JP2005508654A - Sulfatase and uses thereof - Google Patents

Abstract

  The present invention relates to novel alkylsulfatases from actinomycetes and their use, in particular in the case of catalytic reactions of disulfate esters.

Description

  The present invention relates to novel sulfatases from actinomycetes and their use, in particular to the catalytic reaction of disulfate esters.

Sulfatase is an enzyme that catalyzes the degradation of sulfate to inorganic sulfate and the corresponding alcohol by hydrolysis. In doing so, enzymatic hydrolysis of the sulfate ester proceeds according to the following general reaction scheme:
R—O—SO ₃ ⁻ + H ₂ O → R—OH + SO ₄ ²⁻ + H ⁺
At present, a number of sulfatases are known that can be classified in terms of their advantageous substrates into alkyl sulfatases, aryl sulfatases and carbohydrate sulfatases. In higher organisms, sulfatases mediate the hydrolysis of glycolipid sulfate, glycosaminoglycan sulfate or steroid sulfate in particular. In bacteria, on the other hand, providing sulfur-containing compounds that store energy for the assimilation of sulfur from the environment and possibly cell growth plays an important role.

The most characteristic group of sulfatases are highly conserved arylsulfatases that accept aromatics, especially phenolic substrates. Some arylsulfatases, such as arylsulfatases that can be isolated from human cells, certainly do react with carbohydrate sulfates. The is characteristic for arylsulfatase serine - semialdehyde units, C _alpha in the active center of other words enzymes - a formylglycine (Fglyc) unit, which can be formed from a post-translational modification cysteine group or serine group . This post-translational modification is important for the biochemical function of the enzyme. Microbial arylsulfatases belong to the so-called “sulfate starvation-induced proteine” (SSI-proteins), which are cultured while exchanging sulfur sources such as arylsulfonates, alkyl sulfides, thioethers, etc. Can be formed.

  The class of alkyl sulfatases that catalyze the hydrolysis of organic sulfates of primary and secondary alcohols is much less characteristic. This is even more surprising in the context of large amounts of alkyl sulfate being released into the environment every year as an ingredient from household products (eg detergents) and being degraded 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 presence of special 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).

  Conventionally the most characteristic alkylsulfatase is derived from the Gram-negative bacterial strains Pseudomonas C12B (NCIMB 11753 = ATCC 43648) and Comamonas trigena (NCIMB 8193) (Dodgson, KS et al., Sulfatases of Microbial Origin, 2 vols ., CRC Press, Inc., Boca Raton (1982)). Pseudomonas C12B can express five different alkyl sulfatases depending on the culture conditions suitable for hydrolysis of primary or secondary alkyl sulfates (Dodgson, KS, et al., Biochem. J). 138, 53-62 (1974); Bartholomew, B., et al., Biochem. J. 169, 659-667 (1978); Cloves, JM, et al., Biochem. J. 185, 13-21 ( 1980)). In contrast, Comamonas-trigena 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)).

  Further conventionally, Pseudomonas-Putida FLA, filth aerobic bacteria (Payne, WJ, et al., Nature 214, 623-624 (1967)) and possibly also Pseudomonas B-2 (Lijmbach, GWM, et al., J. Microbiol) Serol. 39, 415 (1973)) was able to detect alkylsulfatase activity. Furthermore, alkylsulfatase activity could be confirmed even in mixed culture consisting of microorganisms isolated from active suspension (Vacium, L. et al., Res. Roum. Biochim. 2, 149 (1976)).

  Previously, the only Gram-positive bacterial strains were Bacillus cereus (Singh, KL, et al., World J. Microbiol. Biotechnol. 14, 777-779, (1998)) and coryneform species B1a (Payne, WJ et al. ., Appl. Microbial 13, 698 (1965)) had the first alkylsulfatase activity.

  Previously, it was possible to purify the alkyl sulfatase until it was actually homogeneous only in individual cases. In contrast, there has been little recognition in the past regarding their structure, substrate specificity or their properties. For these reasons, alkyl sulfatase has not been used in organic synthesis, for example, but it is a valuable tool for resolution of racemates, for example, in the enantioselective production of secondary alcohols. obtain. Thus, the technical potential of alkyl sulfatase was hardly developed.

