CN1592783A - Sulphatases and use thereof - Google Patents

Abstract

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

Description

Sulfatase and use thereof

The present invention relates to novel sulfatases from actinomycetes (actinomycetes) and their use, in particular to their use in the enzymatic conversion of secondary sulfates.

Sulfatase is an enzyme that catalyzes the hydrolysis of sulfate esters to inorganic sulfuric acid and the corresponding alcohol. The enzymatic hydrolysis of sulfates proceeds according to the following general reaction scheme:

it is known that a large number of sulfatases can be classified according to their preferred substrates into alkyl sulfatases, aryl sulfatases and carbohydrate sulfatases. In higher organisms, sulfatases mediate in particular the hydrolysis of glycolipids, glycosaminoglycans, or steroid sulfates. On the other hand, in bacteria, it plays an important role in absorbing sulfur from the environment and supplying energy-storing sulfur-containing compounds presumably for cell growth.

The best group of sulfatases described are the very conserved aryl sulfatases that accept aromatic, especially phenolic, substrates. However, some aryl sulfatases, such as those isolated from human cells, preferably convert carbohydrate sulfates. The aryl sulfatase is characterized by having a serine semialdehyde unit, C, at the active center of the enzyme_αA formylglycine (Fglyc) unit, which can be formed by a post-translationally modified cysteine or serine residue. This post-translational modification is essential for the biochemical function of the enzyme. Bacterial aryl sulfatases belong to the so-called "sulfur starvation-induced proteins" (SSI proteins), which can be formed, for example, by using cultures containing alternative sulfur sources such as aromatic sulfuric acids, alkyl sulfides, thioethers, and the like.

Very few alkyl sulfatases have been described which catalyze the hydrolysis of organic sulfates of primary and secondary alcohols. Even more surprising is the annual release of large amounts of alkyl sulfates as constituents of living goods, such as detergents, into the environment and degradation by microorganisms (Y.C.Hsu, Nature200, 1091-1092 (1963); Williams, J. et al, appl. Microbiol.12, 360-362 (1964); White, G.F. et al, environ. polar. Ser.A.37, 1-11 (1985)).

The presence of specific alkylsulfatases was first described in areview by Dodgson, K.S. et al, surfactants of Microbial Origin, Vol.2, CRC Press, Boca Raton (1982).

The alkylsulfatases which are best described at present originate from the gram-negative strains Pseudomonas (Pseudomonas) C12B (NCIMB 11753 ═ ATCC 43648) and Comamonas terrestris (Comamonas terrigena) (NCIMB 8193) (Dodgson, K.S. et al, Sulfatasoff Microbial Origin, Vol.2, CRC Press, Boca Raton (1982)). Pseudomonas sp C12B can express up to 5 different alkylsulfatases depending on the culture conditions, which are suitable for hydrolyzing primary or secondary alkylsulfates (Dodgson, K.S. et al, biochem. J.138, 53-62 (1974); Bartholomew, B. et al, biochem. J.169, 659-667 (1978); Cloves, J.M. et al, biochem. J.185, 13-21 (1980)). In contrast, Comamonas terrestris has only two secondary alkylsulfatases (Fitzgerald, J.W. et al, biochem.J.149, 477-.

In addition, it is now possible to demonstrate the alkyl sulfatase activity in Pseudomonas putida (Pseudomonas putida) FLA, Aerobacter cloacae (Aerobacter cloacae) (Payne, W.J., et al, Nature 214, 623-624(1967)) and to speculate to be present in Pseudomonas B-2 (Lijmbach, G.W.M., et al, J.Microbiol.Serol.39, 415 (1973)). Furthermore, it is possible to detect alkyl sulfatase activity in mixed cultures of microorganisms isolated from active suspensions (Vacium, l. et al, res. roum. biochim.2, 149 (1976)).

Currently, the only gram-positive bacterial strains with primary alkylsulfatase activity are Bacillus cereus (Singh, K.L. et al, World J. Microbiol. Biotechnol.14, 777-779(1998)) and Corynebacterium Bla (Corynebacterium sp. Bla) (Payne, W.J. et al, appl. Microbiol.13, 698 (1965)).

At present, the alkylsulfatase can be purified homogeneously in virtually only individual cases. To date, their structure, substrate specificity or their properties are poorly understood. For these reasons, alkylsulfatases are not currently used, for example in organic synthesis, although these enzymes may be useful tools in enantioselective preparation of secondary alcohols, for example in the cleavage of racemates. The industrial potential of alkylsulfatases has therefore rarely been exploited at all.

Starting from this, it was an object of the present invention to provide novel alkylsulfatases which can be used, for example, as enzymatic tools for cleaving alkylsulfates.

However, most prokaryotic and eukaryotic microorganisms known to have significant secondary metabolism and tested alkylsulfatase do not exhibit activity in hydrolyzing secondary alkylsulfates (table 1).

