WO2003042378A1

WO2003042378A1 - Sulphatases and use thereof

Info

Publication number: WO2003042378A1
Application number: PCT/EP2002/012618
Authority: WO
Inventors: Kurt Faber; Mateja Pogorevc; Thomas Riermeier
Original assignee: Degussa Ag
Priority date: 2001-11-14
Filing date: 2002-11-12
Publication date: 2003-05-22
Also published as: DE10155764A1; EP1444336A1; CN1592783A; JP2005508654A

Abstract

The invention concerns novel alkylsulphatases obtained from actinomycetes, as well as their use, in particular during catalytic conversion of secondary sulphate esters.

Description

SULFATASES AND THEIR USE

The present invention relates to new sulfatases from actinomycetes and their use, in particular for the catalytic conversion of secondary sulfate esters.

Sulfatases are enzymes that catalyze hydrolytic cleavage of sulfate esters to inorganic sulfate and the corresponding alcohols. The enzymatic hydrolysis of sulfate esters follows the general reaction scheme:

RO-SO3 ^" + H ₂ 0 → R-OH + S0 ₄ ^2_ + H ⁺

A large number of sulfatases are now known which can be classified in terms of their preferred substrates into alkyl, aryl and carbohydrate sulfatases. In higher organisms, sulfatases primarily mediate the hydrolysis of glycolipid, glycosaminoglycan or steroid sulfate esters. In contrast, the assimilation of sulfur from the environment and probably the provision of energy-storing sulfur-containing compounds for cell growth play an important role in bacteria.

The best characterized group of sulfatases are the highly conserved aryl sulfatases, which accept aromatic, especially phenolic substrates. Some aryl sulfatases, e.g. B. the isolatable from human cells, but preferably implement carbohydrate sulfate esters. A serine semialdehyde unit, ie a C _α -formylglycine (Fglyc) unit in the active center of the enzyme, which can be formed from a post-translationally modified cysteine or serine residue, is characteristic of aryl sulfatases. This post-translational modification is essential for the biochemical function of the enzyme. Bacterial aryl sulfatases belong to the so-called "sulfate starvation-induced proteins" (SSI poteins), which are used, for example, in a cultivation with alternative sulfur sources such as aryl sulfonates, alkyl sulfides, thioethers, etc. can be formed.

The class of alkyl sulfatases, which catalyzes the hydrolysis of organic sulfate esters of primary and secondary alcohols, is far less well characterized. This is all the more surprising against the background that large amounts of alkyl sulfates are released into the environment as a component of household products (e.g. detergents) and are broken down by microorganisms (Y.-C. Hsu, Nature 200, 1091-1092 ( 1963); Williams, J., et al., Appl. Microbiol. 12, 360-362 (1964); White, GF, et al., Environ. Pollut. Ser. A. 37, 1-11 (1985)) ,

The existence of specific alkyl sulfatases was first described in a comprehensive review by Dodgson, K.S. et al, Sulfatases of Microbial Origin, 2 vols., CRC Press, Inc., Boca Raton (1982).

The best characterized alkyl sulfatases so far come from the Gram negative bacterial strains Pseudomonas C12B (NCIMB 11753 = ATCC 43648) and Comamonas terrigena (NCIMB 8193) (Dodgson, KS et al, Sulfatases of Microbial Origin, 2 vols., CRC Press, Inc., Boca Raton (1982)). Pseudomonas C12B can express up to five different alkyl sulfatases, depending on the cutting conditions, which are suitable for the hydrolysis of primary or secondary alkyl sulfate testers (Dodgson, KS, et al, Biochem. J. 138, 53-62 (1974); Bartholomew, B ., et al. Biochem. J. 169, 659-667 (1978); Cloves, U.M., et al., Biochem. J. 185, 13-21 (1980)). In contrast, Comamonas terrigena has only two secondary alkyl sulfatases (Fitzgerald, JW, et al., Biochem. J. 149, 477-480 (1975); Barrett, CH, et al., Biochem. J., 191, 467-473 (1980 ); Matcham, GWJ, et al., Biochem. J. 167, 723-729 (1977)).

In addition, Pseudomonas putida FLA, Aerobacter cloacae (Payne, WJ, et al. Nature 214, 623-624 (1967)) and probably also in Pseudomonas B-2 (Lijmbach, GWM, et al., J. Microbiol Serol. 39, 415 (1973)) an alkyl sulfatase activity can be detected. Furthermore, in a mixed culture of microorganisms isolated from active sludge Alkyl sulfatase activity can be found (Vacium, L. et al., Res. Roum. Biochim. 2, 149 (1976)).