  Starting from this, the object of the present invention is to provide a novel alkylsulfatase which can be used as an enzymatic tool in the degradation of alkyl sulfates, for example.

  However, the majority of prokaryotic and eukaryotic microorganisms tested for alkyl sulfatases known for significant secondary substance metabolism show no activity for hydrolysis of secondary alkyl sulfates (Table 1).

  Surprisingly, it was found that secondary alkyl sulfatase is expressed in actinomycetes, especially Rhodococcus, in the opposite of other Gram-positive bacteria. Furthermore, these secondary alkyl sulfatases can be purified to homogeneity, in which case the enzyme does not lose its enzymatic activity. A secondary alkyl sulfatase thus obtained from actinomycetes, preferably Rhodococcus sp., Is the subject of the present invention.

Enzymatically active secondary alkyl sulfatases can be isolated from each actinomycete strain by performing the following purification steps:
a) crude purification of cell-free lysate using a phenyl-sepharose column with a sulfate-free decreasing salt gradient;
b) further purifying the obtained fraction having enzymatic alkyl sulfate activity with an ion exchange resin, preferably an anion exchange resin;
c) The fraction having enzymatic alkyl sulfatase activity obtained by ion exchange chromatography is passed through a blue sepharose column, and d) the enzyme obtained from the fraction having enzymatic alkyl sulfatase activity obtained subsequently using a Sperdex200 column. And purify until homogeneous.

  In that case, it is advantageous to treat the cell-free lysate beforehand with DEAE-, TEAE- or Ecteola cellulose or with DEAE-Sephadex A-25 for the separation of non-peptide components. The secondary alkyl sulfatase is then bound to the support and is dissolved from the support for subsequent purification using a salt-containing buffer.

A purification pattern that has proven particularly advantageous for isolating secondary alkyl sulfatases from Rhodococcus comprises the following method steps:
a) treatment of cell lysates of actinomycetes with DEAE cellulose in a batch process to separate carotenoids, lipids, etc.,
b) Elution of the enzyme with a solution consisting of 10 mM Tris / HCl and 0.5 M NaCl,
c) A phenyl-Sepharose column equilibrated with 10 mM Tris / HCl buffer, pH 7.5 (buffer B) containing NaCl and 4 M NaCl added to the eluent to a final concentration of 4 M NaCl. Purification of the resulting solution by column chromatography, using a stepwise NaCl gradient from 4M to 0M for elution of the enzyme. Then for further purification
d) dialyzing the enzymatically active fraction against 10 mM Tris / HCl buffer, pH 7.5;
e) Ion exchange chromatography on a Q6-column (BioRad), where the column is equilibrated with 10 mM Tris / HCl buffer, pH 7.5 (buffer A) and for elution from 0 M to 1 M A NaCl gradient is utilized. The gradient can be performed, for example, by stepwise addition of a buffer mixture consisting of buffer A and buffer C (10 mM Tris / HCl, pH 7.5, 1 M NaCl). Subsequent purification is
f) Apply the enzymatically active fraction to a blue sepharose column (Pharmacia), where the enzymatically active fraction is loaded and eluted in 10 mM piperazine-buffer, pH 6.0. The column is subsequently further washed with Tris / glycine-buffer, pH 8.9.

g) Perform by purification to homogeneity using a Superdex 200 column with a NaCl concentration of 0.15 M using 50 mM NaH ₂ PO ₄ buffer, pH 7.0.

  For the determination of enzymatic activity fractions, enzymatic hydrolysis of secondary alkyl sulfates can be used, with the amount of sulfate or alcohol released being determined according to the enzymatic activity of the individual fractions. Can be measured as a measure for. The first method is described, for example, in Dodgson, K. S., Biochem. J., 78, 312-319, 1961; Tudball, N., et al., Biochem. J. 126, 187-191, 1972.

  The secondary alkyl sulfatase from actinomycetes according to the invention thus isolated preferably has a molecular weight of 30-60 kDa in a modified form and / or a molecular weight of 60-80 kDa in the native state. Particularly advantageous are secondary alkyl sulfatases having a molecular weight of 41-45 kDa in the denatured state and / or a molecular weight of 65-69 kDa in the native state. The molecular weight in the denatured state is measured by gel electrophoresis using the SDS-PAGE method.