On the other hand, it was found that, surprisingly, unlike other gram-positive bacteria, secondary alkylsulfatases are expressed in actinomycetes, in particular in Rhodococcus (rhodococcus). In addition, these secondary alkylsulfatases can be purified homogeneously without the enzyme losing activity. The present invention provides secondary alkylsulfatases from actinomycetes, preferably from Rhodococcus.

Table 1: the results of a search for secondary alkylsulfatases using rac-2-octyl sulfate as detection substrate in gram-positive bacteria and fungi.

Bacterial strains	2-octanonesa	2-octanola	Transformation%b
				Rhodococcus ruber (Rhodococcus ruber) DSM44541	1	1.7	23
Rhodococcus ruber DSM 44539	1	17.8	22
				Rhodococcus ruber DSM 44540	1	15.4	22
Rhodococcus ruber DSM 43338	1	1.1	31
				Rhodococcus equi (Rhodococcus equi) IFO 3730	1	1	35
Rhodococcus (Rhodococcus sp.) R312(CBS 717.73)	1	2.8	33
				Rhodococcus sp NCIMB 11216	n.d.	n.d.	low
Streptomyces lavendulae (Streptomyces lavendale) ATCC 55209	--	--	n.c.
				Bacillus megaterium DSM 32	--	--	n.c.
Mycobacterium paraffinum (Mycobacterium paraffinicum) NCIMB 10420	--	--	n.c.
				Acremonium rubrum (Mycoplana rubra) R14(SM 73)	--	--	n.c.
Arthrobacter sp DSM 312	21	1	4
				Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC 13032	--	--	n.c.
Beauveria bassiana (Beauveria bassiana) ATCC 7159	--	--	n.c.
				Cunninghamella blakesleeana DSM 1806	--	--	n.c.
Helminthosporium sp (NRRL 4671)	--	--	n.c.
				Mortierella alpina (Mortierella alpina alpin)a) ATCC 8979	--	--	n.c.
Mucor plumbeus CBS 110.16	--	--	n.c.
				Syncephalastrum racemosum ATCC 18192	--	--	n.c.

n.d. ->without data

n.c. ═ no conversion

^aRelative molecular weight

^b([ 2-octanol)]2-octanone]) /[ 2-octane sulfate])

The enzymatically active secondary alkylsulfatase may be isolated from a particular actinomycete strain by the following purification steps:

a) crude purification of cell-free lysates was performed by phenyl sepharose column with decreasing sulfate-free gradient,

b) the resulting fraction having alkylsulfatase activity is further purified on an ion exchange resin, preferably an anion exchange resin,

c) passing the fraction having alkylsulfatase activity obtained by ion exchange chromatography through a Blue agarose column, and

d) the enzyme in the obtained active component with the alkyl sulfatase is purified uniformly by a Superdex200 column.

In carrying out the above procedure, it is advantageous to treat the cell-free lysate beforehand with DEAE, TEAE or Ecteola cellulose or with DEAE Sephadex A-25 for removing the non-peptide component. The secondary alkylsulfatase is thus bound to the support and separated therefrom with a saline buffer for further purification.

A purification process which has proven successful, in particular for the isolation of secondary alkylsulfatases from Rhodococcus, comprises the following steps:

a) cell lysates of actinomycete strains are treated with DEAE cellulose in a batch process (a batch process) to remove carotenoids, lipids, etc.,

b) the enzyme was eluted with a solution of 10mM Tris/HCl and 0.5M NaCl,

c) NaCl was added to the eluate to a final concentration of 4M NaCl, and the resulting solution was purified by column chromatography using a phenyl agarose column which had been equilibrated with 10mM Tris/HCl buffer (buffer B) containing 4M NaCl, pH7.5, and the enzyme was eluted stepwise with a gradient of 4M to 0M NaCl. For further purification, the process is continued

d) Dialyzing the enzyme active fraction against 10mM Tris/HCl buffer (pH 7.5), followed by

e) Ion exchange chromatography was performed using a Q6 column (BioRad) equilibrated with 10mM Tris/HCl buffer (buffer A) at pH7.5, and a gradient of NaCl from 0M to 1M was used for elution. The gradient can be obtained, for example, by stepwise addition of a buffer mixture of buffer A and buffer C (10mM Tris/HCl, pH7.5, 1M NaCl). Further purification is

f) The enzyme active fraction was passed through a Blue agarose column (Pharmacia), and the enzyme active fraction was eluted in 10mM piperazine buffer solution of pH 6.0. The column was then washed with an additional Tris/glycine buffer pH8.9,

g) at a NaCl concentration of 0.15M, 50mM NaH, pH7.0 was used₂PO₄The buffer was purified homogeneously on a Superdex200 column.

To determine the enzymatically active components, enzymatic hydrolysis of secondary alkyl sulfates can be used, which can be measured for the enzymatic activity of the individual components by determining the amount of sulfate released or the amount of alcohol released. For example, in Dodgson, k.s., biochem.j., 78, 312-; the first method has been used by Tudball, N.et al, biochem. J.126, 187-191, 1972.

