CA2298069A1

CA2298069A1 - Method for producing and identifying new hydrolases having improved properties

Info

Publication number: CA2298069A1
Application number: CA002298069A
Authority: CA
Inventors: Manfred T. Reetz; Albin Zonta; Klaus Schimossek; Klaus Liebeton; Karl-Erich Jager
Original assignee: Individual
Current assignee: Studiengesellschaft Kohle gGmbH
Priority date: 1997-07-25
Filing date: 1998-07-23
Publication date: 1999-02-04
Also published as: DK1002100T3; WO1999005288A2; ES2202883T3; EP1002100A2; DE19731990A1; WO1999005288A3; DE59808919D1; JP2001512663A; EP1002100B1; ATE244309T1

Abstract

The invention relates to a method for producing and identifying hydrolase mutants having improved properties regarding stereoselectivity and site selectivity, catalytic activity or stability during chemical reactions.

Description

ID: PAGE 2 FEB-24-00 13:57 FROM=
SMB
Meth,Qd for Produdn~nd Idgntr~~ng New H,_ydr2,(ase~ Ha~(,i,~g Improved Prod rtie~
The present invention relates to a process for the preparation and identification of hydrolase mutants having improved properties with respect to stereo- or regioselectivity, catalytic activity or stability in chemical reactions.
P ri r a Hydrolases are among the most wide-spread enzymes in organic syn-thesis. As a subgroup of the hydrolases, esterases and lipases, in particular, catalyze a wide variety of reactions, such as the hydrolysis of carboxylic acid esters, yr the synthesis of esters or transesterifications in organic solvents. Due to their high stereoselectivity, stability and their being readily available, they are interesting for numerous industrial processes. Thus, for example, lipases have been industrially employed for the optical resolution of chiral alcohols, acids or amines, for the preparation of optically pure medicaments, natural substances, plant protective agents or high-grade fats and oils (K. Faber, 8iotransforma-tions in Organic Chemistry, Springer-Verlag, Berlin, 2nd Ed. 1995).
Nevertheless, the enantioselectivity of a lipase or esterase with respect to a given substrate cannot be predicted with certainty, and in many cases, the reactions proceed with only moderate optical yields.
Therefore, there is a need for a process for the preparation of hydro-lases which enables a well-aimed optimization of enantioselectivity FEB-24-00 13:57 FROM= ID: PAGE 3 with respect to a desired product and the special process conditions, such as temperature and solvent. Although effects on the enantiose-lectivity of iipases could be studied using the molecular-biological method of in vitro mutagenesis, which is customary today (K. Hult, M.
Holmquist, M. Martinelle, European Symposium on Biocatalysis, Graz, 1993, Abstracts, L-4), an optimization with respect to a particular substrate which would have led to an enzyme useful in organic syn-thesis could not be achieved.
The most important possible applications of genetic engineering include protein desig, wherein mutations are introduced base-specifically into the gene sequence of the corresponding protein based vn known structural data using in vitro mutagenesis. By selectively substituting amino acids, enzymes having improved catalytical activity or stability could already be prepared in this way {A. Shaw, R. Bott, Current Opinion in Structural Biology, 1996, 6, 546). This technique, the so-called oligonucleotide-directed or site-directed mutagenesis, is based on the substitution of a short sequence segment of the gene coding for the naturally occurring enzyme (wild type) by a synthetically mutagenized oligonucleotide. Subsequent expression of the gene results in a n enzyme mutant which may have advantageous properties. In a method derived therefrom, the so-called cassette mutagenesis, oli-gonucleotides with partially randomized sequences are used. This provides a library of mutants of a limited size, which can then be tested with respect to its properties.
Despite of advantages of these established methods,they are the hardly suitablefor the stepwise optimization enzyme or for of an the generation enzymes having novel properties. The factthat our of understandingof the laws governing protein and the structur'e-folding function relationship of proteins is still incompleteis the main reason for the failingof many projects in the field of the so-called rational Pf~GE 4 ID:
FEB-24-00 13:59 FROM: , protein design. In addition, a stepwise optimization process according to the classical method is relatively labor-consuming and does not ensure a significant improvement of the enzyme properties per se.
More recently, novel molecular-biological methods of mutagenesis have been described (D.W. Leung, E. Chen, D.V. Goeddel, Technique, 19$9, I, 11, and W.P.C. Stemmer, A. Crameri, PCT WO 95/22625) which are based on the polymerase chain reaction known from the literature (R.K.
Saiki, S.J. Scharf, F. Faloona, K.B. Mullis, G.T. Horn, H.A. Erlich, N. Arn-heim, Science, 1985, 230, I3S0). Instead of site-directed mutagenesis, these methods employ combinatorial methods for the generation of extensive mutant libraries which are subsequently screened for mu-tants having positive properties using suitable screening methods. This mimics the naturally occurring evolutive processes of replication and recombination, mutation and selection on a molecular level. This method, described as in ultra evolution for directed evolution), has already proven useful in some cases as a suitable method for obtaining new biocatalysts (W.P.C. Stemmer, Nature, 1994, 370, 389, and F.H.
Arnold, Chemical Engineering Science, 1996, 51, 5091).
In spite of the progress made in this field, this method cannot yet be generally transferred to all classes of enzymes, since suitable test methods for identifying mutants with positive properties are lacking in most cases. Such methods are a sine qua non, however, in view of the large number of mutated enzyme variants to be expected in the production of combinatorial mutant libraries. Especially in the case of the lipases which are interesting for industrial processes, the produc-tion of mutants with improved stereoselectivity by the methods of in vitro evolution has not been successful to date, because an efficient screening method for enantioselectivity testing still does not exist. The classical method for determining the enantioselectivity of a lipase- or esterase-catalyzed reaction is based on the separation of the reaction FEB-24-00 13:58 FROM: ID: PAGE 5 products and educts by iipuid or gas chromatography using chirally modified stationary phases. However, due to the enormous number of samples to be processed in the screening of extensive mutant Libraries, this method is unsuit~Jble since chromatographical separations with chirally modified columns are time-consuming, being only capable of sepuential processing. Another as yet unsolved problem is the diffi-culty, frequently to observe, of expressing functional lipases or es-terases in host organisms with a sufficiently high activity yield. How-ever, this is indispensable to a high-performance screening system since too low enzyme activities are difficult to detect in the determina-tion of enantivselectivity due to the limited sensitivity of a test system.
Obigct~f the inyention Therefore, it has been the object of the present invention to provide a simple process for the preparation of mutated hydrolases, especially iipases or esterases, having improved stereo- or regioselectivity, catalytic activity and stability towards particular substrates (e.g., carboxylic acids, alcohols, amines, or their derivatives), which process additionally enables a rapid identification of positive mutants from extensive mutant libraries, and the use of the enzymes thus prepared in the optical resolution of chiral alcohols, acids and amines, and their derivatives.
Descri~t'on of ~,he inygntion The present Invention relates to (1) a profess for the prepacatlvn and identificaflon of hydrole~sa mutants having improved properric~s with respect to stereo- or regioseiectiv;ty, catalytic activity or stability, charattenxed in that a) a starting hydrofase gene is mutagenized ~y a modified pofymel-ase rttoin 1D: PAGE
FEB-24-00 13:58 FROM:
4a reaction (PCR), wherein the mutation rate and total number of mutations in the amplified DNA is adjusted by adjusting the ~nnCentrations of Mgz*, MnZ+ and of the deoxynucleOttdes and by adjusting tht number of cycles; and/or b) one or more starting hydrolase genes, one or mor-c hyd~olase genes mutated acCVrdtng to step a), or mixtures of one or more starting hydrolese genes and one or mere l~ydroiase genes mutated according to step a) are mutagenized by enxymaticbity fragmenting said genes, followed by enzymatic rer~ssembly of the rtragrrtenzs produced to give complete recombinant hydrolase genes;
c) t>~e mutated hydrolase genes obkained according to step a) or b) are transformed Into a host organism; and d) hydrolast mutants having improved properties, expressed by transformants obtained in step c), are identified by a test method;

(2) a hydrolae mutant obtainable by the process defined under (1):

(3) a oNA sequence e:oding for the hydrolase mutant defined uneer (z);

{4) a vector comprising the DNA sequence defined under (3);

{5) a CranSfvrmant comprising a DNA sequence as det5ned under (3) and/or a vector as defined under (4);
(8) a process for the preparation of hydralas~ mutants hawir~~ in7praved properties, cornprlsing culturing a transfvrmant bccording to (5); and (7) a mesthod for testing catalysts for stereo- or regioselectivity, wherein CquBl amounts of the catalyst are added to a test substrate and to the pure stereo-or reflioisomers of the test substrate, provided with a chrvmvphorous group which Causes a spectrometrically determinable change of absorption or emtsston upon cleavage by the catalyst, in separate test vessels, and the stereo- or regioselectivf~Cy Is defiermined from the ratio of the linear init;at reason rates obtained.
As a rule, the preparation of the new biocatalysts starts wirh the isolation of a lip~rse or esterase gene from the organism of origin. This may be any filCrobial, plant and animal organism which is the carrier oP a lipase or esterase gene.
The isolation of the gene can be effected according to the mothods known Pram the literature (J. Sambrook, E.F. Fritsch, 'f. Maniatts, MOleCUlar Clanlng: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989, New York). Usually, the genwnic bNA Is rtragmented using restriction endanuclee~ses, and the gene fragments obtained are cloned in a host organism i;e.g., ~ co~~')- Then, using otigonucieotides with 5equenCQ homology ic'o a segment of the IipaSG or esterase gene, the gene is identified witt,in the gene libr5ry tn hybridization experiments, followed by isolation thereof.

FEB-24-00 13:56 FROM: ID: PAGE
-Surprisingly, it has been found according to the invention that naturally occurring hydrolase genes can be mutagenized by a modified polym-erase chain reaction (PCR), changing certain reaction parameters, to obtain an extensive mutant library which can be screened for mutants having improved enantioselectivity using a novel test method.
The novelty of the process resides in that an extensive randomized mutant library can be established, starting with a naturally occurring lipase or esterase gene (the so-called wild type gene), using a modi-fied PCR {hereinafter referred to as mutagenizing PCR). It has been found that the mutation rate during the PCR can be adjusted in a well-aimed manner by changing the components of the PCR: The number of mutations in the lipase gene in question (the mutation rate) can be controlled by varying the concentrations of Mgz+ and/or of the deoxy-oligonucleotides and/or the addition of Mn2+ ions. preferably, the following concentrations are used depending on the DNA polymerase employed:
Mg2+: I.5 mM - 8.0 mM
dNTP:0.05mM-I.OmM
Mnz+: 0.0 mM - 3.0 mM
In addition, it has been found that the number of cycles in the PCR
correlates with the number of mutations: the higher the selected number of cycles, the higher is the total number of mutations. By FEB-24-00 13:59 FROM= ID: PAGE 6 means of this parameter, the diversity of the mutant library can be adjusted.
For' determining the mutation rate, the pufified PCR products are sequenced. The mutation rate can be determined by comparing the sequences obtained with the sequence of the wild type gene.
Table 1 shows the mutation rate as a function of the concentration of the above mentioned components of the PCR in the amplification of the lipase gene from P, aeruginosa (IipA).
T_ able 1 Exp. Mg'+ Mn'+ dATP/ dTTP/ Mutation rate (mM) (mM) dGTP dCTp (mutations/
(mM) (mM) 1000 bpl~) 1 ~ 6.1 ~ - ~ 0.2 0.2 1-2 ~ 7.0 l 0.5 I 0,2 ( I.0 ~ 15-20 1~ by = base pairs From the sequencing results, it can further be seen that the transition and transversion types of mutation occur in about the same statistical frequency. In contrast, deletions and insertions are rarely observed. In addition, the mutations are uniformly distributed over the entire lipase gene. Thus, a mutant library with statistically uniformly distributed mutations can be produced by the method described. A mutation rate of 1-2 mutations/hydrofase gene has proven advantageous. Thereby, it is prevented that a negative mutation will mask a mutation with a positive effect, as would be the case if several mutations occurred per one hydrolase gene. In order to obtain a complete mutant library, each with one amino acid substitution per enzyme molecule, 5415 mutants must theoretically be generated in a lipase consisting of 285 amino FEB-24-00 13:59 FROM: ID: PAGE 9 acids {here: lipase from P. aeruginosa). This value results from the following formula:
N = 19 x M x 2$5! / [(2$5 - M)! x M!]
with N = number of mutants, and M = number of amino acid substitu-tions per one lipase molecule. According to the invention, it could be surprisingly shown that positive mutants are found in even substan-tially smaller sized libraries, a mutation rate of 1-2 having been em-ployed.
The mutated lipase or esterase genes obtained by the process de-scribed are ligated into a suitable expression vector and then trans-formed into a host organism, e.g., E. coil. Then, the transformed cells are plated on agar plates and cultured. rf the expression rate is suffrciently high, the colonies obtained can be transferred to microtitra-tion plates provided with a liquid medium and, after growth has started, can be directly employed in a screening test. In the case where only little enzyme is formed in the expression of the lipase gene or the gene product is not correctly folded in the host organism used (inclusion bodies) or incompletely secreted into the culture medium, it will be advantageous to reclone the mutated genes in another host organism, preferably the original organism.
In order to obtain sufficiently high enzyme activities, the individual bacterial clones which contain a mutated lipase or esterase gene are transferred from the agar plates into the wells of commercially avail-able microtitration plates and cultured in liquid medium. Preferably, microtitration plates having 9fi wells per plate are employed. The growth of the bacteria can be monitored by measuring the cell density (~~60o value). It is advantageous to inoculate a second microtitrativn plate in parallel in this way in order to have a reference for the later FEB-24-00 13:59 FROM:i ID: PAGE 10 identification of positive clones. After the growth of the bacteria glyc-erol is conveniently added to the reference plate, which is then stored at -80 °C until used for identification. If the bacteria are secreting the enzyme into the extracellular space (as with the lipase from P. aerugi-nosa), the cells in the microtitration plates are centrifuged off, and the supernatant with the lipase or esterase activity is used for the screening test. In the case where the bacteria (e.g., E. coh~ accumulate the enzyme in the periplasm, a cell wall lysis must be preliminarily done, wherein methods known from the literature, such as lysozyme treatment, can be used.
By culturing the corresponding clones from the reference plate, suffi-cient plasmid DNA can be isolated which can be used for the charac-terization of the mutated lipase or esterase gene. The mutations are localized within the gene by sequencing, One advantage of the inven-tion is the fact that the mutated gene in a positive clone can be further optimized with respect to its properties in further mutation cycles by the process described, even without knowing the exact position of the mutations. Thus, the isolated lipase or esterase gene is again used in a pCR modified according to the above stated conditions {mutagenizing PCR). This procedure may be repeated until the properties of the lipase or esterase mutant meet the requirements of the stereoselective reaction.
For a further optimization of the identified positive mutants, the prod ess described can be extensive in that the DNA of several positive mutants is first fragmented and then can be reassembled into func-tional lipase or esterase genes in a combinatorial process according to W.P.C. Stemmer (Nature, 1994, 370, 389). The thus obtained in vitro recombinant library is subsequently expressed, and the recombinant gene products are examined for improved enantioselectivity using the test methods according to the invention. The advantage of this method FEB-24-00 13:59 FROM: ID: PAGE 11 is that the positive properties of different lipase or esterase mutants may be added in one new recombinant gene due to the recombination, which eventually may result in a further improvement of the lipase or esterase. The course of the method described is as follows:
Using the enzyme DNase I (e.g., from bovine pancreas), the lipase or esterase genes are first cleaved into fragments having a preferably length of between 25 by and 100 bp. The size of the fragments can b a checked by separating them by means of agarose electrophoresis and comparing with corresponding DNA length markers. The DNA fragments thus obtained are purified to free them from adhering DNase. The in vitro recombination is performed under the conditions of a conventional PCR, but without adding any PCR primers. In analogy with conventional PCR, one cycle is comprised of three steps: a) denaturing, b) annealing and c) elongation. During annealing, hybridization occurs of sequence-homologous fragments which may be derived from different mutated lipase or esterase genes. In the subsequCnt elongation step, the strands are completed by the DNA polymerase so that new recombi-nant lipase genes are eventually obtained. The optimum number of cycles is determined in a preliminary experiment. Thus, after every 5 cycles, a small sample of the reaction mixture is separated by agarose gel electrophoresis to determine the cycle in which the maximum of the size distribution of the recombinants in the range of the size of the enzyme gene. A number of cycles of between 30 and 45 is preferably selected. The band obtained in the agarose gel which corresponds in size to the lipase or esterase gene is purified and amplified by a conventional PCR. The PCR product is purified and, Following ligation into a suitable vector (plasmld), transformed into ~ coli. As already discussed in the paragraph dealing with mutagenrzing PCR, it may be required to reclone in another host organism if the lipase activity should be too low after expression in E. coli. The recombinants ob-FEB-24-00 14:00 FROM: ID: PAGE 12 -~.a-tained are grown in microtitration plates for the test for enantioselec~
tivity.
In a variant of the invention, the described methods of mutagenizing PAR and in vitro recombination for the production of mutant or recombi-nant libraries can be performed successively or repeated in any order and frequency desired in order to optimize the enantioselectivity of the lipase or esterase. Preferably, at least one mutation cycle is performed in the beginning using mutagenizing PCR. This may then be followed by an in vitro recombination cycle, wherein the best positive mutant clones are respectively employed. By monitoring the enantioselectivity of the enzyme mutants obtained, the optimization process can be followed.
In another variant of the invention, positive lipase or esterase mutants identified by the screening of mutant or recombinant libraries can be further optimized using classical directed mutagenesis or cassette mutagenesis. Thus, the mutation in the lipase or esterase gene is first localized by sequencing. This gene is subsequently again mutagenized by means of "wobbled" primers at the codons coding for positive mutants. The thus obtained mutant library of a limited size can then be expressed and screened for improved enantioseiectivity.
Positive lipase or esterase mutants identified by the screening of mutant or recombinant libraries can be further optimized using site-directed saturation mutagenesis. Thus, the positive mutation in the lipase or esterase gene is first localized by sequencing. Then, using any method of site-directed rnutagenesis which allows for the ex-change of multiple bases, this gene is changed in such a way that ail possible codons are formed at the site of the gene which codes for the position to be optimized. This provides a library of mutants of a limited size in which mutants the amino acid originally present in the amino acid position to be optimized has been replaced by the remaining 19 pi~lCE 13 FEB-24-00 14:00 FROM=

