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Creation of Enantioselective Biocatalysts for Organic Chemistry by In Vitro Evolution.

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direct evidence for a carbene complex intermediate during a
[2+l] cycloaddition catalyzed by a transition metal complex.
Further studies on the mechanism and the stereochemical
course of the reactions are in progress.
Experimental Section
General procedures: All reactions were performed under argon. Solvents were
dried and degassed according to standard procedures. Yields refer to products
isolated after column chromatography. Compounds 1[16], 2 [17], and 7 (181 were
synthesized according to literature methods.
3-6: A solution of 1(1.14 g, 10 mmol) in CHzCIZ(20 mL) was added dropwise
over 4 h to a stirred solution of 2 (0.15 g,OS mmol, 5 mol%) in the corresponding
alkene (50 mmol), precooled to 5 "C. The reaction mixture changed color from
yellow to green-brown under evolution of N,. After the mixture had been stirred
for a further 8 h at 20°C and the solvent and excess alkene had been removed
under reduced pressure, the residue was purified by column chromatography
(petroleum ether (40160)/Etz02:l).
8-11 A solution of 7 (0.57 g, 3 mmol) in CH2CJ2(30 mL) was added dropwise
over 8 h to a stirred solution of the corresponding alkene (3 mmol) and 2 (0.02 g,
0.06 mmol, 2 mol%) in CH,CI, (10 mL). The reaction mixture changed color
from yellow to brown-violet under the evolution of NZ.After the mixture had
been stirred for a further 8 h at 20°C and the solvent had been removed under
reduced pressure, workup was performed according to one of the following
procedures (see Table 2).
A: Workup by column chromatography.
B: The residue was washed several times with portions of petroleum ether(40160)
(10 mL) and filtered until only bis[9-(9H)-fluorenyIidene]azine could be
detected in the solution (TLC-control, petroleum ether (40/60)/CH2CI2 1:1,
Rf=0.35). The combined filtrates were concentrated, and purified by column
chromatographic workup.
9: 'H NMR (500 MHz, CDCI,): 6 = 7.89 (d, 1H), 7.88 (d, 1H), 7.52 (d, 1 H), 7.41 (t,
1H), 7.39 (t. l H ) , 7.34 (t. l H ) , 7.32 (t, l H ) , 7.18 (d, l H ) , 3.04 (s, 3H), 2.27 (dd,
'Js6.15 HZ, 4J = 0.55 Hz, 1H; H-3), 1.91 (d, '3 ~ 6 . 1 Hz,
5 1H; H-3), 1.82 (d, 4J =
0.49 Hz, 3H; CH,); '.'C NMR (125.6 MHz, CDCI?):6 = 144.9, 144.7, 141.1, 139.9
(4 quart. C), 126.5 (CH), 126.0 (CH), 125.82 (CH), 125.78 (CH), 122.7 (CH), 121.5
(CH), 119.9 (CH), 119.5 (CH), 70.3 (s, 1C; C-2). 54.8 (9.1 C; OCH,), 41.5 (s, 1C;
C-l), 30.4 (t, ' J = 160.8 Hz. 1C; C-3), 16.8 (9,1 C, CH,); IR (KBr): qcm-') = 3059
(m), 2962 (rn), 1473 (s), 1442 (s), 1240 (vs), 1065 (vs), 813 (s), 736 (vs); MS (EI,
70 ev): m / z (%): 236 (95) [M+],
221 (100) [ M - -CH,], 205 (35) [ M i- OCH,],
165 (44) [Cl,Hy+],152 (25) [CIzH8+];
elemental analysis calcd
178 (50) [C14Hlo+],
for CI7Hl60(236.31): C 86.41, H 6.82; found: C 86.11, H 6.82.
[l] Reviews: a) M. P. Doyle, in Comprehensive Organometallic Chemistry 2,
V01.12 (Eds.: E. W. Abei, E G. A. Stone, G. Wilkinson), Pergamon, New
York, 1995, p. 387; b) S. D. Burke, P. A. Grieco, Org. React. (NY) 1979,26,
361; c) M. P. Doyle, Chem. Rev. 1986,86,919; d) A. Padwa, K. E. Krumpe,
Tetrahedron 1992, 48, 5385.