So far, the only gram-positive bacterial strains have been Bacillus cereus (Singh, K.L., et al., World J. Microbiol. Biotechnol. 14, 777-779 (1998)) and Coryneform sp. B1a (Payne, W.J., et al., Appl. Microbiol. 13, 698 (1965)) a primary alkyl activity if atase activity.

Until now, alkyl sulfatases could only be purified to homogeneity in individual cases. On the other hand, there is hardly any knowledge about their structure, substrate specificity or their properties. For these reasons, alkyl sulfatases z. B. not used in organic synthesis, although especially in the enantioselective production of secondary alcohols such enzymes valuable tools such. B. could be in the resolution of racemates. The technical potential of alkyl sulfatases has therefore hardly been tapped.

Based on this, the object of the present invention is to provide new alkyl sulfatases which, for. B. can be used as enzymatic tools in the alkyl sulfate ester cleavage.

However, the vast majority of the pro and eukaryotic microorganisms tested for alkyl sulfatases, which are known for a pronounced secondary metabolism, show no activity with regard to the hydrolysis of secondary alkyl sulfate testers (Table 1).

In contrast, it has surprisingly been found that, in contrast to other Gram-positive bacteria, secondary alkyl sulfatases are expressed in actinomycetes, especially in rhodococci. Furthermore, these secondary alkyl sulfatases can be purified to homogeneity without the enzymes losing their enzymatic activity. The secondary alkyl sulfatases obtained in this way from Actinomycetes, preferably from Rhodococcus sp. are the subject of the present invention. Table 1: Results of the search for secondary alkyl sulfatases in Gram-positive bacteria and in fungi using rac-2-octyl sulfate as the test substrate.

kA = no information kU = no conversion ^a relative molar amount ^b (([2-octanol] + [2-octanone]) / [2-octyl sulfate ester])

Enzymatically active secondary alkyl sulfatases can be isolated from the respective Actinomycetes strain by carrying out the following purification steps: a) rough cleaning of a cell-free lysate using a phenyl-Sepharose column with a falling sulfate-free salt gradient, b) further purification of the fractions obtained, which show an enzymatic alkyl sulfatase activity, with an ion exchange, preferably an anion exchange resin, c) the fractions obtained by ion exchange chromatography, which show an enzymatic alkyl sulfatase activity, are passed through a Blue Sepharose column and d) then this is done Enzyme from the resulting fractions, which show an enzymatic alkyl sulfatase activity, purified with the Superdex 200 column for homogeneity.

It is advantageous to treat the cell-free lysate beforehand with DEAE, TEAE or Ecteola cellulose or with DEAE-Sephadex A-25 to separate non-peptide components. Secondary alkyl sulfatases are bound to the carrier and dissolved with a saline buffer for further cleaning.

A purification scheme that has proven particularly useful for isolating secondary alkyl sulfatases from rhodococci comprises the following process steps: a) treatment of a cell lysate of an actinomycete strain with DEAE cellulose in a batch process for the separation of carotenoids, lipids etc., b) elution of the enzyme with a solution of 10mM Tris / HCl and 0.5M NaCI, c) adding NaCI to the eluate to a final concentration of 4M NaCI and column chromatographic purification of the resulting solution over a phenyl Sepharose column, equilibrated with a 10mM Tris / HCl buffer containing 4 M NaCI, pH 7.5 (buffer B), a step-wise NaCI gradient from 4 M to 0 M being used to elute the enzyme. This is followed by further d) dialysis of enzymatically active fractions against 10 mM Tris / HCl buffer, pH 7.5, followed by e) ion exchange chromatography using a Q6 column (BioRad), the column using a 10 mM Tris / HCI buffer, pH 7.5 (buffer A) is equilibrated and an NaCI gradient of 0 M to 1 M is used for elution. The gradient can e.g. B. by gradually adding buffer mixtures from buffer A. and buffer C (10 mM Tris / HCl, pH 7.5, 1 M NaCl). Further purification is carried out by f) applying the enzymatically active fractions to a Blue Sepharose column (Pharmacia), the enzymatically active fractions being placed in a 10 mM piperazine buffer, pH 6.0, and eluted. The column is then additionally washed with a Tris / glycine buffer, pH 8.9. g) Purification for homogeneity on a Superdex 200 column with a 50 mM NaH ₂ PO ₄ buffer, pH 7.0 at a NaCI concentration of 0.15 M.