ID:
amino acids. The thus obtained mutant library of a limited size can then be expressed and screened for improved enantioselectivity.
In a variant of the method described, the lipase or esterase gene of the wild type enzyme is employed for in vitro recombination together with the positive mutants found. This can result in backcrossings in which mutations having neutral or negative properties can be elimi-nated. Following expression, the recombinant library obtained can be examined for improved enantioselectivity.
in another variant of the method described, hydrolase genes from different organisms are employed for in vitro recombination, provided they possess sufficient sequence homology with the originally em-ployed hydrolase gene.
In a variant of the method, the in vitro recombination is performed under the conditions of the modified PCR described. Thus, the concen-trations of the Mgz' or Mn2+ ions and of the deoxynucleotides (dNTPs) are changed to adjust the mutation rate during in vitro recombination in a well-aimed manner.
The invention further relates to test methods which allow for the identification of enzyme mutants having Improved stereoselectivity or regioselectivity from extensive mutant libraries. Thus, after centrifuging off the bacterial cells, two alipuvts of the enzyme-containing super-natant are transferred to adjacent wells of a new microtitration plate.
After addition of the two enantiomeric pure substrates in the two wells, respectively, the activity of the lipase or esterase is determined by spectrophotometry. The measurements are performed in a commer-cially available spectral photometer for microtitration plates. This allows for a high sample throughput. The selection of the substrate depends on the type of chiral compound for which optimization of the lipase or i, FEB-24-00 14:00 FROM: ID: PAGE 14 esterase is to be effected. The method is particularly suitable for chiral carboxylic acids, alcohols and amines.
In the case of chiral carboxylic acids or chiral COOH-functions( com-pounds, the two corresponding p-nitrophenyl esters of the (R)- and (5)-acids are employed as test substrates. Formula 1 shows the principle of the test method wherein R represents any organic residue having at least one asymmetric center.

FEH-24-00 14:00 FROM: ID: PAGE 15 Form la Scheme of the test method for stereoselectivity for chiral carboxylic acids or COOH-functional compounds Reaction 1:
.~"w..._ Y
ut~ i t H'~ '._ _.'" , .-"~."w r~'~ . ~: .~ I,~.
A ,7 Ft U!i ;C~-' ,w,,~.r~
lR~rtsnGOr~
Reaction 2:
MCS~. n ,~ .,~., "'ny ,, r~:~ ~"
~t ~ ~' i~l~Eryar~ya~t~er Due to the high absorbance of the p-nitrophenolate anion released in the hydrolase-catalyzed ester hydrolysis (a,max = 405 nm, Emax = 14,000), a highly sensitive test method results by which a n activity determination can be performed even for low substrate concen-trations. The enantioselectivity of the hydrolase mutants can be determined with sufficient accuracy from the quotient of the hydrolysis rates Vapp(R) and Vapp(S) for the (R)- and the (S)-ester, respectively.
Since both test reactions contain only one enantiomer (either the R- or the S-ester), the absence of a competing reaction with the other enantiomer must be taken into account when the enanti0selectivity is determined. Although this kinetic effect may lead to the calculation ,of inaccurate enantioselectivities, it has been found that the apparent enantloseiectivities obtained by the presented method (Eapp) are a i FES-24-00 14:01 FROM: ID: PAGE 16 sufficiently telling with respect to the enantioselectivity of the mutated lipases. Eapp is obtained as Vapp(R)/Vapp(s)~ Another advantage is the simple pertormance and good reproducibility of the test, which is also suitable for screening with a high sample throughput.
In the case of chiral alcohols or chiral OH-functional compounds, fatty acid esters of the two optically pure alcohols are employed in the test for stereoselectivity. The chain length of the fatty acids is within a range of from C2 to Cis. As the alcohol component, primary, secondary and tertiary alcohols and their derivatives having at least one asym-metric center can be used. Solutions of the esters of the (R)- and (S)-alcohols are hydrolysed with culture supernatants of the hydrolase mutants in adjacent wells of a microtitration plate. The hydrolysis rates Vapp~R~ and Vapp~s~ for the (R)- and the (S)-ester, respectively, are a measure of the enantioselectivity of the enzyme mutant examined.
Detection is effected through a coupled enzyme reaction (H.U. Berg meyer, Grundlagen der enzymatischen Analyse, Verlag Chemie, Wein-heim, 1977) in which the continuous release of the fatty acid is moni-tored. The dye produced is assayed by colorimetry at 546 nm (c = 19.3 1~mmol'' ~cm-1). The concentrations of the enzymes, cofactors and coenzymes of auxiliary reactions Z and 3 (see Formula 2) and of the indicator reaction 4 must be selected in such a way that the lipase-or esterase-catalyzed reaction to be determined is rate-determining.
The quotient of the hydrolysis rates for the (R)- and the (S)-ester, respectively, corresponds to the apparent enantioselectivity (Eaap). In one variant, the fatty acid amides of chiral amines or NHz~ or IVHR-functional compounds are employed instead of the optically pure esters, Formula Z shows the scheme of the test system.

FEB-24-00 14:01 FROM: ID: PAGE 17 F~~mula 2 Scheme of the test method for stereoselectivity for chiral alcohols; R
represents any organic residue having at least one asymmetric center;
abbreviations: CoA {coenzyme A), ATP (adenosine-5'-triphosphate), AMP (adenosine-5'-monophosphate) 1. ) R w H~1~ ~. + ~
n yo~H
free fatty acid 2. ) free fatty acid + CoA + ATP ,~~ S~ aryl-CoA + AMP + pyrophosphate ~4Gtr!-GaA, O.~art>fas~e 3, ) acyl-CoA + O: --..~,.~:........~.~. enpyl-CoA + HzOz ~~~I~tld~lss 4, ) HxOz + a-aminoantipyrine + 2,4,6-tribromo-3-hydroxybenzoic acid ,-~---.red dys +
2 H20 + HBr In a variant of the method, the corresponding esters and amides of succinic acid can be employed instead of the fatty acid esters or amides. The latter have the advantage, over the fatty acids, of being more soluble in aqueous solutions or aqueous-organic solvents. The measurement is performed by UV spectrometry at 34Q nm (~ = 6.3 l~mmol-l~cm-1). In this test method too, it has to be taken care that the hydrolase-catalyzed reaction 1 be rate~determining. The quotient of the hydrolysis rates Vapp~R) and Vapp(S) for the (R)- and the (S)-ester, respectively, corresponds to the apparent enantioselectivity (Eapp). In one variant, the fatty acid amides of chiral amines are em-ployed instead of the optically pure esters. Both primary and secondary FEB-24-00 14:01 FROM=

amines may be employed as the amine component. The scheme of the test system is represented in Formula 3.
Formula 3 Scheme of the test method for stereoselectivity for chiral aicohols; R
represents any organic residue having at least one asymmetric center;
abbreviations: CoA (coenzyme A), ITP (inosine-5'-triphosphate), IDP
(inosine-5'-diphosphate), NADH/NAD+ (reduced/oxidized nicvtinamide adenine dinucleotide) 1. ) Ht~OC.,~,,r~,~,~~~JR + Hz0 L ~9~ ""'.,.~..~,., . H'!N + R-OH
free succinic acid Sucdr,~rl-GSA. sy~ntt~el~
2, ) Succinic acid + ITP + CQA '~~" IDP + succinyl-CoA + phosphate Py~c~mle knish 3 , ) IOP + phosphaeno( pyruvate ITP + pyruvate LSC~arig ~~ahydroga~sse g., ) Pyruvate + NADH + H''' L-lactate +NAD' The test for the identification of hydro(ase mutants having improved stereoseiectivity may further be performed in such a way that both stereoisomers are contained in the test reaction. Thus, the separated measurements of the (R)- and (S)-enantiomers can be dispensed with.
The test principle starts with binding a racemic mixture of the chiral substrate to a solid phase. Through an ester or amide linkage to this ch(ral compound, a radioactively labeled organic residue is bound. Two cases can be distinguished:

FES-24-00 14:01 FROM: ID: PAGE 19 a) Solid-phase bound chiral carboxylic acid: the carboxy function is esterified with a radioactively labeled alcohol.
b) Solid-phase bound chiral alcohol or chiral amine, or OH- or NHz-functional (or NHR-functional) compounds: the hydroxy or amine function is labeled with a radioactively labeled carboxylic acid.
It is critical that the two enantiomers of the racemic mixture bound to the solid phase be labeled with different isotopes, Preferably, 3H- and laC-labeled compounds are used. As the solid phase, all usual organic functionalized polymers as well as inorganic functionalized supports can be employed. Preferably, solid phases based on polystyrene and silica gel supports are employed. The chiral radioactively labeled compounds are then bound to the solid phase wherein the coupling to the solid phase must be adapted to the chemical nature of the chiral substrate. Formula 4 shows the scheme of the modified solid phase and the principle of the test method.

FEB-24-00 14:02 FROM: ID: PAGE 20 Formula 4 Scheme of the solid-phase screening test for stereoselectivity with a dual radioactively labeled substrate; X = 0, NH; R is a radioactively labeled organic residue Q
t ~_ ~ ~ s-~ .~ x~~ ~o I~
f 1 ~ro~ia H~~ O
I~~-C~-~ l C1 X' '14- -R
Approximately equal amounts of the thus modified support can be dispensed to small reaction vessels (e.g., the wells of microtitration plates) and then admixed with the culture supernatants of the hydro-(ase mutants. In the subsequent reaction, the radioactively labeled components {carboxylic acid or alcohol) are hydrolysed from the solid phase and released into the liquid medium. An aliquote of the medium is then removed and examined for the amount of radioactivity in a scintillation counter. From the ratio between the two different isotopes, the enantiomeric excess and the conversion of the reaction and thus the enantioselectivity of the mutated esterase or lipase can be calcu~
lated. Sy using regioisomeric test compounds, the tests described can also be used for the identification of hydrolase mutants having im-proved regioselectivity. Instead of hydrolase mutants, other catalysts may also be employed for determining the stereo- or regioselectivity:
The test for enantioselectivity of the hydrolase mutants prepared by the process described may also be performed by a capillary-electrophoretical separation using chirally modified capillaries which allow for a direct separation of the enantiomeric substrates or products FEB-24-00 14:02 FROM: ID: PIiGE 21 of the hydrvlase-catalyzed test reaction. Here, the test substrates can be employed as a racemate. The separation may be effected both in capillaries and by the use of prepared microchips which allow for electrophoretical separation and parallel running of the analyses for a high sample throughput. In both cases, it is a precondition that the enantiomers can be separated by capillary electrophoresis.
The invention will now be further illustrated by the following Examples and Figures.
Figure I shows the experimentally obtained measured curves for the determination of the apparent enantioselectivity (Eapp) in the hydrolysis of (R)- and (S~-2-methyldecanoic acid p-nitrophenyl ester with culture supernatants of the lipase mutants P1B O1-E4, PZB 0$-H3, P3B 13-D10, P4B 04-H3, P5B 14-C11, P4BSF 03~G10, and the wild type lipase from P, aeruginosa (the slopes have the unit [mOD/min]).
Figure 2: Comparison of the DNA sequences of the lipase mutants P1$
O1-H1, P1B 01-E4, P2B 08-H3, P3B 13-D10, P4B 04-H3, P5B 14-C11 and P4BSF 03-G10 Si55F with the sequence of the wild type of lipase from P, aeruginosa (the mutated bases with respect to the wild type are boxed, the origin of the mature lipase mutants is at base 163 or at base 162 in the wild type).
In the following Example, the gene of the lipase from P. aeruginosa (isolation according to K.-E. Jager, Ruhr-Universitat Bochum) has been used for an optimization, the substrate for which the enantioselectivity of the lipase was to be improved was (R,S)-2-methyldecanoic acid. A

FEB-24-00 14:02 FROM: ID: PFiGE 22 - as -lipase mutant with a preference for the (S)-enantiorner was to be developed. The screening test was performed with (R)- and (S)-2-methyl-decanoic acrd p-nitrophenyl ester.