  For example, secondary alkyl sulfatases RS1 and RS2 can be isolated from Rhodococcus luber DSM 44541 by the methods described. The molecular weight of the modified secondary alkylsulfatase RS2 is measured by SDS-PAGE and is 43.3 kDa. This enzyme has a molecular weight of 67 kDa as measured by eluting with Superdex 200 in its native state, which may indicate the presence of a dimer of the enzyme. The isoelectric point of enzyme RS2 is 5.1.

For further characterization of the secondary alkyl sulfatase RS2 from Rhodococcus luber DSM 44541, the enzyme was tested by mass spectrometry. In doing so, individual characteristic peptide fragments could be detected using electrospray-tandem mass spectrometry. For this purpose, for example, the sulfatase fraction separated by gel electrophoresis was isolated and digested with trypsin. The resulting peptide mixture was analyzed by ES MS / MS. Thus, the following five peptide fragments can be detected for the second alkyl sulfatase RS2 from Rhodococcus luber DSM 44541:
[L, I] TAT [L, I] [L, I] NN [L, I] QMPPR Sequence I (SEQ ID NOs: 1 to 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 NOs: 23 to 30)
([L, I] = leucine or isoleucine)
In this case, the fragment sequences I and II do not show significant homology to other proteins. Sequences III and V are homologous to a putative thiolase from Streptomyces coelicolor A3 (2), and sequence V is further homologous to a thiolase segment from Mycobacterium tuberculosis. And sequence IV is homologous to a putative acetyl-CoA-acetyltransferase from Streptomyces venezuela. An advantageous subject of the present invention is therefore a second alkyl sulfatase from actinomycetes having at least one amino acid sequence region according to sequences IV.

  The secondary alkyl sulfatase according to the present invention can be isolated not only in an enzymatically active state, but is also superior in its good stability, whereby the sulfatase is capable of enzymatic-organic hydrolysis of sulfate esters. Suitable for use in decomposition. In this case, possible uses are, for example, use in the decomposition of sulfate esters from purification equipment and / or detergents. Alkylsulfatases, for example, maintain their enzymatic activity in the presence of organic solvents over a wide pH value and temperature range. A pH value range of pH 5.5 to pH 10 and a reaction temperature lower than 40 ° C. are advantageous.

  In addition, numerous surfactants, sugars and polyalcohols have little effect on the activity of the enzyme. The effect of most salts also has no significant effect on the hydrolytic activity of the alkyl sulfatase being tested. In contrast, some of these additives have a positive impact on the enzymatic hydrolysis reaction rate or enantioselectivity of alkyl sulfates.

  Furthermore, the enzyme can be ionically immobilized without problems and thereby be removed from the reaction batch and reused, in which case the sulfatase does not lose its enzymatic activity. Particularly suitable for immobilization are anionic ion exchange resins such as DEAD-cellulose, TEAE-cellulose, Ecteola cellulose or DEAE Sephadex A-25. Immobilization takes place particularly effectively at a pH value of 7.5. For example, a 0.1 M Tris buffer can be used as the buffer.

  This results in another subject of the invention, namely the use of the claimed secondary alkyl sulfatase for the enzymatic hydrolysis of alkyl sulfates or for the enzymatic production of secondary alcohols.

  Thus, for example, the secondary alkyl sulfatases RS1 and RS2 from Rhodococcus luber DSM 44541 can be utilized for the production of secondary alcohols. The substrate is preferably reacted with a secondary alkyl sulfate having a carbon chain of 7 to 10 carbon atoms. A carbon chain length of 8 carbon atoms is particularly advantageous. Furthermore, sulfate esters in which the sulfate ester bond is present in the 2-position, 3-position or 4-position of the esterified alcoholic carbon chain are preferred.

  When using a secondary alkyl sulfatase RS2 for the hydrolysis of the sulfate ester, preferably a temperature of 15 ° C. to 36 ° C., particularly preferably 27 ° C. to 32 ° C. in the pH value range of pH 7.0 to pH 8.5. Work with.