The secondary alkylsulfatase isolated from actinomycetes of the present invention preferably has a molecular weight of from 30 to 60kDa in the denatured state and/or from 60 to 80kDa in the native state. Particularly preferred are secondary alkylsulfatases having a molecular weight of from 41 to 45kDa in the denatured state and/or from 65 to 69kDa in the native state. The molecular weight in the denatured state was determined by a gel electrophoresis method using SDS-PAGE.

Using the described method, it is possible to isolate, for example, the secondary alkylsulfatases RS1 and RS2 of Rhodococcus ruber DSM 44541. The molecular weight of the denatured secondary alkylsulfatase RS2 was determined to be 43.3kDa by SDS-PAGE. This enzyme had a molecular weight of 67kDa as determined by Superdex200 elution in the native state, indicating the presence of enzyme dimers. The isoelectric point of the RS2 enzyme was 5.1.

To further characterize the secondary sulfatase RS2 enzyme from Rhodococcus ruber DSM44541, the enzyme was investigated by mass spectrometry. Each characteristic peptide fragment can be detected by electrochsperse-tandem mass spectrometry. Finally, the sulfatase components separated, for example, by gel electrophoresis may be separated and digested with trypsin. The resulting peptide mixture was analyzed by ES MS/MS. Thus, the following 5 peptide fragments can be used for the detection of secondary alkyl sulfatase RS2 of 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, I]═ leucine or isoleucine)

The sequence I and II fragments have no significant homology to other proteins. Sequences III and IV and putative thiolase homology to Streptomyces coelicolor A3 (2). In addition, sequence V is homologous to a thiolase fragment of Mycobacterium tuberculosis (Mycobacterium tuberculosis), sequence IV is homologous to a putative acetyl-CoA acetyltransferase of Streptomyces venezuelae (Streptomyces venezuelae). Thus, the present invention preferably provides a secondary alkylsulfatase of actinomycetes comprising at least one region of the amino acid sequences from sequence I to V.

The secondary sulfatases of the invention not only isolate enzymatically active forms, but, more significantly, their good stability, and thus their suitability for enzymatic organic hydrolysis of sulfates. Possible uses are, for example, for cleaning appliances and/or for degrading sulfates in detergents. Alkyl sulfatases can maintain enzymatic activity, for example, in the presence of organic solvents over a wide range of pH and temperature. Preference is given to a pH range from pH5.5 to pH10 and a reaction temperature below 40 ℃. In addition, many detergents, sugars and polyols have little effect on enzyme activity. Even most of the salts had no significant effect on the hydrolytic activity of the alkylsulfatase enzyme tested. In contrast, some additives show a positive effect on the conversion or enantioselectivity of the enzymatic hydrolysis of alkyl sulfates.

In addition, the enzyme can be easily ionized fixed and thus removed from the reaction batch (reaction batch) and reused without sulfatase loss of enzyme activity. Particularly suitable for immobilization are anionic ion exchange resins, such as DEAE cellulose, TEAE cellulose, Ecteola cellulose or DEAE sephadex A-25 and the like. Fixation is particularly effective at a pH of 7.5. For example, 0.1M Tris buffer may be used as the buffer.

This leads to a further subject matter of the present invention, namely the use of the claimed secondary alkylsulfatases in the enzymatic hydrolysis of alkylsulfates or in the enzymatic preparation of secondary alcohols.

For example, the secondary alkyl sulfatases RS1 and RS2 of Rhodococcus ruber DSM44541 can be used for preparing secondary alcohols. The substrate to be converted is preferably a secondary alkyl sulfate having a carbon chain of 7 to 10 carbon atoms. Carbon chains of 8 carbon atoms are particularly preferred. And preferably sulfate esters with the sulfate ester linkage located at the 2, 3, or 4 position of the esterified carbon chain of the alcohol.

When using secondary alkylsulfatase RS2 in the hydrolysis of sulfates, the preferred working pH ranges from pH7.0 to pH8.5 and the temperature is from 15 ℃ to 36 ℃ and particularly preferably from 27 ℃ to 32 ℃.

When the sulfatase of the invention is used in the hydrolysis of alkyl sulfates, it has proven advantageous to carry out the conversion without sulfate or at very low sulfate concentrations. In practice, for example, a sulfate-free environment may be created by adding barium ions to the reaction batch.

The secondary alkylsulfatase may also enantioselectively catalyze the hydrolysis of secondary alkylsulfates. The enantioselectivity can be increased by adding additives, for example, low concentrations of ferrous or ferric ions and the like. Particularly when using Rhodococcus secondary alkylsulfatase, concentrations of 1mM to 15mM of divalent iron ions and 3mM to 6mM of trivalent iron ions are preferred. The addition of cetyltrimethylammonium bromide (CMAB) also significantly increases the enantioselectivity of the hydrolysis.