FEB-24-00 14:02 FROM: ID: Pi4CE 23 Formula 5 (R,S)-2-methyldecanoic acid Bacterial strains E. cvli JMlp9:
e14-(McrA), re~Al, endAl, gyrA95, thi-_i, hsdRl7(rK-mK+), supE44, relAi, o(lac-proAB), [F' trae36 proAB IacIq ZoM l S]
(Stratagene) P. aeruginosa PABST7.1:
IacUVS/IacIq controlled T7-polymerise gene stably inte-grated in the chromosome of strain P, aeruginosa PASS, which bears a deletion in the structural gene of lipase IipA
(K.-E. Jaeger et al., J. Mol. Cat. Part 8, X997, in press) Plasmid_s pMutS: BamHI/Apal fragment (1045 bp) of the P. aeruginosa lipase gene IipA in the vector pBluescript KSII (Stratagene) pUCPL6A: BamHI/HindlIl fragment (2.8 kbp) comprising the P. aerugi-nosa lipase operon in the vector pUCPKS (Watson et al., Gene 199fi, 172, 163) under the control of the T7 promoter FEB-24-00 14:02 FROM: ID: PAGE 24 Culturing of b~tgri~
E. eoli JM109 is grown over night (16 h) at 37 °C in 5 ml of LB
medium an a test tube roller. For P, aeruginosa PABST7. i, 1 mM IPTG is added to the medium. For the screening test, P. aeruginosa PABST7.1 is grown in microtitration plates on a rotary shaker, the culture volume being 200 NI and the incubation being prolonged to 36-48 h. Antibiotics are added in the following concentrations:
E coli JM109: ampicillin 100 pg/ml; P. aeruginosa PABST7.1: carbenicillin 200 pg/mi, tetracyclin 50 pg/ml Mutagenizina PCR
The lipase gene IipA is amplified using the plasmid pMutS linearized with endonuclease Xmn r as a template and the following PCR primers:
A: 5'-GCGCAATTAACCCTCACTAAAGGGAACAAA-3';
B: 5'-GCGTAATACGACTCACTATAGGGGGAA-~' After purification of the PCR product using a Qiagen Qiaquick Column', it serves as a template in a mutagenic PCR. The reaction conditions are as follows: a 100 NI reaction volume contains 16.6 mM (Nhia)zSOa;
67 mM Tris-HCI {pH 8.8); 6.1 mM MgClz; 6.7 uM EDTA (pH 8.0); O.z mM
dNTPs; 10 rnM mercaptoethanol; 10 pl of DMSO; 10 pmol each of the primers; 0.1 ng of template DNA; and 1 U of Taq polymerase {Goldstar, Eurogentec). The reaction volume is covered with a layer of 100 pl of paraffin. Ten parallel reactions were performed which were combined after completion of the reaction. The cycling protocol is as follows: A
2 min denaturation at 98 °C is followed by Z5 cycles with 1 min at 94 °C, 2 min at 64 °C, 1 min at 72 °C on a Robocycler 40 FEB-24-00 14:03 FROM: ID: PAGE 25 (Stratagene), followed by incubation for 7 min at 72 °C. The Taq polyrnerase is added after the denaturation of the 1st cycle. The sequencing of the PCR products yields an error rate of about 1-2 base substitutions per 1000 bp.
Cloning of the PC products The PCR products are precipitated with ethanol and resuspended in distilled water. After restriction with Apal and BamHI, the 1046 by fragment formed is purified using a Qiagen Qiaquick Column° and ligated into the correspondingly prepared vector pUCPL6A using T4 DNA ligase (MBI Fermentas) for 2 h at room temperature. The reaction volume is diluted 1:5 and transformed into 200 NI of competent cells of E, coli JM109 prepared by the method of Hanahan (J. Mol. Biol. 1983, 166, 557). For this purpose, the DNA and cells are stored on ice for 1 h and incubated with shaking at 42 °C for 2 min and, after the addition of 700 pl of LB medium, at 37 °C for 45 min. The cell suspension is subsequently plated onto LB (ampicillin 100 Ng/ml) plates. Sixty nanograms of the PCR product employed in the ligation reaction will yield about 1500 colonies. All colonies are resuspended in sterile LB
medium, the plasmid DNA is purified and transformed into P. aeruginosa PABST7.i by electroporation according to the method of Farinha and Kropinski (FEMS Microbiol. Lett. 1990, 70, 221). The 96 wells of the microtitration plates are inoculated with one colony each and treated as described in Culturing of bacteria. To obtain the culture super-natant, which is to be employed subsequently in the test for stereo-selectivity, the microtitration plates are centrifuged at 4000 rpm for 30 min.

FEB-24-00 14:03 FROM: ID: PAGE 26 Test for stereoselectivi~,y The lipase-containic~g culture supernatants obtained by centrifugation are pipetted in two aliquots into adjacent wells of a microtitration plate. The test volume is 100 pl and is composed of the following components (Table 2) FEB-24-00 14:03 FROM: ID: PAGE 27 T_",a__b_I_e Z
Composition of the reaction mixture in the test for improved enanti-oselectivity of lipase mutants (R) reaction I (S) reaction 50 NI of culture super- ~ 50 girl of culture supernatant natant 40 NI of 1.0 mM Tris/HCI 40 NI of 10 mM Tris/HCI
buffer, pH 7.5 buffer, pH 7.5 NI of substrate solution 10 NI of substrate solution [10 mg/ml (R)-2-methyl- [10 mg/ml (S)-2-methyl-decanoic acid p~nitrophenyl decanoic acid p-nitrophenyl ester in DMF] ~ ester in DMF]
After the addition of the Tris/HCI buffer to the supernatants, the microtitration plate is incubated at 30 °C for about 5 min. After addition of the substrate solution, the reaction is continuously monitored for IO min by spectrophotometry at 410 nm at 30 °C. From the linear rise of the absorption curve, which is a measure of the constant initial rate of the hydrolysis, the apparent enantioselectivity (Eapp) is determined.
Thus, the slopes measured in the linear region of the initial rates of the reactions for the pair of enantiomers are divided by one another to obtain the value of the apparent cnantioselectivity of the correspond-ing lipase mutant.
Determination of stereoselectivitv by gas chromatograuhy Selected positive clones are grown in 5 ml liquid cultures (LB medium), and after centrifugation and removal of the bacterial pellet, the lipase-containing supernatant is employed for the reaction. As the substrate, FEH-24-00 14:03 FROM: IU

100 pl of a solution of racemic (R,S)-2-methyldecanoic acid p-nitrophenyl ester (10 mg/ml in dimethylformamide) is used. This solu-tion is admixed with 700 NI of 10 mM Tris/HCI buffer, pH 7,5. The reaction is started by adding 100 pl of culture supernatant and per-formed at 30 °C and 1000 rpm in Eppendort reaction vessels. After 2.5 h, samples of 200 ~I each are removed and transferred to an Eppen-dorf vessel filled with 200 NI of dichloromethane. After the addition of 25 N! of 20% aqueous hydrochloric acid, the products and educts are extracted (vortex shaker, 1 min). Finally, the organic phase is used for gas-chromatographic analysis (GC). Separation of the enantiomers of the free 2-methyldecanoic acid is achieved thereby.
Separation conditions of GC:
Instrument: Hewlett Packard 5890 Column: 25 m 2.6 DM 3 Pent ~-CD/80% SE 54 Detector: FID
Temperature: 230 °C inlet; 80-190 °C with 2 °C/min Gas: 0.6 bar Hz Sample quantity: 0.1 ml Results (1st cycle Of the about 1000 clones examined which had been obtained by mutagenizing PGR from the starting DNA (wild type gene of lipase from P. aeruginosa), 12 were identified to have an improved enantioselectiv-ity over that of the corresponding wild type enzyme. Finally, 3 clones were selected and their enantioselectivity determined by GC analysis.

i;
FEB-24-00 14:03 FROM: ID: PAGE 29 - 2? -Ta lei Selected lipase mutants with improved enantioselectivity (1st cycle) M~~ usPP{S) VAPD(R) ~pP'I% ee E value 2}

[rnODtmin] (mODlmin] (by GG)I {calculated conversion from GC) Wild type21.8 14.9 1.5 2.4 I 15.3 1.1 P1 B 01-E4128.4 43.2 3.0 36.1 I 23.2 2.4 P1 B 01-F1278.8 35.7 2.2 14.1 I 30.5 9 .4 P1B01-H1 158.7 56.2 2.8 37.614.5 2.2 ~aPP = R) Vaav(S)IVava~

2) E =
In[1-c(1+eeP)]/In[1-c(1-eeP)]
with c = conversion, eeP =
ee value of the product The DNA of the clone PiB pi-E4 served as the starting point for a new cycle of PGR mutagenization. Thus, the plasmid pUCPL6A was isolated from the clone and transformed into E. coli ~M109 as described above.
After the preparation of the plasmid DNA, the 1045 by fragment was obtained by restriction with Apal and BamHI and subsequent purifica-tion and ligated into the correspondingly prepared plasmid pMutS. After transformation and plasmid isolation, this plasmid served as template DNA in a mutagenizing PCR under the conditions as described above.
The DNA obtained from the mutagenizing PCR served to prepare a new mutant library (2nd generation).
Re_s_ul_ts (2nd c cle From the mutant library of the 2nd generation, about 2200 clones were used for the screening test. Ten mutants with an improved enantiose-lectivity over that of mutant PiB 01-E4 were identified. Two mutants (P2B 04-G11 and P2B 08-H3) were examined more closely by GC
a nalysis.

i~i FEB-24-00 14=04 FROM: ID= PAGE 30 Table 44 Selected lipase mutants with improved enantioselectivity (2nd cycle) Mutant V,Pp(S) V,Pp(R) F_,~')% ee E value Z) [mODlmin][mODlmin~ (by GC)I (calculated conversionfrom GC) P28 04-G11224.9 52.3 4.3 47.8 l 3.4 30.0 P2B 08-H3 310.8 67.4 4.6 56.6 I 4.1 19.3 ~) EaDP - VaPPO)NaPP(R) 2) E = In[1-c(1+eeP)]/In[1-c(1-eeP)] with c = conversion, eeP = ee value of the product -Clone P2B 08-H3 was used for the next mutation cycle (3rd genera-tion) .
Results (3rd cycle) From the mutant library of the 3rd generation, about 2400 clones were used for the screening test. One mutant (P3B 13-DiQ) with an im-proved enantioselectivity over that of mutant P2B 08-H3 was identified.
It was examined further by GC analysis.
Selected lipase mutants with improved enantioselectivity (3rd cycle) Mutant V,~(S) V,pp(R) E,~') % ee E value 2) [mQDlmin][rnODlmin] (by GC)I (calculated conversion from GC) P3B 13-D10240.0 35.2 ~ 6.9 74.8 I 34.610.2 I ~ I

~aPD ~ VaPP~s)NaPP~R~

r i!I
FEB-24-00 14:04 FROM: ID: PAGE 31 2) E x In[1-c(1+eeP)J/Intl-c(1-eev)~ with c = conversion, eeP = ee value of the product Results i(4th r~ycle) From the mutant library of the 4th generation, about 2000 clones were used for the screening test. Four mutants with an improved enanti-oselectivity over that of mutant P3B 13-D10 were identified. They were examined further by GC analysis.
Selected lipase mutants with improved enantioselectivity (4th cycle}
Mutant V,pP{S) Vapp{Rj E,~,')% ee E value 2) [mODlmin][mQDlminj (by GC}I {calculated conversion from GG) P4B 04-H3355.6 26.5 13.4 81.0 I 20.011.2 P48 01-F2162.4 13.8 11.7 82.1 / 5.0 10.6 P4B 15-G1315.4 28.1 11.2 80.0 ! 18.010.7 P4B 15-H7288.0 25.1 11.5 78.4 I 22.010.2 1) ~aPP ~ VaPP~S)/VaaP~R) 2) E = In[1-C(1+eeP)J/In[1-c(1-eep)] with c ~ conversion, eeo = ee value of the product The clone P4B04-N3 was inserted in the next mutation cycle (5~' generation).
Results ,5th cycle From the mutant library of the 5th generation, about 5200 clones were used for the screening test. Two mutants with an improved enanti-oselectivity over that of mutant P4B 04-H3 were identified. They were examined further by GC analysis.

FEB-24-00 14:04 FROM: ID: PAGE 32 Table 7 Selected lipase mutants with improved enantioselectivity (5th cycle) Mutant V,~pp(S) V,pP(R) E,~,')% ee E value 2) [mODlmin][mODlmin] (by GC)I (calculated conversion from GC) P5B 14-C11 275.9 17.3 15.9 77.0143.0 13.7 P5B 08-F2 124.0 8.7 14.3 79.7 ! 40.3 15.1 ~ I