The enzymatic hydrolysis of secondary alkyl sulfate testers can be used to determine enzymatically active fractions, it being possible to determine either the amount of sulfate released or alcohol released as a measure of the enzymatic activity of the individual fractions. The first method is e.g. B. in Dodgson, K.S., Biochem. J., 78, 312-319, 1961; Tudball, N., et al. Biochem. J. 126, 187-191, 1972.

The secondary alkyl sulfatases of actinomycetes according to the invention isolated in this way preferably have a molecular weight between 30 and 60 kDa in denatured form and / or a molecular weight between 60 and 80 kDa in the native state. Secondary alkyl sulfatases with a molecular weight between 41 and 45 kDa in the denatured state and / or between 65 and 69 kDa in the native state are particularly preferred. The molecular weight in the denatured state is determined by gel electrophoresis using the SDS-PAGE method.

With the method described, for. B. secondary alkyl sulfatases RS1 and RS2 from Rhodococcus ruber DSM 44541 can be isolated. The molecular weight of the denatured secondary alkyl sulfatase RS2 was determined using the SDS-PAGE method and is 43.3 kDa. In its native state, this enzyme has a molecular weight of 67 kDa, determined by elution on Superdex 200, which could indicate the presence of a dimeric enzyme. The isoelectric point of the enzyme RS2 is 5.1. To further characterize the secondary alkyl sulfatase RS2 from Rhodococcus ruber DSM 44541, the enzyme was examined by mass spectrometry. Individual characteristic peptide fragments could be detected with the help of electrospray tandem mass spectrometry. For this, e.g. B. the gel electrophoretically separated sulfatase fraction is isolated and digested with trypsin. The peptide mixture obtained is analyzed by means of ES MS / MS. The following five peptide fragments can be detected for the secondary alkyl sulfatase RS2 from Rhodococcus ruber DSM 44541,

[L, I] TAT [L, I] [L, I] NN [L, I] QMPPR Sequence I (Seq ID No. 1-16)

T [L, I] AEGGTAWESPR Sequence II (Seq ID No. 17/18)

PEAV [L, I] VSAAR Sequence III (Seq ID No. 19/20)

NP [L, I] FADAEAR Sequence IV (Seq ID No. 21/22)

E [L, I] G [L, I] TP [L, I] AR Sequence V (Seq ID No. 23-30) ([L, l] = leucine or isoleucine)

the fragments sequence I and II show no significant homology to other proteins. Sequences III and V are homologous to a putative thiolase from Streptomyces coelicolor 3 (2), sequence V is also homologous to a thiolase segment from Mycobacterium tuberculosis, sequence IV is homologous to a putative acetyl-CoA acetyltransferase from Streptomyces venezuelae. Thus, a preferred subject of the present invention are secondary alkyl sulfatases from actinomycetes which contain at least one amino acid sequence region according to sequences I to V.

The secondary alkyl sulfatases according to the invention can not only be isolated enzymatically, but are furthermore distinguished by their good stability, which makes them suitable for use in the enzymatic-organic hydrolysis of sulfate esters. Possible uses here include the use in clarifiers and / or the degradation of sulfate esters from detergents. The alkyl sulfatases keep e.g. B. their enzymatic activity in the presence of organic solvents, in a wide pH and temperature range. A pH range between pH 5.5 and pH 10 and a reaction temperature below 40 ° C. is preferred. In addition, the influence of many detergents, sugars and polyalcohols on the activity of the enzymes is low. The influence of most salts does not show a strong influence on the hydrolysis activity of the alkyl sulfatases tested. However, some of these additives show a positive effect on conversion or enantioselectivity of the enzymatic hydrolysis of alkyl sulfate esters.

Furthermore, the enzymes can be ionically immobilized without any problems and thus removed from the reaction batch and reused without the sulfatases losing their enzymatic activity. Anionic ion exchange resins such as, for. B. DEAE cellulose, TEAE cellulose, Ecteola cellulose or DEAE Sephadex A-25. Immobilization takes place particularly efficiently at a pH of 7.5. As a buffer z. B. 0.1 M Tris buffer can be used.

This results in a further object of the present invention, namely the use of the claimed secondary alkyl sulfatases for the enzymatic hydrolysis of alkyl sulfate esters or for the enzymatic production of secondary alcohols.

So z. B. the secondary alkyl sulfatases RS1 and RS2 from Rhodococcus ruber DSM 44541 can be used for the production of secondary alcohols. Secondary alkyl sulfate esters which contain a carbon chain of 7 to 10 carbon atoms are preferably reacted as substrates. A chain length of 8 carbon atoms is particularly preferred. Furthermore, sulfate esters are preferred whose sulfate ester bond is located at position 2, 3 or 4 of the esterified alcoholic carbon chain.