In addition to the use of free secondary alkylsulfatase in the reaction batch, it is also possible to use immobilized secondary alkylsulfatase. For this purpose, the alkylsulfatase of the invention may be immobilized, for example, on an anionic carrier material, such as DEAE cellulose, DEAE Sephadex A-25, TEAE cellulose or Ecteola cellulose. A particularly suitable support is Ecteola cellulose, since the alkylsulfatase enzyme immobilized thereon exhibits a particularly high enantioselectivity.

Secondary alkylsulfatases are therefore particularly suitable for preparing secondary alcohols from sulfates. Such processes are particularly useful in enantioselective preparation of chiral secondary alcohols, i.e. in racemate cleavage.

Depending on the nature of the enzymatic reaction, the alkyl sulfate undergoes hydrolysis with the configuration either being maintained or reversed. For example, the secondary alkyl sulfatases RS1 and RS2 isolated from Rhodococcus DSM44541, stereoselectively hydrolyze (R) - (-) -2-octyl sulfate with a reversal of configuration to (S) - (+) -2-octanol.

Some examples are described below to illustrate the invention.

Example 1: culture of Rhodococcus ruber

Rhodococcus ruber cell line DSM44541 was aerobically treated with a medium containing 10g/l glucose, 10g/l peptone, 10g/l yeast extract, 2g/l NaCl, 1.5g MgSO₄·7H₂O，1.3g/l NaH₂PO₄And 4.4g/l K₂HPO₄The liquid medium of (4) is cultured. The culture broth was shaken at 130 rpm at 30 ℃ in a culture flask, and cell growth was monitored by measuring optical density at an absorption wavelength of 546 nm.

Example 2: purification of secondary alkylsulfatase

To disrupt the cells, 47g of cells of Rhodococcus ruber DSM44541 (wet weight) were suspended in 120ml of Tris buffer (10mM, pH 7.5). To this suspension was added 120ml of 0.35mm diameter glass beads. The suspended cells were lysed below 4 ℃ using a shaking cell pump with external ice/water cooling. Lysis of the cells was accomplished by 4 cycles comprising a 2 minute shaking procedure followed by 5 minutes of cooling. After filtering the glass beads, the cell lysate was centrifuged at 38000g for 2 hours at 4 ℃.

The centrifuged cell lysate was treated with DEAE cellulose in a batch process (a batch process) to remove carotenoids, lipids, etc., and alkylsulfatase bound to DEAE cellulose carrier. The alkylsulfatase was eluted with a solution of 10mM Tris/HCl and 0.5M NaCl. While stirring, ice-cooling, NaCl was added to the crude enzyme extract until the final concentration reached 4M. The crude enzyme extract was then purified by column chromatography on a phenyl sepharose column (Pharmacia V ═ 20ml) equilibrated first with 80ml of a buffer of 10mM Tris/HCl, 4M NaCl, pH7.5 (buffer B). The NaCl concentration was gradually reduced during the elution. For this, a mixture of buffer B and buffer A (10mM Tris/HCl, pH7.5) was used. Thus two secondary alkyl sulfatases (RS1 and RS2) were found from Rhodococcus ruber DSM44541, sulfatase RS1 eluting at a NaCl concentration of 1M (25% buffer B, 75% buffer A) and the more lipophilic sulfatase RS2 eluting at a NaCl concentration of 0M (0% buffer B, 100% buffer A).

After the resulting sulfatase fractions were dialyzed against 10mM Tris/HCl, pH7.5 buffer, the enzyme active fractions were further purified on a Q6 column (BioRad, V. RTM.6 ml) equilibrated with 10mM Tris/HCl, pH7.5 buffer. The protein content of the fractions used was eluted with a gradient of 10mM Tris/HCl buffer, pH7.5, NaCl from 0M to 1M. Desorption of the secondary alkylsulfatases RS1 and RS2 was carried out with NaCl concentrations of 0.15 to 0.31M. Fractions containing RS1 and RS2 were loaded onto a Blue Sepharose column (Pharmacia) with 10mM piperazine buffer (pH 6.0). Under the given conditions neither RS1 nor RS2 was bound to the column and could therefore be collected immediately. The column was then washed with Tris/glycine, pH8.9 buffer. The resulting enzymatically active fraction was concentrated to 1ml (Centriplus 10, Amicon) and loaded onto a Superdex200 column. With 50mM NaH₂PO₄The column was equilibrated with 0.15M NaCl, pH7.0 buffer and the sulfatase was eluted separately.

The sequence of the individual purification steps is shown in FIG. 1. FIG. 1 shows the results of SDS-PAGE (12%) analysis of protein-containing samples:

lane a shows 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 ion exchange chromatography with Q6.

Lane E shows the sample after chromatography with Blue agarose.

Lane F shows a sample of secondary alkylsulfatase RS2 purified after Superdex200 purification.

Band G shows the molecular weight standards.

The samples were stained with coomassie brilliant blue R.

