Journal of Molecular Catalysis B: Enzymatic 10 Ž2000.

141–149
www.elsevier.comrlocatermolcatb

Enzymatic resolution of diltiazem intermediate by Serratia

marcescens lipase: molecular mechanism of lipase secretion
and its industrial application
Takeji Shibatani a , Kenji Omori b, Hiroyuki Akatsuka b, Eri Kawai b,
Hiroaki Matsumae a,)
a
Product and Technology DeÕelopment Laboratory, Tanabe Seiyaku Co., Ltd., 16-89, Kashima 3-chome, Yodogawa-ku,
Osaka 532-8505, Japan
b
DiscoÕery Research Laboratory, Tanabe Seiyaku Co., Ltd., 16-89, Kashima 3-chome, Yodogawa-ku, Osaka 532-8505, Japan
Received 8 July 1999; accepted 12 October 1999

Abstract

A lipase from Serratia marcescens was selected as an asymmetric hydrolytic enzyme for trans-3-Ž4-
methoxyphenyl.glycidic acid methyl ester wŽ".-MPGMx, a key intermediate in the synthesis of diltiazem hydrochloride that
is useful as a coronary vasodilator. This lipase has high enantioselectivity Ž E s 135. and was applied to the industrial
production of the optically active intermediate of diltiazem using two-phase reaction system of organic solvent–water.
Introduction of enzymatic reaction into the chemical synthetic route of diltiazem reduces the number of processes from nine
to five. Analyses of the secretion mechanism of the lipase from S. marcescens cell membrane revealed that lipase ŽLipA.,
metalloprotease ŽPrtA., cell surface protein ŽSlaA. and flagellin are secreted via ABC-transporter, which is a common
secreting mechanism in Gram-negative bacteria other than N-terminal signal peptide-dependent secreting mechanism.
Molecular cloning of both the lipA gene, which codes the lipase protein, and lipBCD genes, which code the secretion device
proteins, enable the production of the lipase by the self-cloning strain 140-fold as compared to the wild type strain.
Immobilization of the lipase on a hollow fiber type membrane reactor contributes to the repeated use of enzyme and to
efficient separation of the reaction product. Thus, enzymatic reaction and product separation are achieved simultaneously.
q 2000 Elsevier Science B.V. All rights reserved.

Keywords: Enzymatic resolution; Optically active compound; Lipase; Secretion; Hollow fiber type membrane reactor; Diltiazem

1. Introduction effects reside in only one of its enantiomers.

Another enantiomers have different biological
Newly developed drugs have asymmetric effects or toxicity. Their action on human be-
atoms in their chemical structure, and biological ings is competitive and differs in adsorption,
metabolism, degradation and excretion. For the
) production of optically active pharmaceuticals,
Corresponding author. Tel.: q81-6-6300-2575; fax: q81-
6300-2590. it is important to establish the process of optical
E-mail address: matsumae@tanabe.co.jp ŽH. Matsumae.. resolution or asymmetric syntheses.

1381-1177r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved.
PII: S 1 3 8 1 - 1 1 7 7 Ž 0 0 . 0 0 1 2 2 - 3
142 T. Shibatani et al.r Journal of Molecular Catalysis B: Enzymatic 10 (2000) 141–149

Diltiazem is a representative calcium channel itself was very efficient, a simpler and more
blocker and is used throughout the world to economical process was required to reduce the
treat angina pectoris, hypertension and several production cost of diltiazem.
other circulatory disorders w1,2x. We developed recently an enzymatic prepara-
Diltiazem has two asymmetric carbons in its tion of an optically active intermediate, methyl
chemical structure. Among the four possible Ž y . - Ž 2 R ,3S . -3- Ž 4-methoxyphenyl . glycidate
stereoisomers of diltiazem, only the Žq.- wcompound 3, Fig. 1x, and we utilized the enzy-
Ž2 S,3S .-isomer exhibits potent coronary vasodi- matic resolution process in industrial production
lating activity. Therefore, diltiazem has been of diltiazem w3–5x. In our studies, we found a
developed and marketed as a single isomer. new lipase from Serratia marcescens, which
In order to obtain the desired optical isomer, has a high enantioselectivity, E s 135, for the
various methods have been developed and the compound Žq.-3 wFig. 1x and we demonstrated
conventional chemical synthetic route is illus- that the lipase from S. marcescens is different
trated in Fig. 1. That route is composed of nine genetically from other known lipases w4,6x. Thus,
steps and chemical resolution with L-lysine was we achieved the decrease of the number of
carried out at the higher molecular weight com- synthetic processes from nine to five and there-
pound 6. Thus, industrial production of dilti- fore the manufacturing cost of diltiazem de-
azem through this resolution process gave a creased to 2r3 compared to the original method
large quantity of waste and the total processes including chemical resolution. Furthermore,
were much fastidious. Although this resolution overproduction of the lipase was performed
since the cost of enzyme production was some-
what high.
In this review, we describe the new method
for the production of optically active intermedi-
ate, glycidic ester, by the newly found lipase
from S. marcescens w3x, cloning of lipase gene
w6x and also its secretion device genes w7x, over-
production of lipase by self-cloning strain w8–
10x, and its application to the industrial scale
with hollow fiber type membrane bioreactor w5x.