When using the secondary alkyl sulfatase RS2 for the hydrolysis of sulfate esters, a pH value between pH 7.0 and pH 8.5 is preferably carried out at temperatures between 15 ° C. and 36 ° C., particularly preferably between 27 ° C. and 32 ° C. Furthermore, the reaction under sulfate-free conditions or at very low sulfate concentrations has proven advantageous in the hydrolysis of alkyl sulfate esters with the sulfatases according to the invention. Practically sulfate-free conditions can e.g. B. by adding barium ions to the reaction mixture.

The secondary alkyl sulfatases are also able to enantioselectively catalyze the hydrolysis of secondary alkyl sulfate esters. The enantioselectivity can by adding other additives such. B. iron (II) - or iron (III) ions can be increased in a low concentration. Concentrations between 1 mM and 15 mM for iron (II) ions and between 3 mM and 6 mM for iron (III) ions have proven to be preferred, especially when secondary alkyl sulfatases from rhodococci are used. The enantioselectivity of the hydrolysis can also be increased considerably by adding cetyltrimethylammonium bromide (CMAB).

In addition to the use of free secondary alkyl sulfatases in the reaction mixture, immobilized secondary alkyl sulfatases can also be used. For this purpose, the alkyl sulfatases z. B. on anionic carrier materials, such as. B. DEAE cellulose, DEAE Sephadex A-25, TEAE cellulose or Ectoela cellulose, can be immobilized. A particularly suitable carrier is ecteola cellulose, since alkyl sulfatases immobilized thereon show an increased enantioselectivity.

Secondary alkyl sulfatases are therefore particularly well suited for the production of secondary alcohols from their sulfate esters. Such a process is of particular interest in the enantioselective production of chiral secondary alcohols, e.g. B. in the resolution of racemates.

Depending on the type of enzymatic attack, the hydrolysis of the alkyl sulfate esters can take place with retention or inversion of the configuration. The secondary alkyl sulfatases RS1 and RS2 isolated from Rhodococcus ruber DSM 44541 hydrolyze e.g. B. (R) - (-) - 2-octyl sulfate stereoselectively by inversion of the configuration to (S) - (+) - 2-octanol.

Some exemplary embodiments to illustrate the present invention are described below.

Example 1: Cultivation of Rhodococcus ruber

Rhodococcus ruber cells of the strain DSM 44541 are cultivated in a liquid medium composed of 10 g / l glucose, 10 g / l peptone, 10 g / l yeast extract, 2 g / l NaCl, 1.5 g MgS0 ₄ -7H ₂ 0 , 1, 3 g / l NaH ₂ P0 ₄ and 4.4 g / l K ₂ HP0 ₄ under aerobic conditions. The culture solution is swirled in a flask with baffles at 30 ° C and 130 rpm, the cell growth is monitored by measuring the optical density at an absorption wavelength of 546 nm.

Example 2: Purification of the secondary alkyl sulfatases

To break up the cells, 47 g of Rhodococcus ruber DSM 44541 cell paste (wet weight) are suspended in 120 ml of Tris buffer (10 mM, pH 7.5). 120 ml of glass beads with a diameter of approximately 0.35 mm are added to this suspension. The cells of the suspension are broken up with a vibration cell mill with external ice / water cooling at a temperature below 4 ° C. The cells break open in 4 cycles from a two-minute vibration phase and subsequent five-minute cooling. After filtration of the glass beads, the cell lysate is centrifuged at 4 ° C and 38000 xg for two hours. The centrifuged cell lysate is treated with DEAE cellulose in a batch process for the separation of carotenoids, lipids, etc., the alkyl sulfatases being bound to the DEAE cellulose carrier. The alkyl sulfatases are eluted with a solution of 10 mM Tris / HCl and 0.5 M NaCl. NaCl is added to the crude enzyme extract prepared in this way, with stirring and with ice cooling, until the final concentration of 4 M is reached. The crude enzyme extract is then purified by column chromatography using a phenyl Sepharose column (Pharmacia V = 20 ml), the column being first equilibrated with 80 ml of a 10 mM Tris / HCl, 4 M NaCl buffer, pH 7.5 (buffer B). Elution is carried out by gradually reducing the NaCl concentration. Mixtures of buffer B with a buffer A (10 mM Tris / HCl, pH 7.5) are used for this. Two secondary alkyl sulfatases from Rhodococcus ruber DSM 44541 (RS1 and RS2) can be detected, the sulfatase RS1 is eluted at a NaCl concentration of 1 M (25% buffer B, 75% buffer A), the more lipophilic sulfatase RS2 is at one NaCl concentration of 0 M eluted (0% buffer B, 100% buffer A).