Example 3: assays to determine enzyme activity.

a) The amount of sulfate ion released by the enzyme was determined.

To determine the sulfate ion released by the enzyme, the method of Tudball, N et al biochem.J., 126, 187-192, 1972 modified Dogson, K.S. et al biochem.J., 78, 312-319, 1961 was used. In this method, 200. mu.l of an alkylsulfatase solution and 200. mu.l of a 30mM 3-octylsulfate solution are incubated. 0.1M Tris/HCl, pH7.5 buffer was used. The reaction was carried out at 30 ℃ and stopped after 15 minutes by the addition of 15% trichloroacetic acid (w/v). After rapid centrifugation, a 200. mu.l sample of the reaction solution was used to determine the amount of sulfate according to Tudball.

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

One unit of enzyme activity (═ unit) is defined as the release of 1. mu. mol SO per minute₄ ^2-Amount of ionic enzyme.

b) The amount of alcohol released by the enzyme was determined.

To determine the enzymatic activity, 400. mu.l of 30mM substrate solution in 0.1M Tris/HCl, pH7.5 buffer were added to 400. mu.l of a suspension of the sulfatase powder obtained after ion exchange chromatography, or freeze-dried, completely or incompletely purified enzyme solution in the same buffer obtained after phenyl sepharose chromatography. The mixture was shaken at 130 rpm for 6 to 12 hours at 24 ℃ depending on the progress of the reaction.

Example 4: determination of transformation

200 μ l of the reaction mixture was extracted with 200 μ l of ethyl acetate. After mixing the reaction batch well for 0.5 min, the mixture was subsequently centrifuged at 13000 rpm for 5 min and 5 μ l of a stock solution of menthol in ethanol (c 15.4mg/ml) was added as an internal control to 100 μ l of the sample of the supernatant organic phase. The conversion was carried out through an achiral column (HP1301, 6% cyanopropylbenzylpolysiloxane, 30 m.times.0.25 mm.times.0.25 μm (membrane) and HP-1, 30 m.times.0.53 mm.times.0.5 μm; n is a radical of₂) The temperature was maintained at 90 ℃ for 2.5 minutes, and then the temperature was increased to 110 ℃ at a heating rate of 10 ℃/minute. The temperature program indicated corresponds to the measurement of 2-, 3-and 4-octanol.

Example 5: determination of enantiomeric excess

Enantiomeric excess (ee) was determined on a chiral Chrompack CP 7500 column (cyclodextrin-B-2, 25 m.times.0.25 mm. times.25 μm) and a Chrompack Chirasil-Dex CB/G-PN (γ -cyclodextrin propionyl column, 30 m.times.0.32 mm) using hydrogen as a carrier gas. The identification of the (R) -or (S) -alcohol is accomplished by co-injection of the corresponding chiral alcohol.

Derivatization of 2-alkanols

To determine the enantiomeric excess, the alcohol was derivatized with trifluoroacetic anhydride (TFA). Separation of the enantiomers on a chiral GC column is thus improved. Finally, 400. mu.l of the enzyme-converted reaction mixture was extracted with 500. mu.l of dichloromethane and dried over anhydrous sodium sulfate. After addition of 40. mu.l TFA, the solution was heated at 60 ℃ for 20 minutes. After the solution was cooled to room temperature, the sample was extracted twice with 0.5ml of 5% sodium bicarbonate solution. The organic phase was then dried over anhydrous sodium sulfate and the sample was analyzed by gas chromatography.

Alternatively, to avoid the use of sodium bicarbonate solution, the samples can be treated with N-methyl-bis-trifluoroacetamide and dichloromethane (1: 3v/v) at 40 ℃ for 1 hour and then analyzed directly by gas chromatography.

A further alternative procedure is to analyse the corresponding acetate salt by gas chromatography. For this, 400. mu.l of the reaction solution for the enzymatic conversion was extracted with 400. mu.l of ethyl acetate. The organic phase was dried over anhydrous sodium sulfate. After addition of 80. mu.l of acetic anhydride and the catalyst p-dimethylaminopyridine, the solution was shaken overnight at room temperature. The solution was extracted twice with 0.5ml of water, the organic phase was then dried over anhydrous sodium sulfate and analyzed. It is noted that the elution order of trifluoroacetate is reversed compared to acetate.

Example 6: enzymatic hydrolysis of secondary alkyl sulfates with RS2

The conversion of the substrate to be detected is described in example 3. The results of the conversion are shown in Table 2.

Table 2: substrate tolerance of secondary alkyl sulfatase RS2 of Rhodococcus ruber DSM 44541.

