechanical pulping, alone or combined with a gentle chemical treatment, is widely used in the manufacture of pulps. These processes occur at pH in the range 4-9, and the com¬ ponents of the wood undergo relatively small chemical changes. The pulp therefore 20 has a considerable content of triglycerides, esters, and waxes from resin.

Residual resin may cause problems during the subsequent use of the pulp. Thus, agglomerated resin may cause paper breakage during paper manufacture or during printing as well as lowering of the paper quality. It is known that the hydrophobic part of resin contains considerable amounts of triglycerides 5 and other esters. It is, therefore, desirable to hydrolyze these, as the- hydrolysis products are more easily removed in aqueous systems.

Processes for hydrolysis of resins in pulps have been described in e.g. International Patent Application Nos. PCT/DK92/00115 or PCT/DK92/00137, or International Patent Publication Nos. WO 92/07138 or WO 92/13130, and may be o carried out essentially as described herein.

The following examples further illustrate the present invention, and they are not intended to be in any way limiting to the scope of the invention as claimed.

EXAMPLE 1

Cultivation Example A culture of Pseudomonas cepacia, DSM 3401 , on an agar slant was transferred to five 500 ml shaking flasks, each with 100 ml of Bullion-3 medium, and shaken at 30°C for 1 day (200 rpm, amplitude 2,5 cm).

The composition of Bullion-3 medium was as follows:

Peptone _, 6 g/l Trypsin digested casein 4 g/l

Yeast extract 3 g/l

Meat extract 1.5 g/l

Glucose 1 g/l

The medium was autoclaved at 121°C for 40 minutes, The culture broth of these Bullion-3 shake flasks was used as a seed culture for inoculating two hundred 500 ml shake flasks, each with 200 ml of PL-1 medium.

The composition of the PL-1 medium was as follows:

Peptone 10 g/l Tween ^® -80 12 g/l

MgS0 ₄ ;7H ₂ O 2 g/l

CaCI ₂ ;2H ₂ O 0.1 g/l pH before autoclaving 6.0

The medium was autoclaved at 121°C for 40 minutes. Each PL-1 shake flask was inoculated with 0.5-2 ml of Bullion-3 culture broth, and shaken with 200 rpm (amplitude 2.5 cm) at 30°C for 5 days. The culture

broth from the shake flasks was pooled at harvest, totalling 39.5 I with an enzyme yield of 53 LU/ml.

The culture broth was centrifuged for 35 minutes at 4100 xg by means of a Beckman Model J-6 centrifuge. The supernatant was concentrated by filtration s (washed with approximately 1 volume of water) to 1.4 I by a Pellicon ultrafiltration apparatus from Millipore with a 10.000 MW cut off filter sheet. The concentrate was freeze dried, and the yield was 56.2 g of powder with an enzyme activity of 21.500 LU/g.

Assay for Lipase Activity ιo One Lipase Unit (LU) is the amount of enzyme which, under standard conditions (i.e. at30.0°C; pH 7.0; and tributyrine substrate) liberates 1 μmol titratable butyric acid per minute. A folder AF 95/5 describing this analytical method is available upon request to Novo Nordisk A/S, Denmark.

[EXAMPLE 2

s Purification Example

The ultrafiltration concentrate obtained according to Example 1 was suspended in water and applied on a hydrophobic XAD-8 resin matrix, and eluted with 60% (v/v) 96% ethanol.

To the eluate ammonium acetate was added to a final concentration o of 0.5 M. The enzyme was applied on a Toyopearl™-Butyl column and eluted with 0.05 M glycine buffer, pH 9.3.

A molecular weight of approximately 30 kD was determined by SDS- PAGE. Only one fraction appeared on the SDS-PAGE. A pi of approximately 4.0 was determined by isoelectric focusing on LKB Ampholine ^® PAG plates.