[2] Reviews: a) H:U. Reissig, Methoden Org. Chem. (Houben-Weyl) 4th ed.
1952-, Vol. E21c, p. 3179; b) A. Pfaltz, Acc. Chem. Res. 1993, 26, 339; c)
M. P. Doyle in Cafalyfic Asymmetric Synthesis (Ed.: I. Ojima), VCH, New
York, 1993.63; d) T. Aratani, Pure Appl. Chem. 1985,57, 1839.
[3] a) M. P. Doyle, J. H. Griffin, V. Bagheri, R. L. Dorow, Organometaliics 1984,
3, 53; b) M.P. Doyle, R. L. Dorow, W. E. Buhro, J. H. Griffin, W. H.
Tamblyn, M. L. Trudell, ibid. 1984,3,44.
[4] a) W. A. Herrmann, Angew. Chem. 1978, 90, 855; Angew. Chem. Int. Ed.
En@. 1978,17,800; b) W. A. Herrmann, J. L. Hubbard, I. Bernal, J. D. Korp,
B. L. Haymore, G. L. Hillhouse, Inorg. Chem. 1984,23,2978.
[ 5 ] a) I? Schwab, M. B. France, J. W. Ziller, R. H. Grubbs, Angew. Chem. 1995,
107,2179; Angew. Chem. Inf. Ed. Engl. 1995,34,2039; b) P. Schwab, J. W.
Ziller, R. H. Grubbs, J. Am. Chem. SOC.1996,118,100.
161 Review: M. Brookhart, W. B. Studabaker, Chem. Rev. 1987,87411.
[7] a) H. Nishiyama, Y. Itoh, H. Matsumoto, Y. Sugawara, K. Itoh, Bull. Chem.
SOC.Jpn. 199568,1247; b) S.-B. Park, N. Sakata, H. Nishiyama, Chem. Eur
J. 1996,2, 303.
[S] a) M. I? Doyle, R. L. Dorow, W. L. Tamblyn, J. Org. Chem. 1982,47,1538;b)
M. I? Doyle, J. G. Davidson, ibid. 1980,43,1538.
[9] K. H. Dotz, J. Pfeiffer, Chem. Commun. 1996, 895.
[10] The formal carbene dimers diethyl maleate and -fumarate were isolated as
by-products.
1111 1-Hexene, cyclohexene, cyclooctene, furan, and ethyl acrylate were
examined.
[12] 3 , 4 : M. P. Doyle, D. van Leusen, W. H. Tamblyn, Synthesis 1981,787; 5: I.
Reichelt, H.-U. Reissig, Chem. Ber. 1983, 116, 3895; 6 : M. I? Doyle, K.-L.
Loh, K. M. DeVries, M. S. Chinn, Tetrahedron Lett. 1987,28,833.
[13] T. W. Hanks, P. W. Jennings, J. Am. Chem. SOC.1987,109,5023.
1141 The formal carbene dimer 9,9'-bisfluorenylideneand bis[9-(9H)-fluorenylidenelazine were isolated.
[15] The uncatalyzed [2 + 11 cycloaddition of 7 is known, for example, for
reaction with methyl acrylate: L. Homer, E. Lingnau, Liebigs Ann Chem.
1955.591.21.
[16] N. E. Searle, Org. Synth. Coll. Vol. 4 1963,424.