Substrate	Transformation of	e.e.p.％	Ea
				2-heptyl sulfate	4	--	--
2-octyl sulfate	46	82	21
				3-octyl sulfate	65	40	3
4-octyl sulfate	68	Despin	--
				2-nonyl sulfate	23	24	1.7
Sulfuric acid 2-decyl ester	4	--	--
				Sulfuric acid 4-decyl ester	5	--	--
1-octen-3-yl sulfate	48	＜5	--
				2-methyl-3-octyl sulfate	0.3	--	--
1-Phenylethyl sulfate	--b	--	--
				Sulfuric acid 1- β -naphthyl ethyl ester	--b	--	--
1-phenyl-2-propyl sulfate	n.e.a.	--	--
				1-bromo-7-octyl sulfate	Is low in	--	--
6-methyl-5-hepten-2-yl-sulfates	n.e.a.	--	--
				Sulfuric acid 2-dodecyl ester	n.e.a.	--	--
Sulfuric acid 1-phenyl-2-butyl ester	n.e.a.	--	--
				Sulfuric acid 1-cyclohexyl ethyl ester	n.e.a.	--	--

no enzyme activity detected n.e.a ═ n

^aEnantioselectivity is expressed as the "enantiomeric ratio" as determined by Chen et al, j.am. chem.soc., 104, 7294-.

^bUnstable substrates

The results in table 2 show that the secondary alkylsulfatases that can be isolated by the described method are highly specific for potential substrates. In the case of the secondary alkylsulfatase RS2 of Rhodococcus ruber DSM44541, a suitable substrate is linear fat secondary (C) without further substitution₇-C₁₀) -alkyl sulfates. These esters are good in conversion yield and have good enantioselectivity.

RS1 shows similar substrate specificity as RS 2.

Example 7: action of salt

The conversion was carried out according to example 3 with the addition of the particular additive to be investigated. The results are summarized in tables 3 to 5. It is visible and surprisingSurprisingly, the selectivity of the secondary alkylsulfatases of the invention can be increased by adding iron ions, preferably Fe²⁺Ions. For example, the enantioselectivity of RS2 for meso-3-octylsulfate is achieved by the addition of FeCl₂Increasing from E-4 to E-80 (table 3) and increasing selectivity to rac-4-octylsulfate from E-1 to E-10 (table 4). It is furthermore surprising that the addition of EDTA or mercaptoethanol and dithiothreitol has no effect on the activity of the alkylsulfatase.

Table 3: the effect of salt addition on the yield and enantioselectivity of RS2 enzymatic conversion of rac-3-octylsulfate.

Additive material	Concentration of additive	Transformation%	e.e.p％	E
					MgCl2	10mM	41	40	3
CaCl2	10mM	41	46	4
					MnCl2	10mM	39	38	3
FeCl2	10mM	18	97	80
					FeCl2	5mM	35	88	25
FeCl2	2mM	37	66	7
					FeCl3	10mM	0	--	--
FeCl3	5mM	9	99	200
					FeCl3	2mM	27	50	4
CoCl2	10mM	37	50	4
					EDTA	10mM		40	40	3
--a	--	38	52	4

^aComparative measurement without addition

Table 4: the effect of salt addition on the yield and enantioselectivity of RS2 enzymatic conversion of rac-4-octylsulfate.

Additive material	Concentration of additive	Transformation%	e.e.p％	E
					FeCl2	10mM	17	80	10
FeCl2	5mM	32	62	5.6
					FeCl2	2mM	50	14	1.5
FeCl3	10mM	0	--	--
					FeCl3	5mM	19	78	9.7
FeCl3	2mM	48	10	1.3
					--a	--	54	4	1.1

^aComparative measurement without addition

Table 5: the effect of salt addition on the yield and enantioselectivity of RS2 enzymatic conversion of rac-2-octylsulfate.

Additive material	Concentration of additive	Rotating shaftMelting%	e.e.p％	E
					FeCl2	10mM	0	--	--
FeCl2	5mM	0	--	--
					FeCl2	2mM	21	86	16
FeCl3	10mM	0	--	--
					FeCl3	5mM	0	--	--
FeCl3	2mM	23	81	12
					--a	--	25	82	13

^aComparative measurement without addition

Example 8: effect of detergents

The transformation was carried out as in example 3 and with the addition of the particular detergent to be investigated. The results are summarized in table 6. The effect of detergents on the activity of the claimed secondary alkylsulfatase enzymes varies depending on the detergent used. The enzyme had almost completely lost activity due to the addition of SDS or di-n-octyl sulfosuccinate. On the other hand, many detergents have no or only a very weak effect on the transformation, and some detergents even have a positive effect on the enantioselectivity of the enzymatic reaction (Table 6). For example, the enantioselectivity of sulfate hydrolysis can be increased by the addition of CMAB.