[EXAMPLE 3

Assay for Cholesterol Esterase Activity

An enzymatic assay for cholesterol esterase activity was set up and the cholesterol esterase side effect of some lipases examined. The analysis was based on the following three coupled reactions:

1) Cholesterol oleate — (1)— > Cholesterol + Oleic acid

2) Cholesterol + 0 ₂ + H ₂ 0 — (2)— > H ₂ O ₂ + Cholestenone

3) H ₂ 0 ₂ + 4-Aminoantipyrine(red) ---(3)— >

2H ₂ 0 + 4-Aminoantipyrine(ox)

Enzymes involved:

(1) Cholesterol esterase (to be assayed)

(2) Cholesterol oxidase

(3) Peroxidase

The redox reaction of 4-aminoantipyrine is followed by a change in absorbance at 500 nm with an extinction coefficient, E = 6.89.

Conditions and Reagents:

pH = 7.0

Temperature, T = 37 °C

Buffer: 0.4 M KH ₂ P0 ₄ ; pH 7.0; 37 °C (54.4 mg/ml)

Substrate: 0.0086 M cholesterol oleate (5.6 mg/ml). Dissolve 56.0 mg cholesterol oleate in 1.0 ml polyoxyethylene-9-lauryl-ether. Add a small stirring bar and stir gently. While stirring add 9.0 ml of hot (> 50°C) saline solution (0.9 % w/w). Leave the solution at room temperature.

Other: 15 % (w/w) taurocholic acid, Na salt (150 mg/ml) 1.75 % (w/w) 4-aminoantipyrine (17.5 mg/ml)

6 % (w/w) phenol (60 mg/ml)

Coupling enzymes:

Cholesterol oxidase, Sigma ^® C-1512 (1 mg protein/ml). Peroxidase, Sigma ^® P-8250 (39.5 purpurogallin units/ml).

Enzyme: Cholesterol esterase (0.02-0.4 u/ml)

Procedure:

Into a 1 cm standard silica cuvette; buffer, 2.07 ml; taurocholic acid, 0.10 ml; peroxidase, 0.10 ml; and substrate, 0.50 ml; were pipetted and mixed by inversion.

Then phenol, 0.10 ml, was added and mixed by inversion. Next 4-aminoantipyrine, 0.05 ml, and cholesterol oxidase, 0.03 ml, were added, and a baseline at 500 nm was obtained.

Ultimately cholesterol esterase, 0.05 ml, was added, and ΔOD at 500 nm (dA dt) was obtained.

Calculation of Cholesterol Esterase Units: ΔOD/min

Un ^' rts/mg solid =

6.89 x (mg enzyme/ml reaction mixture)

One cholesterol esterase unit is defined as the amount of enzyme that will hydrolyse 1 μmole of cholesterol oleate per minute at pH 7.0 and 37°C.

Enzymes Examined

To ensure only one protein is present, highly purified enzyme preparations must be applied.

The enzymes examined (cf. Table 1) were dosed on an equal OD basis. As reference was used a cholesterol esterase from Sigma ^® with 12.5 units/ml and OD = 0.185. Of this enzyme 0.2 units was used equivalent to OD = 0.062, approximately 0.062 mg/ml.

AH enzymes were scanned for approximately 600 seconds. The spectrophotometric determinations are presented in Fig. 1.

Next the gradient dA/dt, and ultimately the cholesterol esterase activity was calculated using the expression given above. The results are presented in Table 1 , below.

Table 1

Not detectable.

From Fig. 1 and Table 2 it is easily observed that only the reference from Sigma ^® and the P. cepacia 7510A lipase have any significant cholesterol esterase activity.

EXAMPLE 4

Characterization Example

In this example further characterization of the cholesterol esterase acting lipase obtained according to Example 1-2 is presented. The assay for cholesterol esterase activity set forth in Example 3 has been accomplished.

Substrate Specificity

The substrate specificity was examined at pH 7.0, 37°C, with a series of cholesterol esters having hydrocarbon chains from 2 to 18. The results are presented in Table 3.