1171 E W. Grevels, V. Skihbe, J. Chem. SOC.Chem. Commun. 1984, 681.
[18] A. Schonberg, W. Awad, N. Latif, J. Chem. SOC.1951,1368.
of Enantioselective Biocatalysts for
Organic Chemistry by In Vitro Evolution
Creation
lO:'HNMR(5M)MH~,CDCI~):6=7.87(d,1H),7.75(d,1H),7.40(t,lH),7.32
(t. ZH), 7.25 (d, 1 H), 7.24 (t, 1H), 6.80 (d, 1H), 4.65 (d, '3 = 5.76 Hz, 1H; H-l),
4.48-4.39 (m. 2H; H-3), 2.62 (ddd, ?I= 1.76, 5.68, 7.37 Hz, 1 H ; H-9, 2.48-2.31
(m, 2 H ; H-4); 13C NMR (125.6 MHz, CDCI,): 6 = 145.9, 141.6, 141.4, 138.3
(4quart. C ) , 126.8 (CH). 126.2 (CH), 125.9 (CH), 125.8 (CH), 123.0 (CH), 120.2
(CH), 119.5 (CH), 118.4 (CH), 77.0 (t, 1C; C-3). 71.6 (d, ' J = 199.43 Hz, 1C; C-l),
43.8 (s, 1C; C-6), 33.6 (d, ' J = 173.3 Hz, 1C; C-5). 25.6 (t. 1 C; C-4); IR (KBr):
qcm-I) = 3032 (m), 2968 (m), 1438 (vs), 1340 (vs), 1039 (s), 927 (s), 744 (vs); MS
(EI, 70 ev): m/z (%): 234 (78) [M'],205 (85) [M+- C2Hs].178 (100) [Cl,Hil,+],
165 (25) [C,,Hy+];elemental analysis calcd for CI,H140(234.30): C 87.15, H 6.02;
found: C 86.73, H 6.05.
11:'H NMR (400 MHz, CDCI,): 6 = 7.85 (d, 1H), 7.80 (d, l H ) , 7.41 (t. 1H), 7.36
(t. lH),7.30-7.19(m,7H),6.92 (t, 1H),6.15 (d, 1H),3.38 (t,?1=8.41 Hz, 1 H ; H2). 2.22 (d, 3J=8.41 Hz, 2H; H-3); "C NMR (100.6 MHz, CDCIJ: 6=148.2,
144.2, 140.4, 139.6, 137.1 (5quart. C), 130.1 (ZCH), 128.1 (2CH). 126.8 (CH),
126.7 (CH), 126.0 (CH), 125.8 (CH), 125.7 (CH), 121.5 (CH), 119.7 (CH), 119.6
(CH),118.5(CH),35.5(s,lC;C-l),34.9(d,'J=160.6Hz,
lC;C-2),22.3(t,'J=
162.3 Hz, 1 C; C-3); IR (KBr): qcm-I) = 3055 (m),3034 (m), 1496 (m). 1444 (s),
777 (vs), 748 (vs), 696 (vs); MS (El, 70eV): m/z (%): 268 (100) [M+],252 (40)
[M+-CH,], 165 (25) [C13H9+].91 (17) [C,H,+]; MS-HR calcd for CZ1Hl6:
268.1252; found: 268.1249.
Two control experiments in the absence of 2 and in the presence of [Cr(CO),]
(10 mol%), respectively, have been performed for each catalytic reaction under
the reaction conditions described above. A (2 + I] cycloaddition was observed in
the absence of the chromium complexes 1151only in the reaction of ethyl acrylate
with 7.
Received: August 13,1997 [Z108071E]
German version: Angew. Chem. 1997,109,2948-2950
Keywords: carbene complexes
tions
2830
- homogeneous catalysis
- chromium . cyclopropana-
0 WILEY-VCH Verlag GrnbH, D-69451 Weinheim, 1997
Manfred T. Reetz," Albin Zonta, Klaus Schimossek,
Klaus Liebeton, and Karl-Erich Jaeger"
The development of chiral catalysts for the enantioselective
synthesis of optically active compounds is of great acadernid1l
and industrial interest.[*] Inspite of worldwide intensive
research in the area of homogeneous transition metal
catalysis, the number of really efficient enantioselective
catalysts is limited. Owing to a lack of general principles,
the development of a single highly effective chiral catalyst
requires laborious preparation and testing of many ligands.