  Furthermore, it has been found that when hydrolyzing alkyl sulfates with the sulfatase according to the invention, the reaction is advantageous under sulfate-free conditions or at very low sulfate concentrations. Substantially sulfate-free conditions can occur, for example, by adding barium ions to the reaction batch.

  The secondary alkyl sulfatase can further catalyze the hydrolysis of the secondary alkyl sulfate ester enantioselectively. Enantioselectivity can be improved by adding other additives such as iron (II) ions or iron (III) ions at low concentrations. In doing so, concentrations of 1 mM to 15 mM for iron (II) ions and 3 mM to 6 mM for iron (III) ions have proved to be advantageous, especially when using secondary alkyl sulfatases from Rhodococcus. Addition of cetyltrimethylammonium bromide (CMAB) can also significantly improve the enantioselectivity of the hydrolysis.

  Besides using a free secondary alkyl sulfatase in the reaction batch, an immobilized secondary alkyl sulfatase can also be used. For this purpose, the alkyl sulfatase according to the invention can be fixed, for example, on an anionic carrier material such as DEAE-cellulose, DEAE-Sephadex A-25, TEAE-cellulose or Ecteola-cellulose. A particularly suitable carrier is then Ecteola-cellulose. This is because the alkylsulfatase immobilized on the cellulose exhibits high enantioselectivity.

  Secondary alkyl sulfatases are therefore particularly suitable for producing secondary alcohols from sulfate esters. Of particular interest is, for example, a process for enantioselective preparation of chiral secondary alcohols during racemic resolution.

  In this case, depending on the type of attack by the enzyme, hydrolysis of the alkyl sulfate can be performed while maintaining the configuration or inversion. Secondary alkyl sulfatases RS1 and RS2 isolated from Rhodococcus luber DSM 44541 may, for example, stereoselectively configure (R)-(−)-2-octyl sulfate to (S)-(+)-2-octanol. Invert and hydrolyze.

  Several examples are described below to clarify the present invention.

Example 1: Cultivation of Rhodococcus luber The culture of Rhodococcus luber cells of strain DSM 44541 was 10 g / l of glucose, 10 g / l of peptone, 10 g / l of yeast extract, 1.5 g of MgSO ₄ .7H ₂ O, It is carried out under aerobic conditions in a liquid medium consisting of 1.3 g / l NaH ₂ PO ₄ and 4.4 g / l K ₂ HPO ₄ . The culture is shaken in a flask with baffles at 30 ° C. and 130 rpm, and cell growth is followed by measuring the optical density at an absorption wavelength of 546 nm.

Example 2: Purification of secondary alkylsulfatase To destroy the cells, 47 g (wet mass) of Rhodococcus luber DSM 44541 cell paste is suspended in 120 ml of Tris buffer (10 mM, pH 7.5). 120 ml of glass beads having a diameter of about 0.35 mm are added to the suspension. The cells of the suspension are broken at a temperature below 4 ° C. using a vibrating mill with an external ice / water-cooling device. In that case, the destruction of the cells is carried out four times in a cycle consisting of a 2-minute vibration phase followed by a 5-minute cooling. After filtering the glass beads, the cell lysate is centrifuged at 4 ° C. and 38000 × g for 2 hours. The centrifuged cell lysate is treated with DEAE-cellulose in a batch process for separation of carotenoids, lipids, etc., where alkylsulfatase is bound to the DEAE-cellulose support. The elution of alkyl sulfatase is performed using a solution consisting of 10 mM Tris / HCl and 0.5 M NaCl. The crude extract of enzyme thus prepared is added with stirring and ice-cooling until a final concentration of 4M is achieved. Thereafter, the crude extract of the enzyme is purified by column chromatography through a phenyl-Sepharose column (Pharmacia V = 20 ml), with the column first being 10 mM Tris / HCl, 4 M NaCl buffer, pH 7.5 ( Equilibrate with buffer B). Elution is performed by gradually decreasing the NaCl concentration. For this, a mixture of buffer B and buffer A (10 mM Tris / HCl, pH 7.5) is used. In doing so, two types of secondary alkyl sulfatases from Rhodococcus luber DSM 44541 (RS1 and RS2) can be detected, sulfatase RS1 eluting at 1M NaCl concentration (buffer B 25%, buffer A 75%), The more lipophilic sulfatase RS2 elutes at a NaCl concentration of 0M (buffer B 0%, buffer A 100%).