1 J Capp = VaPpW~/ VaPDt K) 2) E = In[1-c(i+eeP))/In[1-c(i-eeP)] with c = conversion, eeP ~ ee value of the product Senuencinq of the positive mutants By sequencing the positive mutants, the mutations could be localized within the lipase genes (see Figure 2), After assigning the base triplets to the corresponding amino acids, the following amino acid substitu-tions result with respect to the wild type lipase from P. aeruginosa:
PiB 01-H1: T103I (Thr103 ~ IIe103), S149G (Ser149 ~ GIy149}
P1B OI-E4: S149G (Ser149 -~ GIy149) P2B 08-H3: S149G (Ser149 ~ G1y149), S155L (Ser155 ~ Leu155) P3B 13-D10: S149G (Ser149 -~ GIy149), S155L (Ser155 -~ Leux55), V47G (Va147 ~ GIy47) P4B 04-H3: S149G {SCr149 ~ GIy149), S155L (Ser155 ~ LeulSS), V47G (Va147 ~ GIy47), S33N (Ser33 ~ Asn33), F259L
(Phe259 ~ Leu259}
P5B 14-Cil: S149G (Ser149 ~ GIy149), 5155L (Ser155 3 Leu155), V47G (Va147 ~ GIy47), S33N (Ser33 ~ Asn33), F259L
(Phe259 ~ Leu259), K110R (Lys110 -~ Arg 110) ~i i I
FEB-24-00 14:04 FROM: ID: PAGE 33 Mutants P1B O1-E4, P2B 08-H3 and P3B 13-D10 were deposited on July 16, 1997, with the DSMZ - Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, D~38124 Braunschweig, Mascheroder Weg ib, under the designations of DSM i 1 658, DSM 11 659 and DSM 1 Z 659, respectively.
Mutants P5B 14-C11 and P4B 04-H3 were deposited on July 20, 1998, with the DSMZ - Deutsche Sammlung von Mikroorganismen and Zellkul~
turen GmbH, D-38124 Braunschweig, Mascheroder Weg ib, under the designations of DSM i2 320 and DSM iz 3Z2, respectively.
. lh a 2 The protocols far the culturing of the bacteria, the mutagenizing PCR
and the test method for enantioselectivity are analogous to those of Example 1. However, in this Example, the preparation of extensive mutant libraries is effected by in vitro recombination.
The DNA used for the in vitro recombination is either generated by mutagenizing PCR or obtained by combining the DNA from any number of clones from one or more clone generations formed by repeated mutageniiing PCR. If the PCR products of a mutagenizing PCR are the starting point for obtaining DNA for the in vitro recombination, the procedure is as follows: The PCR products of the mutagenizing PCR (see Example 1) are purified, cleaved with the restriction endonucleases Apa I and BamH I, ligated into the correspondingly cleaved vector pMUTS
and then transformed into E. coli JM 109. The plasmid DNA from ail transformation clones is isolated. If some number of selected clones from one or more generations of mutant clones are the starting point for obtaining DNA for the in vitro recombination, then the plasmid DNA
of the vector pMUT5 is isolated and combined with the respective variants of the lipase gene of P, aeruginosa. In both cases, the further i~i FEB-24-00 14:05 FROM= ID: PAGE 34 procedure is as follows: Restriction with the endonuclease Pvu II yields a 1430 by fragment which comprises the binding sites of primers A and B already used in the mutagenizing PCR, in addition to the structural gene for the lipase from P. aeruginosa. This fragment is purified and cleaved into randomly generated fragments by incubation with deoxy-ribonuclease I (DNase I from bovine pancreas). The size of the frag~
ments and the error rate of the subsequent reassembling can be influenced by selecting the incubation conditions.
DNase I treatment In a total volume of 100 NI, 3 Ng of Pvu II fragments in 50 mM Tris/HCI, pH 7.5, i0 mM MgCl2 or 10 rnM MnClz, respectively, and 50 Ng/ml BSA is incubated at 23 °C with 0.075 U DNase I for 10-25 min or 1-10 min, respectively. The reaction is terminated by incubation at 93 °C for min. Depending on the reaction time, fragments of smaller than 500 by to smaller than 10 by are obtained. In the Case where only a particular range of sizes is used, these fragments can be obtained from agarose gels by selective electro-blotting on DEAE membrane (accord-ing to F.M. Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley and Sons, 1989). After purification of the fragments by the Qiagen Nucleotide Removal Kit~ (Qiagen), the following reassembling reaction is performed, Reassembling reaction 10-30 ng of the fragments derived from the DNase I restriction a re subjected to the following PCR cycles in 75 mM Tris/HCI, pH 9.0, 20 mM
(NH4)ZSOa, 0.0i°/v (w/v) Tween~ 20, 1.5 mM MgClz, 0.2 mM dNTPs with 2 U Goldstar Taq polymerase (Eurogentec) in a total volume of 50 pl:
2 min at 94 °C, 40 cycles of 1 min at 94 °C, 2 min at 52 °C and 1 min i;i FEB-24-00 14:05 FROM: ID: PAGE 35 at 7Z °C, finally 7 min at 7Z °C. The Taq polymerise is added after the 1 minute denaturing step of the 1st cycle.
i Nl from the reassembling reaction is employed in a subsequent PCR
reaction, which is composed as described for the reassembling reaction with the following differences: instead of the DNase I generated fragments, 1 NI of the reassembling reaction is employed as the template DNA. In addition, primers A and B in a concentration of 0.2 mM
and 10% dimethylsulfoxide are added. The cycling protocol is a s follows:
2 min at 98 °C, 30 cycles of 1 min at 94°C, 2 min at fi4 °C, 1 min at 72 °C and finally 7 min at 72 °C; parallel runs are performed.
The PCR
products formed in these reactions are purified, restricted with the Restriction endonucleases Apa x and Bam Hi and cloned as described in the paragraph Mutagenizing PCR of Example ~..
Results ~(in vitro recombination~w Twelve clones of the 1st generation of the mutant library obtained by mutagenizing PCR (see Example 1) were used for the in vitro recombina-tion. The following clones which had shown improved enantioselectivity in the screening test were used:
P1B 01-AZ, P1B O1-A6, P1B 01-D2, P1B 01-D5, P1B 01-E1, P1B 01-E4, P1B Q1-F3, P1B O1-F11, PIB 01-H1, P1B 01-H3, P1B 01-F12.
The DNA of these clones recombined according to the procedure described above is cloned as stated in the paragraph Mutagenizing PCR, and the culture supernatants are employed in the test for enanti-i r, FEB-24-00 14:05 FROM: ID: PAGE 36 oselectivity. About 1000 recombinant clones were tested. The two identified recombinants S2A OI-EL1 and S2A 02-G3 exhibit a significant improvement of enantioselectivity over the best mutant of the 1st generation (PiB 01-E4) from Example 1.
Tabl~8, Selected lipase mutants with improved enantioselectivity (in vitro recombination) Mutant V,pp(S) V,pp(R) E,~') ~ ee E value 2) [mODVmin]~mODlminJ (by GC)I (calc.f~om conversion GC) S2A 01-E11145.6 41.6 3.5 41.0127.0 2.8 S2A 02-G3 210.8 62.0 3.4 38.0 ! 23.02.5 J APP = VaPGWJI~apP~Kl 2) E = In[i-c(1+ee~))/In[i.-c(i-eeP}) with c = conversion, eeP = ee value of the product x m le,~
Side-directed saturation mutagenesis in the amino acid position 155 of lipase mutant P3B 13-D10:
Plasmids:
pMut5 13D10: BamHI/ApaI fragment (1046 bp) of the gene of mutant P3B 13D10 for the lipase from P. aeruginosa in pBluescript KS II
pMut50AK 13D10: Deletion of the AfIIII/KpnI fragment in pMut5 13D10 FEB-24-00 14:05 FROM: ID: PAGE 37 A fragment of the gene for the lipase from mutant P3B 13D10 is ampli-fied using plasmid pMut5 13D10, linearized by endonuclease XmnI, and the following PCR primers:
A; S'-GCGGAATTAACCCTCACTAAAGGGAACAAA-3' M: 5'-GGTACGGAGAATNNNCTGGGCTCGC-3' where N represents A or C or G or T.
The reaction conditions are as follows: A 50 NI reaction volume contains 75 mM Tris/HCI, pH 9.0 (at Z5 °C); 20 mM {NHa)~S04; 1.5 mM MgCl2;
0.01% (w/v) Tween~ 20; 10% (v/v) DMSO; 10 pmol Of each of the primers; 0.1 ng of the template DNA; and 2 U of Taq polymerase (Goldstar, Eurogentec). The cycling protocol is as follows: A Z min denaturation at 98 °C is followed by 30 cycles with 1 min at 94 °C, 2 min at b4 °C, 1 min at 72 °C on a Robocycter 40 (Stratagene), followed by incubation for 7 min at 72 °C. The Taq polymerase is added after the denaturation of the 1st cycle. After purification of the PCR products by agarose gel electrophoresis and elution of the DNA
from the agarose gel using the Nucleospin Extract Kit (Macherey &
Nagel), it was used as a primer (socalled megaprimer) in a subsequent PCR. Thus, the lipase gene is amplified on the plasmid pMut5aAK
13D10, linearized by endonuclease XmnI, using the following PCR
primers and the above described reaction conditions:
A: 5'-GGGCAATTAACCCTCACTAAAGGGAACAAA-3' B (megaprimer): 5'-GCGTAATACGACTCACTATAGGGCGAA-3' The reaction conditions and the cycling protocol are as described above, except that 1-10 ng of the megaprimer is added to the reaction mixture. The cloning of the PCR products is effected as described under Cloning of the PCR Products.

_. .._. i;i FEB-24-00 14:05 FROM: ID: PfIGE 36 I r i n mu n n r ion P 1 0 From the mutant library of the saturation mutagenesis (3rd generation, P3B 13-D10}, about 900 clones were used for the screening test. One mutant (P4BSF 03-G10) with an improved enantioselectivity over that of mutant P38 13-D10 was identified. Tt was examined further by GC
analysis.
Ta le 9 Selected lipase mutant with improved enantioselectivity (3rd genera-tion, P3B 13~D10}
Mutant VaPO(S) Vpp(R) Ep~') % ee (by E value z) GC)I

(mODlmin][mODlnrinj % conversion(calculated from GC) P4BSF 384.7 25.3 15.2 87.3119.0 20.4 taPP - vaPP~S~IVapD~R~
2) a = in[1-c(1+eeP)]/In[1-c(1-eec)] with c = conversion, eeo ~ ee vane of the product FEB-24-00 14:06 FROM: ID: PAGE 39 Seauencing of the positive mut~n~,sL
By sequencing the positive mutants, the mutations could be localized within the lipase gene (see Figure 2). After assigning the base triplets to the corresponding amino acids, the following amino acid substitution resulted with respect to mutant P3B 13-DiO:
P4BSF 03-G10 : L155F (Leu155 ~ Phe155) Mutant P4BSF 03-G10 was deposited on ~uiy 20, 1998, with the DSMZ -Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, D-38i24 Braunschweig, Mascheroder Weg 1b, under the designation of DSM 12 321.

i;i FEB-24-00 14:06 FROM: ID: PAGE 40 SEQUENCE LISTING
(1) GENERAL INFORMATION:
{i) APPLICANT:
(A) NAME: Studiengesellschaft TCohle mbH
(B) STREET: Kaiser-Wilhelm-Platz 1 (C) CIfY: Muelhei.m an dex Ruhr {E} COUNTRY: Germany (F) QOSTA1~ CODE (ZIP): 45470 (1i) TITLE OF INVENTION: A Process for the Prcperation and Identification of Novel Hxdrolases Having Improved Properties (iii) NUMBER OF SEQUENCES: 71 {iv) COMPUTER RFADAF3LF, FORM:
(A) MEDIUM TYPE: Floppy disk (8) COMPUTER: JAM PC compat~.bJ,e (C) OPERATING SYSTEM: PG-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release X1.0, Version 11.30 (Ef0) f2l INFORMATION FOR SEQ ID N0: 1:
{i) SEQUENCE CH11RACTERI5TIC5:
(A) ~.ENGxH: 30 bass paixs (6) TYPE: nucleic acid (C} STRANDEDNESS: unknown (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /dpsc = "synthetic DNA"
(xil SEQUENCE DESCRIPTION: SEQ ID N0: J.:
GCGCAATTRA CCCTCACTpA ACGGAACAAA 30 (2} INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARP,CTFRISTICS:
(A) LENGTH: 27 base pairs (8) TYPE: nucleic acid (C} STRANDEDNESS: unknown (U) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "synthetic DNA"
(x:i.) SFQUF,NCF nESCR>;PTION: SEQ ID N0: 2:
GCGTAATi4CG AC'fCACTATA GGGCGAA 27 (2) INFORMATION FOR SEQ ID N0: 3:
(i) SEQUENCE Cf~ARACTERISTICS:
(A) LENGTH: 1049 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) ili FEB-24-00 14:06 FROM: ID: PAGE 41 (ix) ~'E.'9TUf~E:
(A) NAME/KEY: CDS
(B) LOCATION:85..1017 (ix) FEATURE:
(A) NAME/KEX: mat~eptide (8) LOCATION:163..1017 (X1) SEQUENCE DESCRIPTION: SEQ ZD N0: 3:
GGATCCCCCG GTTCTCCCGG GCAGGACGCG CCCCTCGGCC

AAGGATTCGG
GCGATGGC'I'G

CCA TCAACCT GAGATGAGAA ATGAAG AAGAAGTAT CTGCTCCCC C1'C 111 CAAC

MetLys LysLysTyr Leul~euPro i~eu Gly LeuAla IleGlyLeu AlaSexLcu AlaAlaSer ProLeuIle Gln Ala SeiThr TyrThrG1~ ThrLysTyx ProIleval LeuAlaHis Gly ATG c'rcGGC TTCGACAAC ATCCTCGGG GTCGACTAC TGGTTCGGC ATT 255 Met LeuGly PheAspAsn IleLeuGly ValAspTyr.TrpPhcGly Ile CCC AGCGCC TTGCGCCGT GACGGTGCC CAGGTCT11CGTCACCGAA G't'C 303 PrA SerAla LeuArgArg AspGl,yA1a GlnValTyr ValThrG,l,uVal.

AGC CAGTTG GACACCTCG GAAGTCCGC GGCGAGCAG 'rTGCTGCAA CAG 351 , Ser GlnLeu AspThrSer GluValArg GlyGluGln .T_,eT.PG1 G1 n n GTG GAGG111iATCGTCGCC CTCAGCGGC CAGCCCAAG GTCAACC:TGATC 399 Val GJ.uGl.uIleValAJ.aLEUSerGly GlnProI,y~ValllsnLeu Ile CCC C11C11GCCE1CGGCGGG CC:~ACCATC CGCTACG1'CGCCGCCGTA CGT 447 G HisSer HisGJ.yG1y prcThrIle Ary~ryrVal AlaAlaVal Arg l y ~ 85 yf.~ 95 O

CCC GAGC;'I'GA'1'CGC1'TCC GCCATCAGC GTCGGCGCC CCGCACAAC CGT 4 Pro A.,pLcu IlclllaSer AlaI1eSer vaiGlyAla ProHisLys G1y TCG GACACC GCCGACTTC CTGCGCCAG ATCCCACCC cGTTcGGcc GGC 513 Ser AspThr Ala.A.cpphP T,pnArgGln IleProPro GlySerAld Gly GAG GCAGTC CTCTCCGCG CTGGTCAAC AGCCTCGGC GcGcTGATC:AGL 591 G.l.uAlaVal Lo_uSerGly LeuVa.lAs SNrT,a~iGl.yAlaLeuIle Ser r~

130 ~3s i~o TTC CTT TGC AGC GGr, c;c;c: Arc: GGT ACG CAG AAT TCA CTG GGC TCG CTG 639 Phc Lcu Scr Ser Gly Vly ~d~hr Gly Thr G1n Asn Sex Leu Gly Ser Leu 145 1~0 155 GAG 'i~CG CTG AAC Aec GAG GGT GCC GCG CGC 'rTC AAC GCC AAG TAC CCG 687 Glu Scr Leu Asn Ser Glu Gly A.la Ala Arg Phe Asn Ala Lys Tyr Pro FEB-24-00 14:06 FROM: ID: PAGE 42 CAGGGC A'1'CCCCACC TCGGCC TGCGGCGAA GGCGCCTAC GTC AAC 735 AAG

Gl.nGly IleProThr SerAla CysGlyGlu GlyAl,aTyr LysVal Asn GGCGTG AGCTATTAC TCCTGG AGCGGTTCC TCGCCGCTG ACCAAC T'1'C7$3 GlyVal SerTyrTyr SerTrp SerGlySer SerProLeu ThrAsn Phe C'TCGAT CCGAGCGAC GCCTTC CTCGGCGCC TCGTCGCTG ACCTTC AAG 831 i,euAsp ProSerAsp AlaPhe LeuGlyAl.aSerScrLeu ThrPhe Lys AACGGC AccGCCAAC GACGGC CTGGTCGGC AccTGCAGT TCGCAC CTG 879 AsnGly 'PhrAJ.aAsn AspGly LeuValGly ThrCysSer 5orIsisLeu GGCATC GTGATCcGC GACAAC TACCGGATG AnccACCTG GACGAG GTG 927 GlyMet ValIleAxg AspAsn TyrArqMet AsnHisLeu AspGlu val AsnG;l.nvatPheGly LeuI'hrSerLeuPhe GluThrSer ProVal Ser 260 265 ?7Q