After dialysis of the sulfatase fraction obtained against 10 mM Tris / HCl buffer, pH 7.5, the enzymatically active fractions are equilibrated on a Q6 column (BioRad, V = 6 ml), with a 10 mM Tris / HCl buffer. Buffer, pH 7.5, further cleaned. The protein constituents of the fraction used are eluted with a 10 mM Tris / HCl buffer, pH 7.5, using a gradual NaCI gradient between 0 M and 1 M NaCI. The secondary alkyl sulfatases RS1 and RS 2 are desorbed at a NaCl concentration between 0.15 and 0.31 M. The fractions containing RS1 and RS2 are then placed in 10 mM piperazine buffer, pH 6.0, on a Blue Sepharose column (Pharmacia) applied. Under the given conditions, neither RS1 nor RS2 binds to the column material and can therefore be collected immediately. The column is then washed out with a Tris / glycine buffer, pH 8.9. The enzymatically active fractions obtained are concentrated to 1 ml (Centriplus 10, Amicon) and applied to a Superdex 200 column. The column is equilibrated and the respective sulfatase eluted with a 50 mM NaH ₂ PO ₄ , 0.15 M NaCl buffer, pH 7.0.

The progress of the individual cleaning steps is shown in Figure 1. 1 shows an SDS-PAGE (12%) analysis of the protein-containing sample:

Lane A shows the crude cell extract.

Lane B shows the sample after treatment with DEAE cellulose.

Lane C shows the sample after chromatography on phenyl Sepharose.

Lane D shows the sample after Q6 ion exchange chromatography.

Lane E shows the sample after chromatography on Blue Sepharose. Lane F shows the sample of the purified secondary alkyl sulfatase RS2 after the

Cleaning with Superdex 200.

Lane G shows molecular weight markers.

The samples were stained with Coomassie Brilliant Blue R.

Example 3: Assays for determining the enzymatic activity.

a) Determination of the enzymatically released amount of sulfate ions.

To determine the enzymatically released sulfate ions, a method from Dodgson, K.S. et al., Biochem. J., 78, 312-319, 1961 by Tudball, N. et al., Biochem. J., 126, 187-192, 1972. 200 μl of an alkyl sulfatase solution are incubated with 200 μl of a 30 mM 3-octyl sulfate ester solution. A 0.1 M Tris / HCl buffer, pH 7.5 is used. The reaction is carried out at a temperature of 30 ° C. and stopped after 15 minutes by adding 15% trichloroacetic acid (w / v). After a short centrifugation, a 200 μl sample of the reaction solution is used for the sulfate determination according to Tudball.

The reaction time is extended to 1 to 2 hours if 2-octyl sulfate ester is used as the substrate for the detection of the enzymatic activity.

An enzyme activity unit (= unit) is defined as the amount of enzyme which releases 1 μmol SO ^{2 "} ions per minute.

b) Determination of the enzymatically released amount of alcohol.

To determine the enzymatic activity, 400 μl of a completely or incompletely purified enzyme solution, obtained after ion exchange chromatography or a resuspended solution of lyophilized sulfatase powder, obtained after chromatography on phenyl Sepharose, in 0.1 M Tris / HCl buffer, pH 7 , 5 mixed with 400 μl of a 30 mM substrate solution in the same buffer. Depending on the course of the reaction, the mixture is shaken at 130 rpm for 6 to 12 hours at 24 ° C. Example 4: Determination of sales

A 200 ul aliquot of the reaction mixture is extracted with 200 ul ethyl acetate. After thorough mixing of the reaction mixture for 0.5 minutes and subsequent centrifugation of the mixture at 13000 rpm for five minutes, a 100 μl sample of the organic phase of the supernatant is mixed with 5 μl of a stock solution of menthol in ethanol (c = 15.4 mg / ml), which serves as an internal standard. The conversion is determined by means of GC on achiral columns (HP 1301, 6% cyanopropylphenylmethylpolysiloxane, 30 mx 0.25 mm x 0.25 μm (film) and HP-1, 30 mx 0.53 mm x 0.5 μm; N ₂ ), the temperature being held at 90 ° C for 2.5 minutes, followed by a temperature increase to 110 ° C with a heating rate of 10 ° C / min. The specified temperature program is tailored to the measurement of 2-, 3- and 4-octanol.