Each time the resulting sulfatase fraction was dialyzed against 10 mM Tris / HCl-buffer, pH 7.5, the enzymatically active fraction was equilibrated with 10 mM Tris / HCl buffer, pH 7.5. Further purification using (BioRad, V = 6 ml). The elution of the protein component of the fraction used is carried out with 10 mM Tris / HCl-buffer, pH 7.5, using a stepwise NaCl gradient from NaCl 0M to 1M. Desorption of the secondary alkyl sulfatases RS1 and RS2 is performed at a NaCl concentration of 0.15-0.31M. The fraction containing RS1 and RS2 is subsequently applied to a Blue Sepharose column (Pharmacia) in 10 mM piperazine buffer, pH 6.0. Neither RS1 nor RS2 binds to the column material under the given conditions and can therefore be recovered immediately. The column is subsequently washed off with Tris / glycine buffer, pH 8.9. The resulting enzymatically active fraction is concentrated to 1 ml (Centriplus 10, Amicon) and loaded onto a Superdex 200-column. Equilibration of the column and elution of the respective sulfatase is performed using 50 mM NaH ₂ PO ₄ , 0.15 M NaCl-buffer, pH 7.0.

The progress of the individual purification steps is shown in FIG. FIG. 1 shows an SDS-PAGE (12%) analysis of a protein containing sample:
Zone A represents a crude extract of cells.
Zone B shows the sample after treatment with DEAE-cellulose.
Zone C shows the sample after chromatography using phenyl-Sepharose.
Zone D shows the sample after ion exchange chromatography using Q6.
Zone E shows the sample after chromatography using blue sepharose.
Zone F shows a sample of purified secondary alkylsulfatase RS2 after purification using Superdex200.
Band G shows molecular weight markers.
Samples were stained with Coomassie Brilliant Blue R.

Example 3: Assay to measure enzymatic activity a) Determination of the amount of sulfate ion liberated by the enzyme Dodgson, KS et al., Biochem. J., 78 to measure the sulfate ion liberated by the enzyme , 312-319, 1961, which was modified by Tudball, N. et al., Biochem. J. 126, 187-192, 1972. In this case, 200 μl of alkylsulfatase solution is incubated with 200 μl of 30 mM 3-octyl sulfate solution. 0.1M Tris / HCl-buffer, pH 7.5 is used. The reaction is carried out at a temperature of 30 ° C. and is interrupted after 15 minutes by addition of 15% trichloroacetic acid (w / v). After a brief centrifugation, a 200 μl sample of the reaction solution is used for sulfate measurement with Tudball.

  When 2-octyl sulfate is used as a substrate for detecting enzymatic activity, the reaction time is extended by 1-2 hours.

Enzyme activity units (= units) are defined as the amount of enzyme that liberates 1 μmol SO ₄ ²⁻ ions per minute.

b) Determination of the amount of alcohol liberated by the enzyme Chromatographic treatment using fully or incompletely purified enzyme solution obtained by ion exchange chromatography or phenyl-sepharose to determine enzymatic activity Add 400 μl of the 30 mM substrate solution in the same buffer to 400 μl of the lyophilized 0.1 M Tris / HCl buffer, pH 7.5 resuspended sulfatase powder. The mixture is shaken at 24 ° C. and 130 rpm depending on the reaction process for 6-12 hours.

Example 4: Determination of reaction rate A 200 μl aliquot of the reaction mixture was extracted with 200 μl of ethyl acetate. After thoroughly mixing the reaction batch for 0.5 minutes and subsequently centrifuging the mixture at 13000 rpm for 5 minutes, a 100 μl sample of the supernatant organic phase was added to a stock solution of menthol in ethanol (c = 15.4 mg / ml) Add 5 μl and use this as internal standard. The reaction rate was measured by GC using an achiral column (HP1301, cyanopropylphenylmethylpolysiloxane 6%, 30 m × 0.25 mm × 0.25 μm (film) and HP-1, 30 m × 0.53 mm × 0. 5 μm; N ₂ ), maintaining the temperature at 95 ° C. for 2.5 minutes, then increasing the temperature to 110 ° C. at a heating rate of 10 ° C./min. The temperature program described is tuned for 2-, 3- and 4-octanol measurements.