Table 6: effect of detergents on the conversion and enantioselectivity of RS2 on the substrate rac-3-octylsulfate

Detergent	Concentration (w/v)	Transformation%	e.e.p％	E
					SDS	0.2	--	--	--
Octane-1-sulfonic acid	0.2	36	56	4.8
					Di-n-octyl sulfosuccinate	0.2	Is very low	--	--
Polyethylene glycol	2.0	36	60	5.5
					Tween 80	2.0	43	48	4.0
CMABb	0.2	25	90	30
					--a	--	41	48	3.9

^aComparative measurement in the absence of detergent

^bHexadecyltrimethylammonium bromide

Example 9: influence of more additives

The reaction is carried out as in example 3 with the addition of the particular additive to be investigated. The results are summarized in table 7. With few exceptions (e.g., CMAB), the effect of various sugars and polyols on the conversion and enantioselectivity of the enzymatic hydrolysis of secondary sulfates by the secondary alkylsulfatases of the present invention is minimal (tables 7 and 8).

Table 7: effect of sugars and polyols on the conversion and enantioselectivity of RS2 on the substrate rac-3-octylsulfate

Additive material	Concentration (w/v)	Transformation%	e.e.p％	E
					DEAE dextran	5	33	74	10
Dextran MW 188,000	5	37	50	4
					Dextran MW 41,272	5	34	64	6
Trehalose	5	38	50	4
					Sucrose	5	37	50	4
Polyethylene glycol 6000	5	41	60	6

Table 8: effect of more additions on the yield and enantioselectivity of the enzymatic conversion of octyl sulfate to RS2

Additive material	Concentration of additive	Substrate	Concentration%	e.e.p％	E
						CMAB	0.2(w/v)	Rac-4-octyl sulfate	35	48	3.6
DEAE dextran	5.0(w/v)	Rac-4-octyl sulfate	35	10	1.3
						DEAE dextran gel Glue	0.1(w/v)	Rac-4-octyl sulfate	36	4	1.0
--a	--	Rac-4-octyl sulfate	54	4	1.1
						CMAB	0.2(w/v)	Rac-2-octyl sulfate	19	86	16
--a	--	Rac-2-octyl sulfate	25	82	13

^aComparative measurement without addition

Example 10: effect of the Carrier on the immobilized enzyme

A sulfatase preparation (11mg/ml) in 0.1M Tris/HCl, pH7.5 buffer was added to 100mg of an immobilization support (DEAE cellulose, TEAE cellulose, Ecteola cellulose (Serva, Heidelberg), DEAE Sephadex A-25) previously washed with 0.1M Tris/HCl, pH7.5 buffer. The mixture (batch) was carefully mixed for 5 minutes and left at 4 ℃ for 30 minutes. The mixture (batch) is then centrifuged, the supernatant removed, and the immobilized vector is washed once with 0.1M Tris/HCl, pH7.5 buffer, and then the alkylsulfatase activity is detected analogously to example 3. The results are summarized in table 9.

Table 9: effect of addition of anionic carrier on yield and enantioselectivity of RS2 enzymatic conversion of rac-3-octylsulfate.

Immobilization carrier	Carrier%	e.e.p％	E
				DEAE cellulose	39	50	4
DEAE Sephadex A-25	32	42	3
				TEAE cellulose	26	66	6
Ecteola cellulose	26	86	17

From this, it can be seen that Ecteola cellulose as an anionic carrier substance for immobilization of secondary sulfatase has a positive effect on the conversion of the alkyl sulfate hydrolysis, in particular on the enantioselectivity.

Example 11: influence of organic solvent

The conversion of rac-2-octylsulfate (5% (v/v)) wascarried out at 30 ℃ using RS2 in different organic solvents. The results are shown in Table 10.

Table 10: RS2 enzymatic conversion of rac-2-octylsulfate (5% (v/v)) in different organic solvents yields and enantioselectivities.

Solvent(s)	Transformation%	e.e.p％	E
				Standard of merit	25	82	13
t-BuOH	24	85	16
				2-propanol	20	73	8
DMSO	23	72	8

It has also been found that the secondary alkylsulfatases of the invention have the further advantage that they lose less stability of their activity in organic solvents. One organic solvent particularly suitable for the enantioselective conversion of secondary alkyl sulfates is t-BuOH.

Example 12: addition of barium ions

Ba was determined using 15mM rac-2-octylsulfate as substrate in 0.1M Tris/HCl, pH7.5 buffer at 24 deg.C²⁺Influence on the enzymatic activity of secondary alkylsulfatase RS 2.

FIG. 2 shows the dependence of the conversion of secondary alkyl sulfates on the reaction time with and without the addition of barium ions to the reaction batch.

Example 13: biochemical properties of the secondary alkylsulfatase RS2 of rhodococcus ruber DSM 44541.

a) Determination of molecular weight

The molecular weight of RS2 was determined by SDS polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight determined by SDS-PAGE was checked on the basis of enzyme size using a Superdex200 column. The calibration was performed using chymotrypsinogen A (25kDa), ovalbumin (43kDa), BSA (67kDa), alcohol dehydrogenase (150kDa) and Blue dextran (2000kDa) as molecular weight standards.