Example 5: Determination of the enantiomeric excess

The enantiomeric excess (ee) is determined on chiral Chrompack CP 7500 columns (cyclodextrin-B-2, 25 mx 0.25 mm x 25 μm) and Chrompack Chirasil-Dex CB / G-PN (γ-cyclodextrin propionyl column, 30 mx 0.32 mm) with hydrogen as carrier gas. The (R) - or (S) -alcohol is identified by co-injection of a corresponding chiral alcohol.

Derivatization of 2-alkanols

To determine the enantiomeric excess, the alcohol is derivatized with trifluoroacetic anhydride (TFA). This results in an improved separation of the enantiomers on the chiral GC column. For this purpose, 400 μl of the enzymatically reacted reaction mixture are extracted with 500 μl dichloromethane and dried over anhydrous sodium sulfate. After adding 40 μl of TFA, the solution is heated to 60 ° C. for 20 minutes. After cooling the solution to room temperature, the sample is extracted twice with 0.5 ml of 5% sodium bicarbonate solution. After the organic phase has finally been dried over anhydrous sodium sulfate, the sample is analyzed by gas chromatography. To avoid processing the sodium bicarbonate extract, the sample can alternatively be treated with a solution of N-methyl-bis-trifluoroacetamide in dichloromethane (1: 3 v / v) at 40 ° C for one hour and then directly examined by gas chromatography.

Another alternative method is the gas chromatographic analysis of the corresponding acetates. For this purpose, 400 μl of the enzymatically reacted reaction solution are extracted with 400 μl ethyl acetate. The organic phase is dried over anhydrous sodium sulfate. After the addition of 80 μl acetic anhydride and cat. p-Dimethylaminopyridine, the solution is shaken overnight at room temperature. The solution is then extracted twice with 0.5 ml of water and then the organic phase is dried over anhydrous sodium sulfate and analyzed.

It should be noted that the trifluoroacetates elute in reverse order to the acetate testers.

Examples 6: Enzymatic hydrolysis of secondary alkyl sulfate esters with RS2

The substrates to be examined are implemented as described in Example 3. The results of the implementation are shown in Table 2.

Table 2: Substrate tolerance of the secondary alkyl sulfatase RS2 from Rhodococcus rube OSM 44541.

n.a = no enzymatic activity ascertainable ^a = enantioselectivity expressed as Εnantiomeric ratio 'determined according to

Chen C.-S. et al. J. Am. Chem. Soc, 104, 7294-7299, 1982. ^b = substrate unstable

The results in Table 2 show that the secondary alkyl sulfatases which can be isolated using the described method have a high specificity with respect to potential substrates. In the case of the secondary alkyl sulfatases RS2 from Rhodococcus ruber DSM 44541, suitable substrates are linear aliphatic secondary (C ₇ -C 10) alkyl sulfate esters without further substituents. Such esters are implemented in good yields and with good enantioselectivity. RS1 shows a substrate specificity similar to RS2.

Example 7: Influence of salts

The reaction follows with the addition of the additive to be investigated in accordance with Example 3. The results are summarized in Tables 3 to 5. It surprisingly turns out that the selectivity of the secondary alkyl sulfatases according to the invention can be increased by adding iron ions, preferably by adding Fe ²⁺ ions. So z. B. the enantioselectivity of RS2 over rac-3-octyl sulfate by adding FeCI ₂ from E = 4 to E = 80 (Table 3) and over rac-4-octyl sulfate from E = 1 to E = 10 (Table 4). This is all the more astonishing since the addition of EDTA, mercaptoethanol and dithiothreithol has no effect on the activity of the alkyl sulfatases.

Table 3: Effect of the addition of salts on the yield and enantioselectivity of the enzymatic conversion of rac-3-octyl sulfate with RS2.

Comparative measurement in the absence of an additive

Table 4: Effect of the addition of salts on the yield and enantioselectivity of the enzymatic conversion of rac-4-octyl sulfate with RS2.

^a Comparative measurement in the absence of an additive

Table 5: Effect of the addition of salts on the yield and enantioselectivity of the enzymatic conversion of rac-2-octyl sulfate with RS2.

Comparative measurement in the absence of an additive

Example 8: Influence of detergents

The reaction follows with the addition of the detergent to be investigated in accordance with Example 3. The results are summarized in Table 6. The influence of detergents on the activity of the claimed secondary alkyl sulfatases differs depending on the detergent used. By adding SDS or di-n-octylsulfosuccinate, the enzymes lose their activity almost completely. In contrast, many detergents show little or no impact on sales, but some detergents even have a positive impact on the enantioselectivity of the enzymatic reaction (Table 6). So z. B. an increase in the enantioselectivity of sulfate ester hydrolysis can be achieved by the addition of CMAB.