Alternatively, biocatalysts can be used, but by nature the
problem of limited substrate specificity per~ists.1~1
In some
cases site-directed mutagenesis can be applied to improve
enzyme activity and selectivity but not in a general way,
[*] Prof. M. T. Reetz, Dr. A. Zonta, Dip1:Chem. K. Schimossek
Max-Planck-Institut fur Kohlenforschung
Kaiser-Wilhelm-Platz1, D-45470 Miilheim an der Ruhr (Germany)
Fax: Int. code + (208)306-2985
e-mail: reetz@mpl-muelheim.mpg.de
Priv.-Doz. Dr. K:E. Jaeger, Dip1:Biol. K. Liebeton
Lehrstuhl fur Biologie der Mikroorganismen der Universitat
D-44780 Bochum (Germany)
Fax: Int. code
(234)7094-425
e-mail: karl-erich.jaeger@ruhr-uni-bOChum.de
+
0570.083319713624-2830 $17.50+.50/0
Angew. Chem. Int. Ed. Engl. 1997.36, No. 24
-
because the process requires detailed knowledge of the threedimensional structure of the enzyme and its catalytic mechanism as well as a certain degree of intuition concerning the
Here we describe
choice of the amino acid to be sub~tituted.[~]
a new approach to developing enantioselective catalysts,
namely in vitro evolution as a method to increase stepwise the
The
enantioselectivity of a given unselective biocataly~t.[~I
underlying principle-"evolution in the test t u b e " 4 o e s not
require any knowledge of the enzyme structure or of its
catalytic mechanism. In vitro
was already used
successfully in efforts directed towards increasing the thermal
Proper molecular-biostability and activity of
logical methods for random mutagenesis and expression of
genes coupled with an efficient screening system for the rapid
identification of enantioselective mutants form the basis of
our strategy.isl
The enantioselective hydrolysis of racemic p-nitrophenyl2methyldecanoate (1)was chosen as the test reaction. The free
(S)-configurated acid belongs to a class of chiral compounds
which are useful as drugs, plant-protecting agents, and chiral
building blocks in organic synthesis.[*]The lipase from the
time, an efficient test system had to be developed; conventional separation based on liquid or gas chromatography was
unsuitable due to the expected number of samples. Accordingly, the 96 wells of commercially available microtiter plates
were loaded painvise with the culture supernatants of the
lipase mutants together with 0 . 0 1 Tris/HCl
~
buffer (pH 7.5;
Tris = tris(hydroxymethy1)aminomethane) followed by the
addition of enantiomerically pure ( R ) and (S) substrafes
dissolved separately in DMF. Thus, up to 48 mutants per
microtiter plate could be screened. The enzyme-catalyzed
hydrolysis of each ( R ) / ( S )hydrolysis pair was monitored by
measuring the absorption of the p-nitrophenolate anion at
410 nm as a function of time. Figure 1 shows the effect of the
a)
'1
(S)-enantiomer
A
(R)-enantiorner
100
bacterium Pseudomonas aeruginosa PAO1, which in its
mature form is composed of 285 amino acids, was used as
the biocataly~t.[~]
The wild-type enzyme shows an enantioselectivity (ee) of only 2 YOin favor of the (S)-configurated acid 2.
The number of mutants for a given enzyme in which one
amino acid per enzyme molecule was substituted by one of the
remaining 19 amino acids is given by Equation (a): N is the
N=19'X!I[(X-- M ) ! M ! ]
(a)
number of mutants, M the number of substituted amino acids
per enzyme (in this case l), and X the total number of amino
acids per enzyme. Therefore, the corresponding library of
l? aeruginosa lipase mutants would contain 5415 members.
Although such a library is already relatively small, our studies
show that upon screening an even smaller number of mutants,
catalysts with clearly improved enantioselectivity can be
obtained.
Using the "error-prone polymerase chain reaction"
(epPCR),[l"I the lipase gene consisting of 933 base pairs was
subjected to random mutagenesis.[lla]By changing the PCR
reaction components and conditions, for example variation of
the Mgz+concentration, the mutation frequency was empirically adjusted so that statistically one to two base substitutions
per lipase gene were introduced; this could lead to substitution of up to two amino acids per lipase molecule. This low
number of mutations per lipase molecule was chosen on the
assumption that the identification of the rarely occurring
mutations with a positive effect would not be prevented by the
more commonly occurring mutations with a negative effect.