Example 5: Determination of Enantiomeric Excess Enantiomeric excess (ee) was measured using a chiral Chrompack CP 7500 column (cyclodextrin-B-2, 25 m x 0.25 mm x 25 μm) and Chrompack Chirasil-Dex CB / G-PN (γ -Using a cyclodextrin propionyl column, 30 m x 0.32 mm) using hydrogen as the carrier gas. The (R)-or (S) -alcohol is identified by co-injection of the corresponding chiral alcohol.

Induction of 2-alkanol The alcohol is derivatized with trifluoroacetic anhydride (TFA) to determine the enantiomeric excess. This achieves improved separation of enantiomers using a chiral GC-column. For this purpose, 400 μl of the reaction mixture reacted with the enzyme are extracted with 500 μl of dichloromethane and dried over anhydrous sodium sulfate. After the addition of 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 carbonate solution. The sample is then analyzed by gas chromatography after the organic phase has been dried over anhydrous sodium sulfate.

  To avoid workup of the sodium bicarbonate extract, the sample was alternatively treated with a solution of N-methyl-bis-trifluoroacetamide (1: 3 v / v) in dichloromethane for 1 hour at 40 ° C. And can subsequently be tested directly by gas chromatography.

  Another alternative is the gas chromatographic analysis of the corresponding acetate. For this purpose, 400 μl of the reaction solution reacted with the enzyme is extracted with 400 μl of ethyl acetate. The organic phase is dried over anhydrous sodium sulfate. After adding 80 μl acetic anhydride and a catalytic amount of p-dimethylaminopyridine, the solution is shaken overnight at room temperature. The solution is then extracted twice with 0.5 ml of water and the organic phase is subsequently dried over anhydrous sodium sulfate and analyzed.

  Note that the trifluoroacetate is eluted in reverse order relative to the acetate ester.

Example 6: Enzymatic hydrolysis of secondary alkyl sulfates using RS2 The substrate to be tested is reacted as described in Example 3. The results of the reaction are listed in Table 2.

The results in Table 2 indicate that the secondary alkyl sulfatase that can be isolated by the described method has a high specificity for potential substrates. In the case of the second alkyl sulfatase RS2 from Rhodococcus luber DSM 44541, a suitable substrate is a linear aliphatic second (C ₇ -C ₁₀ ) alkyl sulfate ester without another substituent. Such esters react with good yield and good enantioselectivity. RS1 has the same substrate specificity as RS2.

Example 7: Effect of salt The reaction described in Example 3 is carried out with the addition of the additives to be tested each time. The results are summarized in Tables 3-5. Surprisingly, it has now been found that the selectivity of the second alkylsulfatase according to the invention can be improved by adding iron ions, preferably by adding Fe ²⁺ ions. Thus, for example, the enantioselectivity of RS2 relative to rac-3-octyl sulfate is E = 4 to E = 80 (Table 3) by addition of FeCl ₂ and for rac-4-octyl sulfate, E = 1 to E = 10 (Table 4). This is surprising as neither the addition of EDTA nor the addition of mercaptoethanol and dithiothreitol affects the activity of alkylsulfatase.

Example 8: Effect of surfactant The reaction is carried out as described in Example 3 with the addition of the surfactant to be tested each time. The results are summarized in Table 6. The effect of the surfactant on the activity of the claimed secondary alkyl sulfatase varies depending on the surfactant used. With the addition of SDS or di-n-octylsulfosuccinate, the enzyme loses its activity almost completely. In contrast, many surfactants have little or no effect on the reaction rate, but some surfactants still have enantioselectivity for enzymatic reactions. Even has an advantageous effect on the table (Table 6). Thus, for example, an improvement in the enantioselectivity of the hydrolysis of the sulfate ester can be achieved by the addition of CMAB.