Table 6: Influence of detergents on the conversion and enantioselectivity of RS2 for the substrate rac-3-octyl sulfate

^a Comparative measurement in the absence of a detergent ^b Cetyltrimethylammonium bromide

Example 9: Influence of other additives

The reaction follows with the addition of the additive to be investigated in accordance with Example 3. The results are summarized in Table 7. The influence of different sugars and polyalcohols on the conversion and the enantioselectivity of the enzymatic hydrolysis of secondary sulfate esters by the secondary alkyl sulfatases according to the invention is, with a few exceptions (such as CMAB), very small (Tables 7 and 8).

Table 7: Influence of sugars and polyalcohols on the conversion and enantioselectivity of RS2 for the substrate rac-3-octyl sulfate

Table 8: Effect of adding other additives on the yield and enantioselectivity of the enzymatic conversion of octyl sulfate testers with RS2.

Comparative measurement in the absence of an additive

Example 10: Influence of a carrier on immobilized enzymes

A solution of a sulfatase preparation (11 mg / ml) in 0.1 M Tris / HCl buffer, pH 7.5 becomes 100 mg of an immobilization vehicle (DEAE, TEAE, Ecteola cellulose (Serva, Heidelberg), DEAE -Sephadex A-25), which was previously washed with a buffer of 0.1 M Tris / HCl, pH 7.5. The mixture is carefully mixed for 5 minutes and left to stand at 4 ° C. for 30 minutes. The mixture is then centrifuged, the supernatant is removed and the immobilization carrier is briefly washed with 0.1 M Tris / HCl buffer, pH 7.5 and tested for alkyl sulfatase activity analogously to Example 3. The results are summarized in Table 9.

Table 9: Effect of the addition of anionic Carriem on yield and enantioselectivity of the enzymatic conversion of rac-3-octyl sulfate with RS2.

It can consequently be shown that ecteola cellulose as an anionic carrier material for the immobilization of secondary alkyl sulfatases has a positive effect on conversion and in particular on the enantioselectivity of the hydrolysis of alkyl sulfate esters.

Example 11: Influence of organic solvents

The reaction of rac-2-octyl sulfate (5% (v / v)) takes place at 30 ° C with RS2 in different organic solvents. The results are shown in Table 10.

Table 10: Yield and enantioselectivity of the enzymatic conversion of rac-2-octyl sulfate (5% (v / v)) with RS2 in different organic solvents.

A further advantage of the secondary alkyl sulfatases according to the invention is shown, namely their stability in the presence of organic solvents without a major loss of activity. A particularly suitable organic solvent for enantioselective reactions of secondary alkyl sulfate esters is t-BuOH.

Example 12: Addition of barium ions

The influence of Ba ²⁺ on the enzymatic activity of the secondary alkyl sulfatase RS2 was determined using 15 mM rac-2-octyl sulfate ester as substrate in 0.1 M Tris / HCl buffer at a pH of 7.5 and a temperature of 24 ° C determined. 2 shows the conversion of secondary alkyl sulfate ester depending on the reaction time with and without the addition of barium ions to the reaction mixture.

Example 13: Biochemical characterization of the secondary alkyl sulfatase RS2 from Rhodococcus ruber DSM 44541. a) Determination of the molecular weight

The molecular weight of RS2 was determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight determined by SDS-PAGE is checked on the basis of the enzyme size using a Superdex 200 column. The calibration is carried out with chymotrypsinogen A (25 kDa), ovalbumin (43 kDa), BSA (67 kDa), alcohol dehydrogenase (150 kDa) and blue dextran (2000 kDa) as molecular weight standards.

The molecular weight determination under denaturing conditions using SDS-polyacrylamide gel electrophoresis showed a molecular weight of 43.3 kDa for the secondary alkyl sulfatase RS2. Elution of the purified enzyme via the Superdex 200 resulted in a molecular weight of approx. 67 kDa, which indicates a non-globular or dimeric structure of the enzyme.

b) determination of the isoelectric point (pl)

The isoelectric point of the secondary alkyl sulfatase RS2 was determined using a BioRad IEF Ready Gel System at a pH between pH 3 and pH 10. The pl determination of RS2 gave a value of 5.1.

c) Determination of the optimum pH

The pH optimum of the sulfatase RS2 was determined using 15 mM rac-2-octyl sulfate ester as substrate in 100 mM Tris / maleate buffer at 24 ° C. over a pH range of 6.0 to 9.0. The activity maximum of the secondary alkyl sulfatase RS2 is between pH 7.5 and 8.0.