GTCTAC CGCCAGCAC GCCAAC CGCCTGRAG AACGCCAGC C'!'G 101.7 ValTyr ArgGlriHi.sAlaAsn ArgLeuLys AsriAlaSer Leu TAGGACCCCG GCCGGGGCC1' CGGCCCGGGC CC 1049 (2) INFORMATION FICR SEQ J:D N0: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 311 amino acids t8) TYPE: amino acid (D) TOPOLOGY: Zinear (ii) MOLECULE TYPE: protein (xi) SEQUk~NCE DESCRJPTION: SEQ ID NO: 4:
Mpt Lys Lys Lys Tyr Leu Leu Pro Leu Gly Leu Ala Ile Gly Leu Ala Ser Leu Ala AJ.a Ser Pro Leu Ile Glri Ala Ser Thr. Tyr Thr Gln Thr Lys Tyr Pro Ile V~t i~Pls Ala His GIy Met Leu Gly Phe Asp Asn Ile Leu Gly Vat Asp Tyr Trp Fhe Gly Ile Fro Scx l~la Leu Arg Arg A.sp Gly Ala Gln VaJ. Tyr val Thr Glu Val Ser, Gln Leu Asp Thr Ser Glu Val llrg Gly Glu Gln Leu Leu Gln Gln Val Glu G7,u Ile Val Ala Leu Ser Gly Gln Pxo Lys Val Asn Leu Ile Gly Ftis Sex. His Gly Gly Pro Thr. Il.e Arg Tyr val Ala iSla Val Arg Pz'o Asp Leu Ile Ala Ser Ala FEB-24-0O 14:06 FROM: ID: PAGE 43 ile Ser Val Gly Ala Pro His Lys Gly Ser Asp Th,r. Ala Asp Phe Leu Arg Gln lle Pro Pro Gly Scr Ala Gly Glu Ala Va.7. Leu Ser ~Gly Leu Val Asn Ser Leu Gly Ala Leu Ile Ser Phe Lcu Ser Ser Gly Gly '!'hr Gly Thr Gln Asn Sex Lcu Gly Ser Leu Glu Ser Leu Asn Scr Glu Gly 155 7.60 165 Ala Ala Arg Phe Asn Ala Lys Tyr Faro Gln GJ.y .T.3.e Pro Thr Ser Ala Cys Gly Giu Gly Ala Tyr Lys Val Asn Gly Val Ser Tyr Tyr Ser Trp Ser G,ly Ser Ser Pro Leu Thr Asn Phe Leu Asp Pro Ser Asp Ala Phe Leu G:l,y Ala ser Ser Leu '!'hr Phe Lys Asn Gly Thr Ala Asn Asp Gly Leu Val Gly Thr Cys Ser Sc:r His Leu Gly Met VaJ. I1e Arg Asp Asn 235 240 2~5 Tyr Arg Met Asn His Leu Asp Glu Va,~, Asn Gln Val phe Gly Leu Thr Ser heu Phc Glu Thr Ser Pro Val Sex val Tyr Arg Gln His Ala Asn Arg Leu Lys Asn Ala Ser Leu (2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) Lk:NGTH: 1049 base pairs (B) TYPE: nucleic acid (C) STRANUEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: DN11 (genomic) (ix) FEATURE:
(A) NAME/KEY: CDS
(H) LOCATION:85..1017 ( a. x ) FEATURE
(A) NAME/KEY: znat-peptide (Bl LOCATION:163..101~
(xi) SEQUk:NCE DESCRIPTION: SEQ ID N0: 5:
GGATCCCCCG GTTCTCCCGG 1~GGA1~TCGG GGCATGGCTG GCAGGACGCG CCCCTCGGCC 60 CCATCAACCT GAGATGAGAA CAAC ATG AAG ~1AG AAG TC'J;' CTG CTC CCC CTC 111 Met Lys Lys Lys Sir Leu Leu Yro Leu Gly Leu Ala Tl.e Gly Lcu 111a Ser Leu Ala Ala Ser Pro Leu Ile Gln -15 -10 ~5 FEB-24-00 14:07 FROM: ID: PAGE 44 '1'AC

AJ,aSer ThrTyrThr Gln'rhrLys TyrPro IleValT.euAlaHisGly 1 5 1.0 15 ATGCTC GGCTTCGAC A11C1~.TCC'1'CGGGGTC GACTACTGG TTCGGCA'TT 7_55 MetLeu G1yPheAsp AsnI1eLeu Glyval AspTyr~I'rpPheGlyIle CCCAGC GCCTTGCGC CGTGACGGT GCCCAG GTCT'ACCTC ACCGAAGTC 303 Proser AlaLeuArg ArgAspGly AlaGJ.nVa.I.Tyrval ThrGluVal AGCCAG TTGGACACC TCGGAAGTC CGCGGC GAGcAGTTG CTGCAACAC; 351 SerGln LeuAspThr Ser,GluVa1 ArgGly GluGlnLeu LeuGlnGln ValGJ,uGluIleVal AlaLeuSex GlyGln proLysVal AsnLeuIle GlyHis SerHisGly G.l.yProThr lleArg TyrvalAla AlaVaiArg P.ro,AspLeuIleAla SerAlaThr SerVal GlyAlaPro HisLysGly TCGGAC nCCGCCGAC TTCCTGCGC CAGATC CCACGGGGT TCGGCCGGC 593 SerAsp ThrAlaAsp PheLeuArg GlnIle ProProGly SexAlaGly GAGGCA GTCCTCTCC GGGCTGGTC 11110AGC CTCGGCGCG CTGATCAGC 591.

GluAla ValLeuSer GlyLeuVal AsnSer LcuGlyAla LeuzleSFr 130 135 1.40 1'TCCTT TCCAGCGGC GGCACCGGT ACGCAG AAT'1'CACTG CGCTCGCTG 639 PheLeu ser5erGly Gl,yThrGly ThrGln AsnSerLEU GlySerLeu GAG'PCGCTGAACACC GAGGGTGCC GCGCGC TTCAACGCC AAGTAGCCG 687 GluSer LeuAsnSer GluGlyAla Alal~lrgPheAsnAla LysTyrPro 160 165 1~i0 1'75 Gln Gl.y IJ_e Pro Thr Ser Ala Cys Gly Glu Gly Ala Tyr Lys val Asn Gly Val Sex Tyr Tyr Ser Trp Ser Gly Ser Ser Pro Taeu Thr Asn Phe Leu Asp Pro Ser Asp Ala Phe Leu Gly Ala Ser Ser ,heu Thr Phe Lys AAC GGC AcC GCC AAC GAC GGC cTG GTC GGC ACC TGC AGT TCG CAC cTG 879 Asn Gly Thr Ala Asn Asp GJ_y hpu Val Gly Thr Cys Ser Ser His Leu GGC ATG GTG A'i'C CGC GAC N1C TAC CGG ATG AAC C~1C CTG GAC GAG GTG 927 Gly Met Val Ilc llrg Asp Asn Tyr Arg Met A.sn His L~ru As,p Glu Val FEB-24-00 14:07 FROM: ID: PAGE 45 Asn Gln Val Phe Gly Leu Thr Ser Leu Phe Gl,u Thr Ser Pro Val Scr Val Tyx' Arg Gln His Ala Asn Arg Leu Lys Asn Ala Ser Leu (2) INFORMATrON ~'OR SEQ ID N0: 6:
(i) SEQUENCE CHARACTF,RISTICS:
(AI LENGTH: 31.1 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TXPE: protein (ri) SEQUENCE DESCRIPTrON: SEQ ID N0: 6:
Met hys J.,ys Lys Ser Leu Leu Pro Leu Gly Leu Ala IJ.e Gly Lcu Ala Ser Leu Ala Ala Ser P,ro J,eu Ile Gln Ala Ser Thr Tyr Thr Gln Thr -J.0 -5 1 5 Lys Tyr Pro Ile Va1 Leu Ala His Gly Met Leu Gly Phe Asp Asn I1~

Leu Gly vaJ. Asp Tyr Trp Phe Gly Ile Pro Sex AJ.a J.eu Arg Arg Asp Gly Ala Gln val Tyr val Thr Glu val Ser G1n l,eu Asp Thr Scr G1u VaJ. Arg Gly Glu Gln Leu Leu Gln Gln val Glu Glu Ile Val Ala Leu 55 60 65 ?0 5er Gly Gln Pro Lys val Asn Leu Ile Gly His Ser Ha.s Gly G1y Pro Th.r. Ile Arg Tyr val Ala Ala Val Arg Pxo Asp Leu Ile Ala Ser Ala Thr Ser val Gly Ala Pro His Lys GJ.y Ser Asp Thr Ala Asp Phe Leu Arg Gln IlE Pro Pro Gly Ser AJ.a Gly Glu Ala val Leu Ser Gly Lev, 120 7.25 130 Val Asn Ser Leu Gly Ala T~eu Ile Ser Phe Leu Ser Ser Gl~y GJ.y Thr Gly Thr Gln Asn SEr Lcu Gly Ser Leu Glu Ser Leu Asn 5er Glu Gly 155 160 7.65 Ala Ala Arg Phe Asn Ala Lys Tyx Fro Cln Gly Ile Pro 'i'hr Ser Ala 1~0 175 180 C;ys Gly Glu Gly Al.a Tyr Lys val Asn Gly val Ser Tyr Tyr. SQL T~~
l90 ~~s Ser Gly Ser Sez Pzo Leu Thr Asn Fhe Leu Asia Fro Ser Asp Ala Phe FEB-24-00 14:07 FROM: ID: PACE 46 Leu Gly Ala Scar Ser Leu Thr Phe Lys Asn Gly 'rhr Ala Asn Asp Gly 21S ?.20 225 230 Leu Val Gly Thr Cys Ser Ser H.is Leu Gly Met Val I.le Arg Asp Asn Tyr Arg Met Asri His Leu Asp Gl.u Val Asn Gln Val Phe Gly Leu Thr 250 755 .7, 60 Sex Leu Phe Glu Thr Sex Pro Val Ser Val Tyr Arg G1n His Al.a Asn Arg I,eu Lys Asn Ala Ser Leu (2) INFORMA'T'ION FOR SEQ ID N0: 7:
(~.) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1099 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (i.i) MOLECULE TYPE: DNA (genomic) (ix) FEATURE:
{A) NAME/KEY: CDS
(B) LOCATION:85..1OZ~
(ix) FEATURE:
(A) NAME/KEY: mat~Ept:i.de {H) LOCAT10N:163..1017 (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 7:
GGATCCCCCG GTTCTCCCGG AAGGA'iTCGG GCGATGGCTG GCAGGACGCG CCCCTCGGCC 60 CCATCAACCT 11.7.
GAGA'1GAGAA
CAAC
ATG
AAG
AAG
AAG
T'C'1' CTG
CTC
CCC
CTC

Met Lys Lys Lys Se,r.
Leu Leu Pro Leu ~26 -20 GGCcTGGCC ATCGGT CTCGcCTCT CTCGC'CGCC AGCCCTCTG ATCCAG 159 G1yLeuAla IleGly LeuAla5er l~euAlaAla SerProLeu IleGln GCCAGCACC TACACC CAGACCA~1ATACCCCATC GTGCTGGCC CACGGC 20'~

AlaSexThr TyrThr GlnThrLys TyrProIle V3.).LeuAla H.isGly ATGCTCGGC TTCGAC AACATCCTT GGGGTCGAC TACTGGTTC GGCAT'1'255 MetLeuGly PheAsp AsnIleLeu GlyVallispTyr"1'rpPhe GlyIle ProSerAla LeuArg Ar.gAspGly AlaGlnVal TyrValThr GluVal AGCcAGT'rGGACACC TCGGAAGTC CGCGGCGAG CAGTTGCTG calACAG 351 SerGlnLeu AspThr ScrGluVal ArgGtyGlu GlnLeuLeu GlnGln ValGluGlu IleVal Alaz~euSer GlyGlnPro LysvalAsn LeuIle FE8-24-00 14:0A FROM: ID: PAGE 47 GGCC.ACAGC CACGGCGGG CCGACC CGCTAC GTCGCCGCC GTA 497 A'fC CGT

GlyHisSer HisGlyGly ProThr Il,eArgTyr ValAlaAla val.Arg CCCGACCTG ATCGCTTCC GCCACC AGCG'T'CGGC GCCCCGCAC AAGGGT 495 ProAspLeu IleAlaSer AlaThr ServalGly AlaProH,i.sLysGly 100 10.5 lI0 SerlispThr AlaAspPhe r..euArg Gl,nIlcPro ProGlySer AJ.,aGly GluAlaVal heuSerG1y LeuVal,AsnSerLeu GlyAlaLeu TleSer 1,30 135 140 PheLeuSer SerGlyGly ThrGly Thr.GlnAsn LeuLcuGly ScxLeu GAGTCGCTG AACAGr:GAG GGTGCC GCGCGCTTC AACGCCnAG TACcCG 68~

GluSerLeu AsnSerGlu GlyAla AlaArgPhe AsnAlaLys TyI;Pro 160 7.65 170 175 CAGGGCATC CCCACCTCG GCCTGC GGCGAAGGC GCCT~1CAAG GTCAAC 735 G.l,nGlyIle ProThrSer nlaCys GlyGl,uGly AlaTyrLys ValAsn GGCGTGAGC TATI'ACTCC TGGAGC GGTTCCTCG CCGCTGACC AACZ"1'C783 GlyValSex' TyrSer TrpSer GlySerSer ProLeuThr AsnPhe z95 200 205 CTCGATCCG AGCGACGGC TTCcTC GGCGccTCGTcG CTGACC TTC 831 AAG

LeuAspPro SerAspAla PheLeu GlyAlaSerSer LeuThr PhcL.ys 21.0 215 220 AACGGCACC GCCAACGAC GGCCTG G'1'c:GGCACCTGC Ac:'rTCG CACCTG 879 AsnGiyThr AlaAsnAsp GJ,xLcu ValGIyThrCys SexSer HisLeu 2~~ 230 235 GGCATGGTG ATCCGCGAC AACTAC GGc;A'1'~AAGCAC CTGc:ACGAG~TG 9~7 GlyMetVat T1_AArgAsp AsnTyr ArgMctAsnIti9LrCLiAsp G1uvdl .~,ACGAGGTG rTCGGCCTC ACCAGC C'i'GTTCGAGACC AGCCCG GTCA~;C97 h AsnGlnVal Phpc1_yLeu ThLSer LeuPheGIu'1'hrSPrFro ValSe.c GTCTnCCGC cAGc:ACGCC AACCGC CTGAAG~C GCC HGCCTC; I01?

valTyrArg GlnHisAla AsnArg L.euLysAsnAIa SerLeu 2~~ zao TAGGACCCCG C:C 10q GCCGGGGCCT y CGGC;CC;UGGC

(%) INFORMATION FOR SEO Ip NO: 8:
(i) SEQUENCE CHARAC'1'~:RISTIf:S:
(A.) LENGTH: 311 amino acids (B) TYPI~:: amino acid (D) TOPOLOGY: linear (ii) MOLECUhF. TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ZD N0. 8:

ID: PAGE 49 FEB-24-00 14:09 FROM:

Met Lys Lys i,ys Ser Lcu Leu Pro Leu Gly Leu Ala I;Le Gly Leu Ala Ser Leu ia.la A13 Ser Pro Lcu Ile Gln Ala Scr Thr Tyr Th.r Gln 'I~hr Lys Tyr Pro Ile val Leu Ala His Gly Met Leu Gly Phe Asp Asn I.le Leu Gly Val Asp Tyr Trp Phe Gly Ile Pro Ser Ala I~eu Arg Arg Asp Gly Ala Gln Val Tyr Val Thr GIu Val Ser Gln Leu Asp Thr Ser Glu.