Example 9: Effect of other additives The reaction is carried out with the respective additive to be tested as described in Example 3. The results are summarized in Table 7. The effects of different sugars and polyalcohols on the reaction rate of secondary sulfates by secondary alkyl sulfatases according to the invention and the enantioselectivity of enzymatic hydrolysis are very slight, with some exceptions (eg CMAB) (Tables 7 and 8).

Example 10: Effect of carrier on immobilized enzyme A solution of a sulfatase preparation (11 mg / ml) in 0.1 M Tris / HCl-buffer, pH 7.5 is buffered with 0.1 M Tris / HCl, pH 7.5. Add to 100 mg of immobilization carrier (DEAE-, TEAE-, Ecteola-cellulose (Servia, Heidelberg), DEAE-Sephadex A-25) pre-washed with liquid. The batch is carefully mixed for 5 minutes and left at 4 ° C. for 30 minutes. The batch is then centrifuged, the supernatant removed, and the immobilized support washed briefly with 0.1 M Tris / HCl-buffer, pH 7.5, and tested as in Example 3 for alkylsulfatase activity. . The results are summarized in Table 9.

  Therefore, Ecteola-cellulose has an advantageous effect on the reaction rate, and in particular on the enantioselectivity of the hydrolysis of alkyl sulfates, as an anionic carrier material for immobilizing secondary alkyl sulfatases. Prove.

Example 11 Effect of Organic Solvent The reaction of rac-2-octyl sulfate (5% (v / v)) is carried out with RS2 in various organic solvents at 30 ° C. The results are listed in Table 10. ing.

  It has been found that another advantage of the secondary alkyl sulfatase according to the present invention is stable without significant loss of activity in the presence of organic solvents. An example of an organic solvent that is particularly suitable for the enantioselective reaction of secondary alkyl sulfates is t-BuOH.

Example 12: Barium ion addition The effect of Ba ^{2+ on} the enzymatic activity of the secondary alkyl sulfatase RS2 was determined using rac-2 as a substrate in 0.1 M Tris / HCl-buffer at a pH value of 7.5 and a temperature of 24 ° C. -Measure using 15 mM octyl sulfate ester.

  FIG. 2 shows the reaction rate of the secondary alkyl sulfate ester depending on the reaction time with and without addition of barium ions to the reaction batch.

Example 13: Biochemical characterization of secondary alkyl sulfatase RS2 from Rhodococcus luber DSM 44541 a) Determination of molecular weight The molecular weight of RS2 was determined by SDS-polyacrylamide gel-electrophoresis (SDS-PAGE). A reexamination of the molecular weight confirmed using SDS-PAGE is performed based on the size of the enzyme using a Superdex 200 column. Calibration is performed using chymotrypsinogen A (25 kDa), ovalbumin (43 kDa), BSA (67 kDa), alcohol dehydrogenase (150 kDa) and blue dextran (2000 kDa) as molecular weight standards.

  Measurement of molecular weight by SDS-polyacrylamide-gel electrophoresis under denaturing conditions resulted in a molecular weight of 43.3 kDa for the second alkylsulfatase RS2. Elution of the enzyme purified by Superdex 200 resulted in a molecular weight of approximately 67 kDa, suggesting a non-spherical or dimeric structure of the enzyme.

b) Measurement of isoelectric point (pl) The isoelectric point of the second alkylsulfatase RS2 was measured using a BioRad IEF Ready Gel System at a pH value of pH 3 to pH 10. A pi measurement of RS2 yielded a value of 5.1.

c) Determination of the optimum pH The optimum pH of sulfatase RS2 is measured in a pH range of 6.0-9.0 at 24 ° C using 15 mM rac-2-octyl sulfate as substrate in 100 mM Tris / maleate buffer. It was measured. The maximum activity value of the secondary alkylsulfatase RS2 is a pH value of 7.5 to 8.0.

  FIG. 3 shows the enzymatic activity of sulfatase RS2 depending on the pH value.

d) Measurement of optimum temperature The second alkylsulfatase RS2 in a temperature range of 20 ° C. to 40 ° C. at a pH value of 7.5 in 100 mM Tris / maleate buffer using 15 mM rac-2-octyl sulfate as substrate. The effect of temperature on the enzymatic activity of was measured. The enzymatic activity of RS2 for 2-octyl sulfate shows an optimum value at about 30 ° C.