The molecular weight determination by SDS-polyacrylamide gel electrophoresis under denaturing conditions gives a molecular weight of 43.3kDa for secondary alkylsulfatase RS 2. Elution of Superdex200 purified enzyme gave a molecular weight of approximately 67kDa, indicating a non-spherical or dimeric structure of the enzyme.

b) Determination of isoelectric Point

The isoelectric point of secondary alkylsulfatase RS2 was determined using the BioRad IEF Ready Gel system at a pH range of from pH3 to pH 10. The pI value of RS2 was determined to be 5.1.

c) Determination of the optimum pH

The optimum pH of the sulfatase RS2 was determined in 100mM Tris/maleate buffer at 24 ℃ with 15mM rac-2-octyl sulfate as substrate in the pH range 6.0 to 9.0. The secondary alkylsulfatase RS2 has an optimum activity at pH values between 7.5 and 8.0.

FIG. 3 shows the pH dependence of the enzyme activity of sulfatase RS 2.

d) Determination of optimum temperature

The effect of temperature on the enzymatic activity of secondary alkylsulfatase RS2 was determined in a buffer of 100mM Tris/maleate, pH7.5, using 15mM rac-2-octylsulfate as substrate at a temperature range of 20 ℃ to 40 ℃. RS2 showed the best enzyme activity on 2-octyl sulfate at about 30 ℃.

FIG. 4 shows the temperature dependence of the enzyme activity of sulfatase RS 2.

Example 14: substrate Synthesis, general Process Instructions

According to the synthetic instructions described by White, G.F., et al, biochem.J., 187, 191, 1980Book by adding triethylamine-SO₃The compound sulfurizes corresponding secondary alcohol to prepare sodium sec- (+/-) -alkyl sulfate. 0.132g of sodium hydride (5.52mmol, 60% dispersion in mineral oil, washed with petroleum ether) are suspended in 2ml of dioxane under an argon atmosphere. 2.48mmol of secondary alcohol were slowly added dropwise to this suspension through a septum. The mixture was stirred at room temperature for 1 hour. Sulfur trioxide-triethylamine complex (0.5g, 2.76mmol) was dissolved in 4ml of anhydrous dioxane with heating. The resulting solution was slowly added dropwise to the sodium ethoxide solution and stirred overnight. Distilled water was added to terminate the reaction. The solution was completely concentrated by evaporation in a rotary evaporator. The residue was taken up in 15ml of distilled water and extracted 5 times with 10ml of ethyl acetate. The water was removed by freeze drying. The resulting powder was taken up in 30ml of methanol and filtered. The filtrate released the solvent at low pressure.

1-Bromocin-7-ol was prepared synthetically according to the method of Yadav, J.S. et al, Tedrahedron, 45, 6263-Strobenzo 6270, 1989 and sulfurized as described above.

Sequence listing

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Claims (15)

1. Secondary alkyl sulfatases from actinomycetes (actinomycetes).

2. The sulfatase of claim 1, which is derived from Rhodococcus sp.

3. The sulfatase of claim 1 or 2, which is obtained from Rhodococcus ruber (Rhodococcus ruber), Rhodococcus equi (Rhodococcus equi) or Rhodococcus R312(Rhodococcus sp.R 312).

4. Sulfatase according to any one of the preceding claims, characterized in that the sulfatase has a molecular weight in the denatured state of 30kDa to 60 kDa.

5. Sulfatase according to claim 4, characterized in that the sulfatase has a molecular weight in the denatured state of 41kDa to 45 kDa.

6. The sulfatase enzyme according to any one of the preceding claims, characterized in that the sulfatase enzyme has a molecular weight of 60kDa to 80kDa in nature.

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

8. Use of a sulfatase according to any one of claims 1 to 7 in the preparation of a secondary alcohol or in the degradation of an alkyl sulfate.

9. Use of a sulfatase enzyme according to any one of claims 1 to 7 for the cleavage of an enantioselective racemate.

10. Process for the preparation of secondary alcohols, characterized in that alkyl sulfates are enzymatically hydrolyzed with a secondary alkylsulfatase of any of claims 1 to 7.

11. The process according to claim 10, characterized in that a ferrous or ferric salt and/or CMABis added to the reaction batch as an additive.

12. The method of claim 10 or claim 11, characterized in that the secondary alkylsulfatase is applied in immobilized form on the Ecteola cellulose.

13. Process 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. Process according to any one of claims 10 to 13, characterized in that the secondary (C) is hydrolysed with a secondary alkylsulfatase according to any one of claims 1 to 7₇-C₁₀) -alkyl sulfates.

15. The secondary alkylsulfatase of any one of claims 1 to 7, obtained from a cell-free actinomycete lysate by:

a) purification by phenyl sepharose column with decreasing sulfate-free gradient followed by

b) Purifying the obtained component with alkylsulfatase activity by chromatography on an ion exchange column,

c) subjecting the fraction having alkylsulfatase activity obtained by ion exchange chromatography to chromatography on a Blue agarose column, and

d) the resulting fraction with alkylsulfatase activity was filtered with Superdex 200.

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