Val llrg Gly Glu Gln Leu Leu Gln Gln Val Glu Glu Ile Val Ala Leu 55 60 65 j0 Ser Gly Gln Pro Lys VaI Asn Leu Ile Gly Hi.s Ser His Gly G1y Pro Thr Ile Arg Tyr Val AIa Ala Val Arg Pro hsp Leu Ile J~l,a Ser Ala Thr Ser Val Gly Ala Pro His Lys Gly sex Asp Thr A.la Asp Phc Leu Axg Gln Ilc Pro Pxo Gly Ser Ala Gly Glu Ala Val Leu Ser Gly Leu ~. 20 125 130 Val Asn Ser Lcu Gly Ala Leu Ile Ser Phe Leu Ser Ser Gly Gly Thr Gly Thr Gl_n Asn Leu Leu Gly Ser Leu G.lu Ser Leu Asn Sez' Glu Gly 7.55 160 165 Ala ALa ~lrg phe Asn Ala Lys Tyr Pro Gln Gly Ile Pro '!'hr Ser Ala Cys Gly Glu Gly 111a Tyr Lyg Va.l, Asn Gly Val Ser Tyr Tyr Ser Trp Ser Gly Ser 5er Pro Leu Thr Asn Phe Leu Asp L~ro Ser Asp Ala Phe Leu Gly Ala Ser Ser Leu Thr Phe Lys Asn G7.y Thr Ala Asn Asp Gly Lcu Val Gly Thr Cys Ser Ser. His Leu Cly Met Vai Il.e Arg Asp Asn Tyr Arg Met Asn His Leu Asp Glu Val Asn G.7.,n Val J'he Gly Lcu Thr Ser Leu Phe Glu Thr Ser Pr.o Val. Ser Val Tyr Arg Gln His Ala Asn Arg heu Lys Asn Ala Sex Leu (2) INFORMATION FOR SEQ ID NO: 9:
(i} SEQUENCE CHARACTERISTICS:
(A} LENGTH: 10!7 ba;;e pairs FEB-24-00 14:06 FROM: ID: P14GE 49 {B) TYPE: nucleic acid {C) STRANDEDNESS: unknown (D) TOPOJ,OGY: unknown {ii) NJOLFCULE TYPE: vNA (genomic) (iX) FEATURE:
{A) NAME/KEY: CDS
(R) LOCATION:84..1016 (.ix) FEATURE:
tA) NAME/KEY: mat~pept~.de {8) LOCA'I'ION:162..1016 {xi) SEQUENCE DESCRINTION: SEQ ID N0: 9:
GGA'1'CCCCGG TTCTCCCGGA AGGATTCGGG CGATGGCTGG CAGGACGCGC CCCTCGGCCC 60 CATCAACCTG AGATGAGAAC AAC ATG AAG AAG AAG TCT CTG CTC

Met Lys Lys Lys Ser Leu Lcu Pro Leu GGC CTG GCC ATC GGT CTC GCC TCT CTC GCT GGC AGC CCT CTG

ATC CAG 1.58 Gl,y Leu Ala Ile Gly Leu Ala Ser Leu Ala Ala Se P

r ro Leu Ile Gln -IO _g GCC AGC .ACC TAC ACC GIG ACC AAA TAC CCC ATC GTG

Ala Ser Thr Tyr Thr Gln Thx Lys '1'yr Pro I1 l e Va Leu Ala Flis G1y 'i ' 7. 5 ATG CTC GGC TTC GAC AAC ATC CTC GCG GTC GAC TAC TGG

TTC GGC ATT 25~
Met Leu Gly Phe Asp Asn Il.e Leu Gl Val A
T
~

y sp yr, 1 rp Phe Gly Ile CCC AGC GCC TTG CGC CGT GAC GGT GCC CAG GTC

' ~C G
TC ACC GAA GTC
Pro Ser Ala Leu Axg Arg Asp Gl Ala GI
V
l y n a Tyr va,l Thr Glu Val AGC CAG TTG GAC ACC TCG GAA GTC CGC GGC GAG CAG TTG

Ser Gln Leu Asp Thr Ser Glu Val Arg G~
y GIU Gl , n Leu Leu Gln Gln GTG GAG GAA ATC GTC GCC CTC AGC GGC CAG CCC AAG GTC A

AC cTG ATC 398 Val Glu Glu IIe Val 111a Leu Ser Gl Gl p y n ro I,ys val Asn Leu Tle GGC C.~C AGC CAC GGC GGG CCG ACC ATC CGC TAC GTC GCC

G.ly His Ser His G7.y Gly Pro Thr Ile Arg T
r V
l Al y a a Ala Val Arg CCC CAC CTG ATC GCT TCC GCC ACC AGC GTC GGC GCC CCG CA

Pro Asp Leu Ile Ala Ser ala Thr Ser 'VaJ
Gl Al P

, y a ro His Lys Gly 105 17.0 TCG cAC ncC GCC GAC TTC CTG CGC CAG IaTC CCA CCG

Sex Asp Thr Ala Asp Phe Leu Axg Gln Il P

e ro Pro Gly Ser Ala Gly GAG CCA GTC cTC Tcc GGG CTG GTC AAC AGC CTC Gf;r GC

G CTG ATC J~CC ;:90 GJ.u Ala Val Leu Ser Gly Leu Val A
S

an ex Leu C;ly Ala Leu lle Se.r 1_i5 190 TTC cTT TCC AGC GGC AGC Acc GGT ACG cAG AAT Tr A eT
~

_. 638 G GGC
rcG c:TG
Phe Lou Ser SNr Gly Ser ~rl,.r Cly Thr Gl A

n EI= J~_Y Leu Gly Ser Leu 195 15c) FEB-24-00 14:06 FROM: ID: PAGE 50 GAGTCGCTG AACAGCGAG GGTGCC GCGCGCT'TCAACGCCAAG TAGCCG 686 GluSerLeu AsnSerGiu GlyAla AlaArgPhe AsnAlaLys TyrPro ~5 GAGGGCATC CCCACCTCG GCCTGC GGCCAAGGC GCCT11CAAG G'fCAAC 734 G,lnGiyIle ProThrSer AlaCys GlyGluGly AlaTyrLys ValAsn GGCGTGAGC TATTAGTCC TGG1~GCGGTTCCTCG CCGCTG11CCAACTTC 782 GlyValSer TyrTyrSer TrpSer GlySerSer ProLeu'ThrAsnPhe CTCGATCCG ACCGAGGCC T'TCCTC GGCGCCTCG TCGCfGACC TxCAAG 830 LeuAspPro SerAspAla PheLeu GlyAlaSer SerLeuThr pheLys z,.
o 215 220 AsnGl,yThr AlaAsnAsp G.lyLeu ValGlyThr CysSerSer HisLeu AAC

GiyMetVal IleArgAsp AsnTyr ArqMetF.snHisLeuAsp G1uVal AACGAGGTC TTCGGCCTC ACCAGC CTGTTCGAG ACCAGCCCG GTCAGC 97~

AsnGlnVal PheClyLeu ThrSer LeuPheGlu ThrSerPro Val5er GTCTAGCGC GAGCACGCC AACCGC CTGAAG1~4CGCCAGCCTG x026 ValTyrArg GinHisAla AsnArg LQULysAsn AlaSerLeu 27g 280 285 TAGGACCCCG C
GCCGGGGCC'f CGGCCCGGGC

(2) INFORMATION fl~R Sk;Q ID NO: 10:
(i.) SEøUENCE CHARACTERISTICS:
(A} LENGTH : 311 am:i rrc, acids (8) TYPE:; amino BCid (U) ToPCLOGY: linaar (ii) MOL~;CULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEø ID N0: 10:
Met Lys Ly3 Ly3 Sci Leu hev pro Leu Gly Leu At.tt Ile fly Leu Ala Ser Leu Ala Ala Ser Pro Lcu Ile Glr~ Aid Spr Thr '.t'yr Thr Gln Thr ~la ~J ~ 5 l.ys Tyr Pro Ilo Val Leu A.le His Gi5 Met Leu Gly Fhe A3p Asn Ile ~n 2U
Leu Gly Val A3p Tyr Trp phe Gly Ile rro Ser Al:r Leu Arg Arg 1s.3,p GJ.y Ala Gln Val Tyr val Thr Glu Val Ser Gln Leu Asp Thr Ser Glu Val Arg Gly Glu Gln Leu heu Gin Gln Vai Glu Glu Ile Val Ala Leu 55 60 65 'I O
Ser Gly Gln Pro Lys Val, Asn Leu Il,e G1y His Ser H;~.s Giy Gly f'ro FEB-24-00 14:09 FROM: ID: PACE Sl Thr Ile .Ard Tyr Val Ala A.la Val A95 Pro lap Leu Ile ~A.7.a Ser Ala g0 100 Thr Ser Val Gl~r Ala pro His i~s0 Gly Scr Asp Thr A7,a Asp Phe Leu Arg Gln Ile Pro Pro Gly Ser Al,a Gly Glu A.la Val Leu Ser Gly Leu 1.20 Val Asn Ser Leu Gly Ala Leu Ile Ser Phe heu Ser Ser. Gly Ser Thr Gly Thr Gln Asn Ser Leu Gly Ser Leu Glu Ser Leu Asn Ser Glu Gly i55 a60 7.65 Ala Ala Arg Phe Asn Ala Lys Tyr Pro Gln Gly Ile Pxo Thr Ser Ala Cys Gly Glu Gly Ala ~'yr Lys vat Asn Gly Va.~. Ser Tyr Tyx Ser Trp 185 190 1.95 Ser Gly ser Ser Pro Lau Thr Asn Phe Leu Asp Pro Ser Asp Ala Phe Leu Gly Ala Sex Ser Lcu Thr Phe Lys Asn Gly Thr AJ.a Asn Asp Gly 215 220 2z5 230 Leu Val Gly Thr Cys Scr Ser His Leu Gly Met val .rle Elrg Asp Asn Tyr Axg Met Asn His Leu Asp Glu Val Aan Gln Val Phc Cly Leu Thr Ser Leu Phe Glu Thr Sex Fro val Ser Val Tyr Axg Gln His Ala Asn Arg L~u Lys Asn Ala Se:r Leu (2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERTSTrCS:
(A) LENGTH. 1049 base pai.xs (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (,ii) MOLECULE TYPE: DNA (genomic) (.ix) YEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:85..1017 ( i.X ) FEATURE
(A) NAME/KEY: mat-peptide (8) LOCATION:163..101'7 (xi.) S~;QUENCE DESCRTPTION: SFQ ID N0: 11:

CCATCAACCT GAGATGAG~ CAAC ATG AAG AAG AAG L'CT CTG CTC CCC CTC 111 "~.,,.
FEB-24-00 14:09 FROM: ID: PAGE 52 Met Lys Lys Lys Ser L~u T,eu Pro L,eu Gly Leu AlaIle GlyLeuAla SexLeuAlc~AlaSerP.roLeuIleGln GCC AGC ACCTAC ACCCACACC AlIATACCCC ATCG'PGCTG GCCCACGGC 207 Ala Ser ThrTyr ThrGlnThr LysTyrPro 11eVa1Leu AlaHisGly ATG CTC GGCTTC GACAACATC CTTGGGGTC GACT11CTGG TTCGGCATT ?55 M2t Leu GlyPhe AspAsriIle LruG,lyval AspTyrTrp PheGIyI).e Pra Ser AlaLeu ArgArgAsp G7.yAlaGln ValTyrval ThrGluGly Sex Gln LeuAsp ThrSerGlu ValArgGly GluGlnLeu LeuG.lnGln Val Glu GluIlc VaIAlaLeu SerGlyGln ProLysVal AsnLeuIle GGC cAC AGCCAC GGCGGGCCG AcCATCCGc TACGTCGCC GcCGTACGT 447 Gly His 5orHis GlyGlyPro ThrIlcArg TyrvalAla AlavalA,rg Pro Asp Leu11e AlaSerAla ThrSc:rVal GlyAlaPro HisLysGly Ser Asp ThrA1a AspPheLeu ArgGlnIle ProProGly SexAlaG1y 115 17.0 125 Glu Ala ValLeu SerGlyLau ValAsnSer i,euGlyAla LeuIleSer TTC CTT TCCAGC CCCCCCACC GGTACGCAG AATTTACTG GGCTc:GCrG 639 Phe Leu SerSex GlyGlyThr GlyThrGln AsnLeuLcu GlySerLeu 145 1:p0 155 GAG TCG CTGAAC AGCGAGGG'PGCCGCGCGC 'T"1CAACGCC AAGTACCCG 68'~

Glu Ser LeuAsn SerGluGly AlaAa.aArg PheAsnAia LysTyrPro cAG GGC ATCccc ACCTCGGcC TGCGGCGAA GGCGCCTAC AAGGTCAAC 735 Gln Gly IlePro ThrSerA,7,aCysGlyGlu GlyAlaTyr LysValAsn 3.80 185 190 GGC GTG AGCTAT TACTCCTGG AGCGG?TCC TCGCCGCTG ACCraACTTC 783 Gly vat SerTyr TyrSexTrp SerGlySex SerproLeu ThrAsnPhe CTC GAT CCGAGC GACGCCTTC CTCGGCGCC 'I'CGTCGCTG ACC'rTCAAG 831 Leu Asp ProSer AspAlaPhe LeuGlyAla SerSerLeu ThrPheLys Asn GJ.yThrRla AsnAspGly LeuvalGly ThrCysSer SerHisLeu FEB-24-00 14:09 FROM: ID: PlIGE 53 GGC ATG GTG ATC CGC GAC lfAC TAC CGG ATG AAC CAC CTG GAC GAG GTG 927 Gly Met Val Ile Arg Asp Asn Tyr Arg Met Asn His Lcu Asp Giu Val AAC cAG GTC TTC GGC CTC ACC AGC CTG TTC GAG ACC AGC cCG GTC AGC 975 Asn Gln Val Phe Gly Leu Thr Ser Leu Phe Glu Thx Ser Pro Val Ser GTC TAC CGC CAG CAC GCC AAC CGC CTG AAG A1~C GCC AGC CTG 1027 Val Tyr Arg Gln His Ala Asn Arg Leu Lys Asn Ala Ser l,eu (2) INFORMATION EAR SE(~ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 311 amino acids ($) TYPE: amino acSd (D) TOPOLOGY: linear {ii) MOLECULE TYPE: protein (Xi) SEQUENCE DESCRIPTION: SEQ ID N0: 12:
Met Lys Lys Lys Ser Leu Leu Pro Leu Gly Leu Ala Ile Gly Leu Ala Sex Leu Ala Ala Ser Pro L,eu Ile Gln Ala Ser Thr Tyr Thr Gln Th:t ~10 -5 1 5 Lys 'fyr Pro Ile Val Leu Ala His Gly Met Leu 61y Phe Asp Asn Ile Leu Gly Val Asp Tyr Trp Phe G.l.y Ile Pro Ser Ala Leu Arg A,r.g Asp Gly Ala Gln Val Tyr Val Thr Glu Gly Ser Gln Leu Asp Thr Scr Giu Vai Arg Gly Glu GIn Leu Leu G,l.n Gln Val Clu Glu Ile Val Ala Leu Ser Gly Gln Pro Lys Val Asn LQU Ile G80 His Ser His Gly Gl,y Pro Thr Ile Arg Tyr Val Ala Ala Val Arg Pro Asp Leu Ile 111a Ser Ala Thr Ser, val G1y Aia Pro His Lys Gly Ser Asp Th.r. AIa Asp Phe L,eu Arg Gln Ile Pro Pro Gly Ser Ala Gly Glu Ala Val Leu Spr Gly Leu Val. A3n Ser Leu Gly Ala Leu Iae Ser Phe Leu Ser Ser Gly Gly Thr Gly Thr Gln Asn Leu Leu Gl,y Ser Leu Glu Se.r. Leu Asn Ser Glu Gly Ala A1a Axg phe Asn Ala Lys Tyr Pro Gln Gly I1Q Pro Thr Ser Ala FEB-24-00 14:09 FROM= ID: PAGE 54 Cys Gly Glu Gly Ala Tyr Lys Vai Asn Gly Val Ser Tyr Tyr Set Trp Ser Gly Ser Ser Pro Leu Thr Asn Phe Leu Asp Pro Ser Asp Ala Phe 200 205 21.0 Leu Gly Ala Scr Ser Leu Thr Phe Lys Asn Gly Thr 111a Asn Asp Gly 77.5 220 225 230 Leu Va,a_ Giy Thr Cys Ser Ser His Leu Gly Met Val Ile llrg Asp Asn Tyr Arg Mgt Asn His heu Asp Glu Val Asn G7.n Val Phe Gly Leu Thr Se.r. Leu Phc G~_u Thr Ser Pro Val Sex Val Tyr Arg Gln IIis Ala Asn Arg Leu Lys Asn Ala Ssr Leu (?) YNFORMATION FOR SEQ TD NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) hENGTH: 1050 base pazrs (H) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (ix} FEATURE:
(A} NAME/KEY: CDS
(B) LOCATION:85..1017 ( 1X ) r'EATURE
(A) NAME/KEY: mat~eptide ($) LOCATION:163..L017 (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 13:
GGATCCCCCG GTTCTCCCGG AAGGAT'TCGG GCGATGGCTG GCAGGACGCG CCCCTCGGCC 6U