  FIG. 4 shows the temperature-dependent enzymatic activity of sulfatase RS2.

Example 14: Substrate synthesis, general method definition Sodium sec- (±) -alkyl sulfate is prepared according to the synthesis method described for example in White, GF et al., Biochem. J. 187, 191, 1980. By adding a triethylamine-SO ₃ -complex and sulfating the corresponding secondary alcohol. In that case, 0.132 g of sodium hydride (5.52 mmol, 60% dispersion in mineral oil, washed with petroleum ether) is suspended in 2 ml of dioxane under an argon atmosphere. To this suspension, 2.48 mmol of secondary alcohol is slowly added dropwise via a septum. The mixture is stirred at room temperature for 1 hour. Sulfur trioxide-triethylamine complex (0.5 g, 2.76 mmol) is dissolved in 4 ml of anhydrous dioxane under heating. The resulting solution is subsequently slowly added dropwise to the sodium alcoholate solution and stirred overnight. The reaction is interrupted 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. Subsequently, water is removed by lyophilicity. The resulting powder is taken up in 30 ml of methanol and filtered off. The solvent is removed from the filtrate under reduced pressure.

  1-Bromooctane-7-ol is prepared synthetically by Yadav, J. S. et al. Tetrahedron, 45, 6263-6270, 1989 and sulfated as described subsequently.

Show the progress of individual purification steps Shows the reaction rate of secondary alkyl sulfates depending on the reaction time with and without addition of barium ions to the reaction batch Shows enzymatic activity of sulfatase RS2 depending on pH value Shows temperature-dependent enzymatic activity of sulfatase RS2

[Sequence Listing]

Claims (15)

  A secondary alkylsulfatase obtained from actinomycetes.   The sulfatase according to claim 1, obtained from Rhodococcus sp.   A sulfatase according to claim 1 or 2 obtained from Rhodococcus luber, Rhodococcus equi or Rhodococcus sp. R312.   The sulfatase according to any one of claims 1 to 3, which has a molecular weight of 30 kDa to 60 kDa when the sulfatase is denatured.   The sulfatase according to claim 4, which has a molecular weight of 41 kDa to 45 kDa when the sulfatase is denatured.   The sulfatase according to any one of claims 1 to 5, wherein the sulfatase has a molecular weight of 60 to 80 kDa in a native state.   The sulfatase according to any one of claims 1 to 6, wherein the sulfatase has at least one amino acid sequence region selected from the group of SEQ ID NOs: 1 to 30 (sequences I to V).   Use of a sulfatase according to any one of claims 1 to 7 in the production of secondary alcohols or for the degradation of alkyl sulfates.   Use of a sulfatase according to any one of claims 1 to 7 for enantioselective racemic resolution.   A method for producing a secondary alcohol, wherein the secondary alkyl sulfatase according to any one of claims 1 to 7 is used for hydrolyzing an alkyl sulfate by an enzyme.   11. The process according to claim 10, wherein iron (II) salt or iron (III) salt and / or CMAB are added as additives to the reaction batch.   The method according to claim 10 or 11, wherein the secondary alkyl sulfatase is used by being fixed to Ecteola cellulose.   13. A process according to any one of claims 10 to 12, wherein the reaction is carried out in the presence of an organic solvent. Claims 1 to the second alkyl sulfatase as described in any one of up to 7 second (C 7 _-C 10) _- used for the hydrolysis of alkyl sulfates, any of claims 10 to 13 1 The method described in the paragraph. From a cell-free actinomycete lysate a) Purification with a phenyl-sepharose column without sulfate, with a decreasing salt gradient and subsequently b) The resulting fraction with enzymatic alkylsulfatase activity using an ion exchanger C) purification by chromatography, followed by c) chromatography of the fraction with enzymatic alkylsulfatase activity obtained by ion exchange chromatography with blue sepharose, and d) having enzymatic alkylsulfatase activity The second alkylsulfatase according to any one of claims 1 to 7, which is obtained by filtering the obtained fraction using Superdex200.

Images