Table 3

Substrate

Cholesterol-acetate -butyrate -laurate -myristate -palmitate -oleate

pH Profile

The pH profile was determined at 37°C between pH 4 and pH 10, and with cholesterol oleate as substrate.

The results are presented in Table 4.

Table 4

Temperature Profile The cholesterol esterase activity was measured as a function of the temperature. Cholesterol oleate was used as substrate, and the measurements were made at pH 7.0.

The results are presented in Table 5.

Table 5

Temperature induced changes in the substrate emulsion made experiments above 45°C impossible. This temperature experiment is, therefore, non- conclusive.

SEOJENΈ LISTING

(i) SE__JEN_E CHaR ERlSTICS:

(A) LEϊsGϊH: 318 anino acids

(B) TYPE: anino acid (C) SIFWsCEEN-SS: single

(D) TCK-UDGY: I inear

(i r) DLEDULE TYPE: protein

(vi) CRIGINAL SORE:

(A) CHa'ΗISvl: Pseudαπ ias cepacia (B) STRMN: DEM 3401

(C) INDIVIDUAL ISOLATE: 751QA

(xi) SFQ.FNCECE9CRIPTICN:

AlaAlaGly Tyr Ala Ala Thr Arg Tyr Pro lie Ile Leu Val His Gly 1 5 10 15 Leu Ser Gly Thr Asp Lys Tyr AlaGly Val Leu Glu Tyr Trp Tyr Gly 20 25 30 lie Gin Glu Asp Leu GinGin Asn GlyAla Thr Val Tyr Val Ala Asn 35 40 45

Leu Ser Gly Phe Gin Ser Asp AspGly Pro Asn Gly Arg Gly Glu Gin 50 55 60

Leu LeuAla Tyr Val Lys Thr Val Leu AlaAla Thr Gly Ala Thr Lys 65 70 75 80

Val Asn Leu Val Gly His Ser Gin GlyGly Leu Ser Ser Arg Tyr Val 85 90 95 AlaAla Val Ala Pro Asp Leu Val Ala Ser Val Thr Thr Ile Gly Pro 100 105 110

AlaAspArg Gly Ser Glu Phe AlaAsp Phe Val Gin Asp Val Leu Ala 115 120 125

Tyr Asp Pro Thr Gly Leu Ser Ser Ser Val Ile Ala Ala Phe Asn Asn 130 135 140

Val Phe Gly Ile Leu Thr Ser Ser Ser His Asn Thr Asn Gin AspAla 145 150 155 160

Leu Ala Ala Leu Gin Thr Leu Thr Thr Ala Arg Ala Ala Thr Tyr Asn 165 170 175 in Asn Tyr Pro Ser Ala Gly Leu Gly Ala Pro Gly Ser Cys Gin Thr 180 185 190

Gly Ala Pro Thr Glu Thr Val Gly Gly Asn Thr His I le Leu Tyr Ser 195 200 205

Trp Ala Gly Thr Ala I le Gin Pro Thr Leu Ser Val Gly Val Thr Gly 210 215 220

5 Ala Thr Asp Thr Ser Thr Leu Pro Leu Val Asp Pro Ala Asn Val Leu 225 230 235 240

Asp Leu Ser Thr Leu Ala Leu Phe Gly Thr Thr Val M_t I le Asn Arg 245 250 255

Gly Ser Gly Gin Asn Asp Gly Leu Val Ser Lys Cys Ser Ala Leu Tyr o 260 265 270

Gly Lys Val Leu Ser Thr Ser Tyr Lys Trp Asn His Leu Asp Glu lie 275 280 285

- Asn Gin Leu Leu Gly Val Arg Gly Ala Tyr Ala Glu Asp Pro Val Ala 290 295 300 s Val I le Arg Thr His Ala Asn Arg Leu Lys Leu Ala Gly Val 305 310 315