GAGATGAGAlI
CAAC
ATG
AAG
AAG
AAG
TCT
CTG
CTC
CCC
CTC

Met Lys Lys Lys Ser Leu Leu Pro Leu GGCCTGGCC ATCGGTCTC GCcTcTCTC GCTGCCAGC CCTcTGATC CAG 159 GlyLeuAla IleGlyLcu AlaSerLcu AlaAlaSer ProLeuIlc GJ_n AlaSe.r.1'hrTyrThrGln ThrLysTy1'ProIlcVa.l..T,euAlaHis Gly ATGCTCCCC TI'CGACAAC ATCC'i'TGGG GTCGACTAC TGGTTCGGC ATT 255 MetLeuGly PheAspAsn IleLeuGly ValAspTyr TxpPheGly Ile CCCAACGCC TTGCGCCGT GACGGTGCC CAGGTCTAC G'J'CACCGAA GGC 303 P.roAsnA:laLeuArgArg AspGl,yAla GlnVaJ.,'TyrValThrGJ.uGly 35 40 q5 SerGlnLeu AspThrSe.rGluValArg GlyGluGln LeuLeuGln Gln FE8-24-00 14:10 FROM: ID: PACE 55 GTGG11GGAA ATCGTCGCC CTC1~GCGGCCAGCCCA1~GGTCAAC CTGATC 399 ValGluGlu IleValAla LeuSer GlyGlnProLys ValAsn L~uIle GGCCACAGC CACGGCGGG CCGACC ATCCGCTACGTC GCCGCC cTACGT 447 GlyHisSer HisG,lyGly ProThr IleArgTyrVal AlaAl.aValArg Prohspheu IleAlaSer AlaThr 5erValGlyA1a l~rotiffsLysGly 100 lOS 110 SerAspThr AlaAspPhe LeuA.rgGlnIleProPro Gl.ySer AlaGly 17.5 120 a?,5 GAGGCnGTC CTCTCCGGG CTGGTC AACAGCCTCGGC GcGCTG ATCAGC a91 GluAlaVal LeuSerGly I,euVal AsnSerLeuGt,yA1aLcu IJ.eSer PheLeuscr SerGlyGly ThrGly ThrGlnAsnLeu LeuGly SerLeu GluSerLeu AsnSerGlu GlyAla AlaArgPheAsn AlaLys '1'yrPro lfi0 165 170 175 C11CCGCATC CCCACC'i'(:GGCCTGC GGCG11AGGCGCT TACI~AVG'1CAAC 735 GlnGlyIle ProThrSar AlaCys GlyGluGlyAla TyrLys ValAsn GGCGTGAGC TATTACTCC TGGAGC GGTTCCTCGCCG CTGACC 1~ACTTC 783 GlyValSer TyrTyrSer TrpSer GlySerSerPro LeuThr AsnPhe LeuAspPro SerAspAla PheLeu GlyA.l.aSerSer Leu'1'hrPhcLys AACGGCACC GCCAACGAC GGCc'rGGTCGGCnCCTGC AGTTcG CACCTG 879 AsnGlyThr AlaAsnlispGlyLeu Va1G.7,y'1'hrCys SerSer HisLeu 7?.5 230 235 Gl.yMe~CVal IJ.eArgAsp AsnTyr ArgMetAsnHis LeuAsp GluVal 1~CCAGGTC CTCGGCC'1'CACCAGC C'TGTTCGAGACC AGCCCG GTCAGC 975 AsnGlnVal i.euGly~,euThrSer l~euPheGluThr SerPro Va1Ser GTCTACcGC CAGCACGCC AACCGC CTGAAAAACGCC AGCCTG 1017 ValxyrArg GlnHisAla AsnArg LeuLysAsnAla SerLPu 'i'r~GGACCCCG CCG 10~U
GCCGGGGCCT
CGGCCCGGGC

(7 INFORMATION E'ORSEQ In 4:
) N0:

FEB-24-00 14-10 FROM: ID: PAGE 56 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 311 amino acids (H) TYPE: am~,no acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (.ci) SE(~UENCE DESCRIPTION: SEQ ID N0: 14:
Met Lye Lys Lys Ser Leu Leu Pro Leu Gly Leu 111a Ile Gly Leu Ala ~26 -25 -20 -15 Ser Leu Ala Ala Ser Pro Leu Ile Gln Ala Ser Thr Tyr Thr Gln Thx Lys Tyr Pro Ile Val Leu Ala His Gly Met Leu Gly Phe Asp Asn Ile Leu Gly Val Asp 'fyr Trp Phe Gly Ile Pro Asn 111a Leu Arg Arg Asp ~5 30 35 Gly Ala Gln Val Tyr Val Thr GJ.u Gly Sex. Gln Leu Asp Thr Ser Glu Va,l Arg Gly Glu Gln Leu Leu G.l,n Gln Va7. Glu Glu Tle Val Ala Leu Scr Gly Gln Pro Lys Val Asn Leu Ile Gly His Ser His Gly Gly Pro 75 80 ~ 85 Thr Ile Arg Tyr Val Ala Ala Val Arg Pro Rsp Leu Ile Al.a Ser Ala Thr Ser val Gly A1a Pro His Lys Gly Ser Asp Thr Ala Asp Phe Leu lOS lI0 115 Arg Gln Ile P.ro Pro Gly Ser Ala Gly Glu Al.a Val Leu Ser Gly Leu Val Asn Ser Leu Gly Ala J~eu Ile Ser Phe Leu Sex Ser Gly Gly Thz~

Gly Thr Gln Asn Leu Leu Gly Ser Leu Gl.u Ser Leu Asn Ser G.i.u Gly Ala Ala Arg Phe Asn Ala L~ys Tyr Pro Gln Gly Ile Pro Thr. Ser l~la Cys G.7_y Glu Gly Ala Tyr Lys Val Asn Gly Val Ser Tyr Tyr Ser Trp 1,85 190 195 Ser Gly Ser Ser Pro Lau 'fhr 7lsn Phe Leu lisp Pro Ser Asp Ala Phe Leu Gly Ala Ser Ser Lau Thr hhe Lys Asn Gly Thr Ala Asn Asp Gly Leu Va.l. Gly Thr Cys Ser Scr His Leu Gl.y Met Val Ile Arg Asp Asn '1'yr Arg Met Asn His Leu Asp Glu Val Asn GJ.n Val Leu G7.y Leu Thr 2a0 255 260 Ser Leu Phe Glu Thr Ser Pro Val Ser Val Tyr Arg G1n His Al~a Asn FEB-24-00 14:10 FROM= ID= PAGE 57 llz;g Leu Lys Asn Ala Ser Leu (2) INFORMATION FOR SEQ ID N0: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1099 base pairs (B) TYPE: nucleic aced (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATI:ON:85..1017 (ix) FEATURE:
(A) NAME/KEY: mat~epti,de (B) LOCATION:163..1017 (xi) SEQUENCE DESCRIPTION: SEQ ID N0: I5:

CC11TCA,A.CCT GAGATCAGAA AAG TCTCTG CTCCCC CTC 111 ATG AAG

Met Lys LysSe.r,heu LeuPro Leu Lys -z6-25 -20 GGCCTGGCC11TCGGTCTC GCCTCTCTC GCTGCCAGCCCT CTGATC CAG 1$9 GlyLeuAlaIle GlyLeu AlaSerLeu AlaAlaSerPro LeuIle Gln '15 -10 -5 GCCAGCACCTAC AccCAG ACCAAATAC CCCATCGTGCTG GcccAC GGC 207 l~laSerThrTyr ThrGln ThrLys'TyrProIIevalLcu AlaHis Gly AT'GcTCGGCTTC GACAAC ATCCTTGGG GTCGACTACTGG TTCGGC ATT z55 MetLeuGlyPhe AspAsn IIeLeuGly valAspTyrTrp PheGly Ile ProSerAlaLeu ArgArg AspGlyAla GlnVal'1'yrvat ThrGlu Gly SerGlnLeuAsp ThrSpx GluvalArg G.tyGluGlnI,euLeuGln Gln GTCGAGGAAATC GTCGCC CTCAGCGGC CAGCCCAAGGTC AACC~'GATC 399 valGluGluzle Val.Ala LeuScarG,lyGlnProLysVal l~snLeu Tle GGC CAC AGC CAG GGC GGG CCG ACC ATC CGC TAC GTC GCC GCC GTA CG'T 497 Gly His Ser His Gly Gly Pro Thr Ilc Arg Tyr val A13 Ala Val Arg CCC GAC CTG ATC GCT TCC GCC ACC AGC GTC GGC GCC CCG CAC AGG GGT g95 Pro Asp I,eu Ile A1a Ser. Ala Thr Ser vat Gly Aln Pxo His Arg Gly Ser Asp Thr Ala Asp Phc heu Arg Gln Ile Pro Pro Gly Ser Ala G1y FEB-24-00 14:10 FROM: ID: PAGE 5A
56 _ GAGGCA GTCCTCTCC GGGCTG G'tCAACAGC CTCGGCGCG CTCATC AGC 591 GluAla ValLPUSer GlyLeu ValAsnSer LeuGlyAla LeuI1e 5cr PheLeu SCrSerGly GlyThr.GlyThxGln AsnLeu>'.,euGlySer Leu GAGTCG CTGAACAGT GAGGGT GCCGCGCGC TTCAACGCC AAGTAC C:CG68~

GluSex'LeuAsnSer GluGly AlaAlaArg PheAsnAla I,ysTyr Pro CAGGGC ATCCCCACC TCGGCC TGCGGCGAA GGCGCTT.~CAAGcTC A~aC735 GlnGly IlcProThr SerAla CysGlyGJ.uGlyAlaTyr LysVal Asn GGCGTG AGCTATTAC TCCTGG AGCGGTTCC TCGCCGCTG ACCAAC TTC -~83 GlyVal SerT'yrTyr SerTrp SerGJ.ySer SerProLeu 't'hrAsn Phe 7.95 200 205 LeuAsp ProSerAsp AlaPhe L~uGlyAla SerSerJ.,euThrPhe Lys 77.0 215 220 AACGGC ACCGCCAnC C11CGGC CTGGTCGGC ACCTGCAGT TCGC:aCCTG 879 AsnGly ThrAlaAsn AspGly LeuValGly ThrCysScr SerHis Leu GGCATG G'1'GATCCGC GACAAC TACCGGATG AACCACCTG GACGAC G2'G927 G.lyMet VallleArg AspAsn TyrArgMet AsnIlisLeu AspGlu Val AsnGln ValLeuGJ.yLeuThr SerLcuPhe GluThrSer ProVal Ser 260 265 27p GTCTAC CGCCAGCAC GCCAAC CGCCTGAAG .SACGCCACC CTG 1017 ValTyr ArgGl.nHis AlaAsn ArgheuLys AsnAlaSex Leu GCCGGGGCCT
CG

(2) INFORMATION FOR SEQ ID NO: 16:
(.i? S~;QUENCE CHARACTFRISTZCS:
(A) LENGTH: 311 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (i.i.) MOLECULE 'TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 16:
Met Lys hys Lys 5el~ Leu Leu Pro Leu Gly i,eu Ala Ile Gly Leu Ala -26 -75 -?.0 -7.5 Ser Leu Alx Ala Ser Pro Leu Zle Gln Ald Ser Thr Tyr Thr Gln 'Phr -10 -g 1 5 Lys Tyr Pro IJ.e Val Leu Ald His G25 Met Lcu Gly Phe A..~,p Asn I:le Leu Gly Vdl Asp Tyr Trp Phe Gly 71e Pro Ser A7a r,~" Arg Arg Asp i ID: PAGE 59 FEB-24-00 14:11 FROM:
..
GJ.y Ala Gln val Tyr val Thr Glu Gly Sex Gln Leu Asp Thr Ser Glu 40 q5 50 Val Arg Gly Glu Gln Le:u Leu Gln Gln val Glu Glu Ile Va.l Ala Leu Ser Gly Gln Pro Lys val Asn Leu Zle Gly His Ser His Gly Gly Pro Thr Ile Arq Tyr Val Ala Ala val Arg Pro Asp Leu Ile Ala Sex Ala Thr Ser VaJ. Gly A1a Pro His Arg Gly Ser Asp Thr Ala Asp Phe Leu 105 110 1.15 Arg Gln Ile Pro Pro Gly Ser Ala Gly Gl.u Ala val Leu Ser Gly Leu Val Asn Ser J~eu Gly Ala Leu Il.e Ser Phe I,eu Sor Ser Gly Gl,y Thr Gly Thr Gln Asn Leu Lcu Gly Ser Leu Glu Ser Leu Asn Ser Glu Gly Ala Ala Arg Phe Asn Ala Lys Tyr Pro Gln Gly Ile Pro Thr Ser Ala 1.75 7.80 Cys Gly Glu Gly Ala Tyr Lys Val Asn Gly Val Ser Tyr 'fyr Se.r Trp Ser Gly Ser Ser Pro Leu Thr Asn Phe Lcu Asp Pro Sex Asp 111a Phe Leu Gly Ala Ser Ser I,eu Thr Phc hys Asn Gly Thr Ala Asn Asp Gly Leu Val Gly Thr Cys Ser Ser His Leu Gly Met Val Tle Arg Asp Asn Tyr Arg Met Asn His l,eu Asp Glu Val Asn Gln Val Leu Gly Leu Thr Ser Leu Phe Glu Thr Ser Pro Val Sex Val Tyr Arg Gln His Al.a Asn 265 2.'70 2 ~5 Arg Leu Lys Asn ALa Ser Leu (2) INFORMATION FOR SEQ ID N0: 17:
(i) SFQU~;NCE CHARAC1'I;ItISTICS:
(A) J.ENGTH: 1049 base pairs (H) TYPk'.: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown ( i.i ) MOLECULE TYPE : DNA ( genom ic; ) (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:85..1017 (ix) FEATURE:
(A) N11ME/KEY: mat peptide FEB-24-00 14:11 FROM= ID: PACE 60 58 ~-(B) LOCATION:163..1017 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7.7:
GGA~i'CCCCCG GrTCTCCCGG CCCCTCGGCC

AAGGATTCGG
GCGATGGCTG
GcAGGACGCG

CAAC MetLysLys LysSerLau LeuProLeu -26-25 ~20 GGCCTG ATC CTCGCC 'rCTCTC GCCAGC CTG CAG 159 GlyGCC GGT LeuAla SerGCT AlaccT ATC Gt7.n Leu Ile -10Lcu Ser Leu Ala Gly Ala Pro Ile GCCAGC ACCTACACC cACACC AAATACCcc ATCGTGcTG GCCcACGGC 207 Al,aSer ThrT'yrThr GlnThr LysTyrPro IloValLeu A.l,aHisGay 1 5 J.