The mutated genes were ligated into a suitable expression
vector, amplified in E. coli, and transformed into l? aeruginosa.[5,"hl The bacterial clones were then cultivated, each
theoretically producing a mutant lipase.
About 1OOOmutants isolated in this manner had to be
screened for their enantioselectivity in the test reaction. To
accomplish this difficult feat within a reasonable period of
Angew. Chem. Inr. Ed. Engl.
1997,36,No. 24
4
2M)
1
I
(S)-enantiomer
A
tls
---D
Figure 1. Course of the lipase-catalyzed hydrolysis of the R- and S-ester 1 as a
function of time. a) Wild-type lipase from fl aeruginosa, b) improved mutant in
the first generation.
original wild-type lipase as well as the typical reaction profile
of an improved mutant. Although the relative slopes of the
two straight lines can serve as a basis for calculating the
corresponding enantioselectivity, we used them only to obtain
a qualitative indication due to the possibility of various
sources of error. Of 1OOOmutants of the first generation
tested, 12 showed an increase in enantioselectivity. The exact
enantioselectivity was determined by hydrolyzing the racemate 1 in the presence of the corresponding mutant lipases
and analyzing the reaction products by gas chromatography
on chiraily modified capillary columns. The best mutant,
PlBOl-E4, showed an enantioselecivity of 31 YOee.
In each generation the clone with the highest enantioselectivity was chosen for the subsequent mutagenesis cycle
(Table 1). Figure 2 shows that it was possible to increase the
enantioselectivity from 2 % ee (wild-type enzyme) to 81 Yoee
in only four generations.
Table 1. Results of screening.
Generation
1
2
3
4
Number of
clones tested
Number of
uositive clones
Designation of
the chosen clone
1000
12
10
PlBOLE4
P2B08-H3
P3B13-Dl0
P4B04-H3
22M)
2400
2000
0 WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1997
1
6
0570-083319713624-2831 $ 17.50+.5010
2831
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7=fl
81%
i
i
i
i
i
Q
mutant generation
Figure 2. Sequential increase of enantioselectivity in the test reaction of 1in the
course of the mutagenesis experiments. (The ee values refer to the corresponding
best mutant in a reaction with a conversion range of 20-30%; corresponding E
values: 1.00, 2.10, 4.40, 9.40, and 11.3, respectively.)
In view of the fact that only four mutant generations were
produced and only relatively few mutants were actually
screened, the results obtained so far are remarkable. To
increase the enantioselectivity even more we are currently
exploring four possibilities: 1) more comprehensive screening, 2) production of further generations, 3) DNA-shuffling[6b,7]
as an alternative mutagenesis method, and 4) saturation mutagenesis[l21(systematic substitution at the mutated
positions with the remaining amino acids). At present it is not
possible to predict which of the four strategies will lead most
rapidly to an even further increase in enantio~electivity.[*~~
Investigations concerning sequence determination of the
mutant genes as well as modelling the three-dimensional
structure of the mutant proteins are also under way.
Lipases from different organisms serve routinely as catalysts in the enantioselective synthesis of chiral esters, alcohols,
amines, diols, diesters, diamines, amino alcohols, a- and pamino acid derivatives, and cyanohydrins, albeit not always
with acceptable ee va1ues.P 141 Therefore, our method, which is
presumably not restricted to lipases, opens up new perspectives in organic chemistry. The evolutive optimization of
enantioselectivity described here can now be combined with
the search for higher enzyme stability and activity."') All
efforts in this new area depend upon the development of
further screening methods.15,l61
Received: October 30, 1997 [Z11106IE]
German version: Angew. Chem. 1997,109,2961 -2963
-
Keywords: asymmetric catalysis enzyme catalysis
mutagenesis screening methods
-
-
- lipases
[I] See for example a) D. J. Berrisford, C. Bolm, K. B. Sharpless, Angew. Chem.