3 shows the enzymatic activity of the sulfatase RS2 as a function of the pH.

d) Determination of the optimum temperature The influence of temperature on the enzymatic activity of the secondary

Alkyl sulfatase RS2 was made using 15mM rac-2-octylsu ifate ester as

Substrate in 100 mM Tris / maleate buffer at pH 7.5 in one

Temperature range from 20 ° C to 40 ° C determined. The enzymatic activity of RS2 against 2-octyl sulfate shows an optimum at approx. 30 ° C.

4 shows the enzymatic activity of sulfatase RS2 as a function of

Temperature.

Example 14: Substrate synthesis, general procedure

Sodium sec- (±) -alkyl sulfates are obtained by sulfating the corresponding secondary alcohols with the addition of triethylamine-SO ₃ complexes in accordance with the synthesis instructions as described in White, GF et al., Biochem. J., 187, 191, 1980. 0.132 g of sodium hydride (5.52 mmol, 60% dispersion in mineral oil, washed with petroleum ether) are suspended in 2 ml of dioxane under an argon atmosphere. 2.48 mmol of the secondary alcohol are slowly added dropwise to this suspension through a septum. The mixture is stirred for one hour at room temperature. The sulfur trioxide-triethylamine complex (0.5 g, 2.76 mmol) is dissolved in 4 ml of anhydrous dioxane with heating. The solution obtained is then slowly added dropwise to the sodium alcoholate solution and stirred overnight. The reaction is terminated by adding distilled water. The solution is then completely evaporated in a rotary evaporator. The residue is taken up in 15 ml of distilled water and extracted 5 times with 10 ml of ethyl acetate. The water is then removed by lyophilization. The resulting powder is taken up in 30 ml of methanol and filtered off. The filtrate is freed from the solvent at reduced pressure.

1-Bromoctan-7-ol is synthesized according to Yadav, J.S. et al., Tedrahedron, 45, 6263-6270, 1989 and then sulfated as just described.

Claims

claims

1. Secondary alkyl sulfatases available from Actinomycetes.

2. Sulfatases according to claim 1 obtainable from Rhodococcus sp.

3. sulfatases according to claim 1 or 2 obtainable from Rhodococcus ruber, Rhodococcus equi or Rhodococcus sp. R 312.

4. sulfatases according to any one of the preceding claims, characterized in that the sulfatases in the denatured state have a molecular weight between 30 kDa and 60 kDa.

5. sulfatases according to claim 4, characterized in that they have a molecular weight of 41 to 45 kDa in the denatured state.

6. sulfatases according to any one of the preceding claims, characterized in that the sulfatases in the native state have a molecular weight between 60 and 80 kDa.

7. sulfatases according to any one of the preceding claims, characterized in that the sulfatases selected at least one amino acid sequence region from the group Seq ID No. 1 to 30 (sequences I to V) included.

8. Use of sulfatases according to any one of claims 1 to 7 in the manufacture of secondary alcohols or for the degradation of alkyl sulfate esters.

9. Use of sulfatases according to one of claims 1 to 7 for enantioselective resolution.

10. A process for the preparation of secondary alcohols, characterized in that secondary alkyl sulfatases according to one of claims 1 to 7 are used for the enzymatic hydrolysis of an alkyl sulfate ester.

11. The method according to claim 10, characterized in that iron (II) or iron (III) salts and / or CMAB are added to the reaction mixture as an additive.

12. The method according to any one of claims 10 or 11, characterized in that the secondary alkyl sulfatase is used immobilized on ecteola cellulose.

13. The method according to any one of claims 10 to 12, characterized in that the reaction is carried out in the presence of an organic solvent.

14. The method according to any one of claims 10 to 13, characterized in that a secondary alkyl sulfatase according to one of claims 1 to 7 is used for the hydrolysis of secondary (C-7-Cιo) alkyl sulfate esters.

15. Secondary alkyl sulfatases according to one of claims 1 to 7, obtainable from a cell-free actinomycete lysate by a) purification on a phenyl-Sepharose column with a falling sulfate-free salt gradient and subsequent b) chromatographic purification of the fractions obtained which show an enzymatic alkyl sulfatase activity, on an ion exchanger, after which c) the fractions obtained by ion exchange chromatography, which show enzymatic alkyl sulfatase activity, are chromatographed on Blue Sepharose, and d) the resulting fractions, which show enzymatic alkyl sulfatase activity, are filtered with Superdex 200.