MetLeu GlyPheAsp Asnza.eLeuGlyVal lispTyrTrp PheGlyLle CCCAGC GCCTTGCGC cGTGAC GGTGCCCAG GTCTACGTC ACCGAAcCC 303 ProSer Al,aLeuArg ArgAsp GlyAlaGln ValTyrVa,~ThrGluGly 35 40 ~!5 AGCCAG TTGGACACC TCGGAA GTC:CGCGGC GAGCAGTTG CTGCAl1CAG 351 SerGln LeuAspThr SerG7.uValArgCly GluGlnLeu LcuGJ.nGln Va,1_Glu GluIleVal AlaLeu SerG.tyGln ProJaysVal Asn,LeuI1e GGCCAC AGCCACGGC GGGCCG ACCATCGC TACGTCGCC GCCGTACGT 4~7 GlyHis Se'rHisGly GlyC ThrIleArg TyrValAla AlaValArg 80 85 Pro 90 95 CCCGAC C'i'G11TCGCT TCC AGCGTC.GGCGCCCCG CACAAGGG'r 4 ProAsp J,euIleAla GCG Serval GlyAlaPro H:isLysGly 95 Ser Ala Thr TCGGAC nCCGCCGAC TTCGTGC;GCCAGATC.CCA CCGGGT TCGGCC:GGC

SerAsp ThT~AlaAsp PheLeuArg GlnIlePro P,-oGly SerAlaGl y GluAla ValT~euSex GlyLeuVa,~,AsnSerLeu GlyAla LeuIleSer TTCCTT 'i~CC11GCGGC GGCATCGGT ACGC11GAAT TTTCTG GGCTCGCTG

PheLeu SerSerGly GlyIleGly ThrGlnAsn PheLeu GlySerLeu GluSer LeuAsnSer GluG:LyAla AlaArgPhe AsnAa.aLysT P
1 r r o 60 165 170 y .
.

Gl n Gly IleProThr SerAlaCys GlyGluG~.yAlaTyr LysVall\sn GGCc;TGAGrTATTAC TCCTGGAGC GG'1'TCCTcc c:CGCTr AccnACTTC

GlyVaI 5r:rTy.Tyr SerTxpaer GlySers~r PrnLeu ThrAsnPhe FEB-24-00 14:11 FROM: ID: PAGE 61 CTCGAT CCGAGCGAC GCCTTC C'1'CGGCGCC '1'CGTCGCTG ACC'fTCAAG 837 L

.
eu Asp Pro5erAsp AlaPhe LeuGlyAla SerScr.Leu ThrPheLys AACGGC ACCGCCAAC GACGGC CTGGTCGGC ACC'fGCAGT TCGCliCCTG $i9 A ' sn Gly 1 AlaAsn AspGly LeuValGly ThrCysSer SerH;isLe hr u 24 Met ValZleArq AspAsn TyrArgMet AsnHisLcu AspGluVal y AACCAG GTCT'PCGGC CTCACC AGCcTGTTC GAGnCCAGC CCGGTCAGC 975 AsnGln ValPheGly LeuThr SerLeuPhe GluThrSer ProValSer GTCTIaCCGCCAGCAC GCCAAC CGCCTGAAG 11ACGCCAGC CTG

x.017 Va,lTyr ArqGInHis AIailsnArgLeuLys AsnA1aScx'Leu TAGGACCCCG CCGGGGCC T GGGCCC
G CGGCCC

(2) INFORMlITION FOR SEQ ID N0: 1$:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 311 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOhECULE TYP);: protein (xi) SEQUENCE DESCRIPTION: SFQ ID N0: 18:
Met Lys Lys Lys Sex Leu Leu Pro Leu Gly Leu Ala Ilc Gly Lpu Ala -?6 ,25 -20 -15 Ser Leu Ala Ala Ser Pro Leu lle Gln Ala Ser Thr Tyr Th.r. Gln 7.'hr -10 _5 1 5 Lys Tyr Pro I1P Val Leu Ala His Gi5 Met Leu Gly Phe Asp Asn Iie Leu Gly Val, Asp Tyr Trp Phe Gly Ile Pxo Ser Ala Leu Arg Arg Asp Gly Ala Gl,n Val Tyr Val Thx Glu C~,l.y Ser G7 n T~ru Asp Thr Ser Glu 40 ~5 50 Val Arg G1y Qlu Gln Leu Leu Cln Gln Vr~l Glu Glu Ile Val Ala l;,eu 55 b0 55 70 Ser Gl,y Gln Pro Lys Val Asn Leu Ile Gly Kic Cor Hi.s Gly Gly Pro 75 00 $5 'i'hr lle Arg Tyr Val Ala Ala Val llrg pro Asp Leu Ile Ala Ser Ald Thr Scr val Gly Ala PL'U His LVS Gly Ser Asp Thr Al.s~ Asp Phe Leu Arg C;ln Ile Pr.~ Prn Gly Sor Al,~ GIy CJ.u Ala Val LCU Ser Gly Leu Val lien Ser Leu Gly Ala Leu Ile 58r the Leu Ser Spr c:l.y ,ly Zls FEH-24-00 14:11 FROM: ID: PACE 62 ' 60 GlyThr GlnAsnPhe T,euGly SerLeuGlu SerLcuAsn SprGlu Gly 7.55 160 165 AlaAla ArgPhoAsn nlaLys TyrFaroG.lnGlyIlePro 'PhrSer Ala CysGly C~7.uGlyAla Tyr,Lys ValAsnG7,yValSerTyr TyrSer Trp SerGly SerSerPro LeuThr AsnPheLeu AspPxoSer AspAla Phe LeuGly AlaSerSc~xLeuThx PhcLysAsn GlyThrAla AsnAsp Gl.y 715 2z0 225 230 LeuVa.lGlyThrCys SerSer HisLcuGly MctVdlIle ArgAsp Asn TyrArg MetAsnHis LeuAsp GluValAsriGlnValPhe GlyLcu 'L'hr SerLou PheGluThr SexPro ValSerVaJ.'TyrArgGln HisAla Asn AxgLeu LysAsnAla SerLeu (2) rNFORMATION FOR SE:O ID N0: 19:
(i} SEQUENCE CHARACTERISTICS:
(A) L~;NGTH: 30 base pairs (g) TYPE: nucJ.eic acid (C) STRANDEDNESS: unknown (D} TOPC?LOGY: linEar (i..i) MOLECULE TYPE: other nucleic acid {A) DESCRIPTION: /dese = "synthetic DNA"
(xi) SEQUENCE DESCRIPTION: SF.Q ID NO: 19:
GcGC:AATTAA ccCTCACTAA AGGGAncAAA 30 (2) INFORMATION FOR SEQ ID N0: 20:
(i) SEQUENCE CHARACTERISTICS:
{A) LENGTH: 25 base paixs {B) TYPE: nucleic acid (C) STRAhDEDNESS: unknown (D) TOPOLOGY: linear (.ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTTON: /desc = "synthetic DNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 20:
GGTACGCAGA 1~x'NNNCTGGG C TCGC
(2} INFORMA':CrON FOR SEQ ID N0: 21:
(i} SEQUENCE CHARACTERISTICS:
{A) LENGTH: 27 base pairs ii b FEB-24-00 14_12 FROM: ID: PAGE 63 (B) TYPE: nucleic acid (G) STRANDEDNESS: unknown (b) TOPOLOGY: linear (ii) MOLECUhE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "synthetic DNA"
(xi) SEQUENCE DESCRIPTION: SEQ ZD N0: 21:
GCGTAATACG ACTCACTA1'A GGGCGAA

Claims

CLAIMS:

1. A process for the preparation and identification of hydrolase mutants having improved properties with respect to stereo- or regloselectivity, characterized in that a) a starting hydrolase gene is mutagenized by a modified polymerase chain reaction (PCR), wherein the mutation rate and total number of mutations in the amplified DNA is adjusted by adjusting the concentrations of Mg2+, Mn2+ and of the deoxynucleotides and by adjusting the number of cycles;
b) optionally one or more hydrolase genes mutated according to step a), or mixtures of one or more starting hydrolase genes and one or more hydrolase genes mutated according to step a) are mutagenized by enzymatically fragmenting said genes, followed by enzymatic reassembly of the fragments produced to give complete recombinant hydrolase genes;
c) the mutated hydrolase genes obtained according to step a) or b) are transformed into a host organism; and d) hydrolase mutants having improved properties, expressed by transformants obtained in step c), are identified by a test method.

2. The process according to claim 1, wherein an average mutation rate of 1-2 base substitutions, per one hydrolase gene to be mutagenized, is adjusted in the PCR in step a) by adjusting the concentrations of Mg2+, Mn2+ and of the deoxynucleotides.

3. The process according to claim 1, wherein a hydrolase gene mutagenized in a PCR previously performed according to claim 1 is used as the starting hydrolase gene in step a).

4. The process according to claim 1, wherein the enzymatic fragmentation of the hydrolase genes in step b) is performed using a de-oxyribonuclease.

5. The process according to claim 1, wherein the reassembly of the fragments in step b) is effected enzymatically by means of a thermostable DNA polymerase using temperature cycles in which the parameters of temperature and duration of cycles are adjusted.

6. The process according to claim 1, wherein the mutation rate is adjusted during the enzymatic reassembly in step b) by adjusting the concentrations of Mg2+, Mn2+ and of the deoxynucleotides.

7. The process according to claim 1, wherein the completely recombined hydrolase genes are amplified by a polymerase chain reaction in step b) after completion of the reassembly reaction.

8. The process according to claim 1, wherein either modified hydrolase genes obtained from step a) according to claim 1 or 2 or several hydrolase genes mutagenized according to claim 3 are subjected to fragmentation and reassembly in step b).

9. The process according to claim 1, wherein synthetically prepared gene fragments are additionally used for the reassembly in step b).

10. The process according to claim 1, wherein hydrolase gene fragments from different organisms sharing a sequence homology of at least 60% can be used for the reassembly in step b).

11. The process according to claim 2 or 6, wherein the hydrolase mutants are lipase or esterase mutants, and the concentration of the magnesium ions is from 1.5 to 8.0 mM, preferably from 5.8 to 6.4 mM, and the concentration of the manganese ions is from 0.0 to 3.0 mM, preferably < 0.3 mM.

12. The process according to claim 2 or 6, wherein the hydrolase mutants are lipase or esterase mutants, and the concentration of the deoxynucleotide triphosphates is from 0.05 to 1.0 mM, preferably 0.2 mM.

13. The process according to claim 1, wherein for the test for stereo- or regioselectivity of the hydrolase mutants in step d), a test substrate is provided with a chromophorous group which causes a spectrometrically determined change of absorption or emission upon cleavage by the catalyst, and equal amounts of the hydrolase mutants are added to the pure stereo- or regioisomers of the test substrate in separate test vessels, and the stereo- or regioselectivity can be determined from the ratio of the linear initial reaction rates obtained.

14. The process according to claim 13, wherein the stereo- or regioisomers of a compound with a UV/VIS-active or fluorescence-active molecular group bound through a carboxylicacid ester or carboxylic acid amide linkage are used as the test substrate.

15. The process according to claim 14, wherein said UV/VIS-active molecular group is a p-nitrophenyl residue.

16. The process according to claim 1, wherein the test for stereo- or regioselectively in step d) is effected through determination of the change of concentration with time of free fatty acids or succinic acid, wherein the corresponding stereo or regioisomeric carboxylic acid esters or amides are hydrolyzed in separate vessels by means of the hydrolase mutants to give free fatty acids or succinic acid.

17. The process according to claim 1, wherein the test for stereo- or regioselectively in step d) is effected through measuring the radio-activity, wherein the hydrolase mutants are reached with stereo- or regioisomers having different radioactive labels in one functional group, and wherein the mixture of the stereo- or regioisomers is fixed on a support.

18. The process according to claim 17, wherein one of the stereo- or regioisomers of the support-bound mixture of isomeric compounds is labeled with the radioisotope 3H, and the other stereo- or regioisomer is labeled with the radioisotope 14C.

19. The process according to claim 1, wherein the test for stereoselectively in step d) is effected through the capillary-electrophoretic determination of the reaction products and educts of a test reaction, the separation of the stereoisomeric reaction products and educts being performed in chirally modified capillaries.

20. The process according to claims 13 to 19, wherein several reactions are performed in parallel in microtitration plates.

21. The process according to claim 1, wherein the position of the codon coding for the changed amino acid is localized by sequencing in the mutants having improved properties identified in step d), followed by generating a set of hydrolase genes with all possible codons for this position by means of site-directed saturation mutagenesis, and the mutated hydrolase genes thus obtained are further treated in analogy with steps c) and d) of claim 1.

22. The process according to claim 21, wherein the localization of the position of the codon coding for the changed amino acid is effected through DNA sequencing.

23. A hydrolase mutant obtainable by a process according to one or more of claims 1 to 22.

24. The hydrolase mutant according to claim 23 which is a lipase mutant.

25. The hydrolase mutant according to claim 23 which is an esterase mutant.

26. The hydrolase mutant according to claim 24 which is a lipase mutant of the starting lipase from the strain P. aeruginosa.

27. The hydrolase mutant according to claim 26 which is obtainable by expression from the transformants P1B 01-E4 (DSM 11 658), P2B 08-H3 (DSM 11 659), P3B 13-D10 (DSM 11 660), P4B 04-H3 (DSM I2 322), P5B 14-C11 (DSM 12 320) or P4BSF 03-G10 (DSM
12 321).

28. The hydrolase mutant according to claim 24 which has the amino acid sequence of the mature proteins shown in SEQ ID NOS. 4, 6, 8, 12, 14, 16 or 18.

29. A DNA sequence coding for a hydrolase mutant according to one or more of claims 23 to 28.

30. The DNA sequence according to claim 29 which comprises a DNA
sequence shown in SEQ ID NO5. 3, 5, 7, 11, 13, 15 or 17.

31. A vector comprising a DNA sequence according to claim 29 or 30.

32. A transformant comprising a DNA sequence according to claim 29 or 30 and/or a vector according to claim 31.

33. The transformant according to claim 32 which is transformant P1B
01-E4 (DSM 11 658), P2B 08-H3 (DSM 11 659), P3B 13-D10 (DSM
11 660), P4B 04-H3 (DSM 12 322), P5B 14-C11 (DSM 12 320) or P4BSF 03-G10 (DSM 12 321).

34. A process for the preparation of hydrolase mutants having improved properties, comprising culturing a transformant according to claim 32 or 33.

35. A method for testing catalysts for stereo- or regioselectivity, wherein equal amounts of the catalyst are added to a test substrate and to the pure stereo- or regioisomers of the test substrate, provided with a chromophorous group which causes a spectrometrically determinable change of absorption or emission upon cleavage by the catalyst, in separate test vessels, and the stereo- or regioselectivity is determined from the ratio of the linear initial reaction rates obtained.