1995,107,1159-1171 ;Angew. Chem., Inr. Ed. Engl. 1995,34,1059-1070;
b) R. Noyori, Asymmetric Catalysis in Organic Synrhesis, Wiley, New York,
1994; c) A. Pfaltz, Acc. Chem. Reg. 1993,26,339-345.
[2] a) Chirality in Industry: The Commercial Manufacture and Applications of
Optically Active Compounds (Eds.: A. N. Collins, G. N. Sheldrake, J.
Crosby). Wiley, Chichester, 1992; b) Chirality in Indilstry 11: Developments
in the Commercial Manufacture and Applications of Opticolly Active
ComDounds (Eds.: A. N. Collins. G. N. Sheldrake. J. Crosbv). Wilev.
Chit.heste1, &?.
C)
a) H. G. Davies, R. H. Green, D. R. Kelly, S. M. Roberts, Biofransformalions in Preparative Organic Chemistry: The Use of Isolated Enzymes and
Whole Cell Systems in Synthesis, Academic Press, London, 1989; b) C. H.
Wong, G. M. Whitesides, Enzymes in Synthetic Organic Chemistry, Pergamon, Oxford, 1994; c) Enzyme Catalysis in Organic Synthesis (Eds.: K.
Drauz, H. Waldmann), VCH, Weinheim, 1995; d) K. Faber, Biorransformalions in Organic Chemistry, 3rd ed., Springer, Berlin, 1997.
a) A. J. Russell, A. R. Fersht, Nature (London) 1987,328,496-500; b) M.
Holmquist, I. G. Clausen, S. Patkar. A. Svendsen, K. Hult, J. Protein. Chem.
1995, 14, 217-224; c) H. D. Beer, G. Wohlfahrt, J. E. G. McCarthy, D.
Schomburg, R. D. Schmid. Protein Eng. 1996,9, 507-517; d) Y. Hirose, K.
Kariya, Y. Nakanishi, Y. Kurono, K. Achiwa, Tefrahedron Lett. 1995, 36,
1063- 1066; e) M. J. Haas, R. D. Joerger, G. King, R. R. Klein, Ann. N Y
Acad. Sci. 1996,799,115-128.
M. T. Reetz, A. Zonta, K. Schimossek, K. Liebeton, K.-E. Jaeger, patent
application D E A 19731990.4.
a) K. Chen, F. H. Arnold, Proc Natl. Acod. Sci. USA 1993, 90,5618-5622;
b) W. P. C. Stemmer, Nature (London) 1994,370, 389-391.
a) A. Crameri, G. Dawes, E. Rodriguez, Jr., S. Silver, W. P. C. Stemmer, Nut.
Biotechnol. 1997, 15,436-438; b) W. P C. Stemmer, Proc. Natl. Acad. Sci.
USA 1994, 91, 10747- 10751; c) J.-H. Zhang, G. Dawes, W. P C. Stemmer,
Proc. Narl. Acad. Sci USA 1997,94, 4504-4509.
a) J. C. Moore, F. H. Arnold, F. H. Nut. Biotechnol. 1996, 14,458-467; b)
F. H. Arnold, Chem. Eng. Sci. 1996,51,5091- 5102; c) Z. Shao, F. H. Arnold,
Cum Opin. Struct. Biol. 1996,6,513-518; d) L. You, F. H. Arnold, Protein.
Eng. 1996, 9, 77-83; e) A. Shinkai, A. Hirano, K. Aisaka, J Biochem.
(Tokyo) 1995,120,915-921.
a) K.-E. Jaeger. B. Schneidinger, K. Liebeton, D. Haas, M.T. Reetz, S .
Philippou, G. Gerritse, S. Ransac, B. W. Dijkstra in Molecular Biology of
Pseudomonads (Eds.: T. Nakazawa, K. Furukawa, D. Haas, S. Silver), ASM
Press, Washington, 1996, pp. 319-330; b) K.-E. Jaeger, K. Liebeton, A.
Zonta, K. Schimossek. M. T. Reetz, Appl. Microbiol. Biotechnol. 1996, 46,
99- 105.
a) D. W. Leung, E. Chen, D. V. Goeddel, Technique (Philadelphia) 1989.1.
11- 15; b) K. A. Eckert, T. A. Kunkel, PCR Merhods'Appl. 1991, 17-24;
c) R. C. Cadwell, G. E Joyce, ibid 1992.2, 28-33.
[I11 a) The lipase gene cloned into pBluescript I1 KS (Stratagene) was mutagenized by epPCRlba01using the following primers: A: 5'-GCG CAA TTA
ACC CTC ACT AAA GGG AAC AAA-3' and B: 5'-GCG TAA TAC GAC
TCA CTA TAG GGC GAA-3'. PCR was performed under the following
conditions: 2 min at 98 "C (1 x ); 1 min at 94"C, 2 min at 64"C, 1 min at 72°C
(25 x ); 7 min 72°C (1 x ); b) PCR products were ligated into plasmid
pUCPL6A derived from pUCPKS (A. A. Watson, R. A. Aim, J. S . Mattick,
Gene 1996, 172, 163-164). Plasmids were propagated in E. coli JM 109,
isolated, and used to transform P. aerugrnosa PABST7.1 (K.-E. Jaeger, B.
Schneidinger. E Rosenau, M. Werner. D. Lang, B. W. Dijkstra, K.
Schimossek, A. Zonta, M. T. Reetz, J. Mol. Catal. B: Enzym. 1997, 3, 312). For each new cycle of mutagenesis the lipase gene of the best perfoming
clone for a given mutant generation was isolated and recloned into
pBluescript I1 KS.
(121 a) H. Flores, J. Osuna, J. Heitman, X. Soberon, Gene 1995,157,295-301; b)
M. S. Warren, S. J. Benkovic, Protein Eng. 1997, 10, 63-68.
I131 Our current strategy of using the best mutant of a given generation is not
necessarily optimal. It is conceivable that a less selective mutant may lead to
a better result in the subsequent mutagenesis round.
1141 a) W. Boland, C. Frossl, M. Lorenz, Synthesis 1991,1049-1072; b) F. Theil,
Chem. Rev. 1995,95,2203-2221; c) Lipases, Part A : Biotechnology (Eds.: B.
Rubin, E. A. Dennis), Academic Press, San Diego, 1997 (Methods Enzymol.
1997, 284); d) Lipases, Part B: Enzynze Characterization and Ufilization
(Eds: B. Rubin, E. A. Dennis), Academic Press, San Diego, 1997 (Methods
Enzymol. 1997, 286); e) E. N. Vulfson in Lipases (Eds.: P. Woolley, S. B.
Petersen), Cambridge University Press, Cambridge, 1994, pp. 271 -288; f)
Engineering orwith Lipuses (Ed.: F. X . Malcata), Kluwer, Dordrecht, 1996
(NATO AS1 Ser: Ser E. 1996.317).
1151 In our studies this also occurred to some extent, since the mutants having a
low activity (which in some cases may lead to higher enantioselectivities) are
"sorted out" upon screening. Only those mutants are considered which
induce a notable conversion within ten minutes under the reaction
conditions. Indeed, many mutants show practically no activity, which is
not surprising.
[16] L. E. Janes, R. 3. Kazlauskas, J. Org. Chem. 199762,4560-4561.
i,
A. Pers'dis, Nat. Biotechnol. 1997, 75, 594-
595; d) R. A. Sheldon, Chirotechnology: Industrial Synthesis of
Optically Active Compounds, Dekker, New York, 1993; e) tndustrial
Enzymology (Eds.: T. Godfrey, S. West), 2nd ed., Macmillan Press, London,
19%.
2832
0 WILEY-VCH Verlag GmbH, D-69451 Weinheim. 1997
0570-0833/97/3624-2832$ 17.50+.50/0
Angew. Chem. Int. Ed. Engl. 1997.36. No. 24
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