2. Molecular mechanism of lipase secretion

2.1. Secretion of lipase by S. marcescens

Fig. 1. The synthetic process for diltiazem. Ž1. 4-Anisaldehyde;
Ž2. trans-3-Ž4-methoxyphenyl.glycidic acid methyl esterwŽ".-
MPGMx; Ž3. Ž2 R, 3S .-3-Ž4-methoxyphenyl.glycidic acid methyl
N-terminal signal peptide-dependent protein
esterwŽy.-MPGMx; Ž4. threo-2-hydroxy-3-Ž4-methoxyphenyl.-3- secretion is abundant in procaryotes and eucary-
Ž2-nitrophenylthio.propanoic acid methyl ester; Ž5. threo-2-hy- otes. This universal mechanism consists of
droxy-3-Ž4-methoxyphenyl.-3-Ž2-nitrophenylthio.propanoic acid;
Ž6. Ž2 S, 3S .-3-Ž2-aminophenylthio.-2-hydroxy-3-Ž4-methoxy-
SecA, SecB, SecD, SecE, SecF, SecY, signal
phenyl.propanoic acid; Ž7. Ž2 S, 3S .-2,3-dihydro-3-hydroxy-2-Ž4- peptidase and signal peptide peptidase. Gram-
methoxyphenyl.-1,5-benzothiazepin-4-Ž5H .-one; Ž8. Ž2 S, 3S .-3- negative bacteria has outer membrane and
acetoxy - 5 - w2- Ždimethylamino.ethylx - 2,3 - dihydro -2-Ž4-methoxy- periplasm between inner and outer membrane
phenyl.1,5-benzothiazepin-4Ž5H .-one hydrochloride wdiltiazemx;
Ž9. Ž2 S, 3 R .-3-Ž4-methoxyphenyl.glycidic acid; Ž10. 4-methoxy- accumulates proteins secreted through Sec sys-
phenyl acetaldehyde. tem. Gram-positive bacteria has no outer mem-
 T. Shibatani et al.r Journal of Molecular Catalysis B: Enzymatic 10 (2000) 141–149 143

brane and secretes proteins, which is secreted

through N-terminal signal system into culture
medium. Thus, Gram-positive bacteria are use-
ful for industrial production of proteins, which
is secreted into culture medium. Otherwise,
Gram-negative bacteria also secretes some pro-
teins into culture medium. For example, S.
marcescens secretes lipase and proteinase into
culture medium and these proteins have no N- Fig. 3. ABC-transporter for Gram-negative bacteria. ABC-protein:
terminal signal peptide. ATP-binding cassette protein; MFP: membrane fusion protein;
OMC: outer membrane component protein.
From the cloning and sequence analyses of
Serratia lipase gene Ž lipA. , LipA has no N-
ions, secretion system other than Has system
terminal signal peptide and has short consensus
was suggested for the secretion of lipase.
sequences between some other secreting pro-
Cloning of the genes of secretion system was
teins, which is secreted through N-terminal sig-
tried by the formation of hallo on tributyrin-agar
nal peptide-independent secretion mechanism.
and lipB, lipC and lipD genes, which code the
These proteins are hemolysin in Escherichia
proteins composed of 588, 443 and 464 amino
coli, adenylcyclase in Bordetella pertussis, met-
acids, respectively, were found. These genes
alloprotease in Erwinia chrysanthemi and so on
forms operon. As a result of homology analysis,
as shown in Fig. 2. In general, this N-terminal
transporter protein LipB, LipC and LipD have
peptide-independent secretion mechanism con-
55%, 46% and 42% homology with PrtD, PrtE
sists of three kinds of proteins as shown in Fig.
and PrtF, which forms Prt system for the metal-
3. Among these three proteins, the nearest pro-
loprotease transporter in E. chrysanthemi w13x
tein to the cytoplasm is called ABC-protein and
and have 54%, 45% and 26% homology with
has ATP-binding motif. Therefore, this secre-
HasD, HasE and HasF, which forms Has sys-
tion system composed of these three proteins is
tem for the HasA protease transporter in S.
classified as one of the ABC transporter fami-
marcescens w14x.
lies. Letoffe et al. w11,12x reported that metallo-
protease and heme-binding protein are secreted 2.2. Mechanism of Lip system
into culture medium through the Has system in
S. marcescens and expression of transporter gene In S. marcescens, lipase, metalloprotease,
is repressed by iron ions. Since lipase is se- flagellin and heme-binding protein are secreted
creted by S. marcescens in the presence of iron into culture medium. On the medium for the

Fig. 2. Alignment of homologous regions. The nonapeptide repeat regions of the S. marcescens lipase are aligned with the repeats from the
extracellular secreted proteins, Serratia sp. protease ŽPrtA., E. chrysanthemi protease ŽPrtB., E. coli hemolysin ŽHlyA., P. haemolytica
leukotoxin ŽLktA., B. pertussis cyclolysin ŽCyaA., and R. leguminosarum Ca2q-binding protein ŽNodO.. The numbers refer to the positions
of the amino acid residues in the protein sequences. Identical residues and alterations ŽI–V–M–L–F, D–E, N–Q, R–K and S–T., which are
conserved in more than 60% of the proteins are boxed and nonapeptide repeat units are indicated by arrows.
144 T. Shibatani et al.r Journal of Molecular Catalysis B: Enzymatic 10 (2000) 141–149

production of lipase, lipase and metalloprotease

are synthesized abundantly and these two pro-
teins compete for secretion. In fact, secretion of
protease is depressed under the expression of
lipase gene transformed into the host S.
marcescens with plasmid vector.
In general, genes of secreted protein forms
cluster with those of transporter. However, genes
of lipase and protease in S. marcescens does not
form a cluster with those of transporters, lipB,
lipC and lipD. On the downstream of lipBCD,
the gene of lipopolysaccharide-related synthase Fig. 4. Proteins in the cultured media and cell surface fraction of
is coded and on the upstream of lipBCD, the wild-type strain and Lip system-dependent mutants of S.
marcescens 176. The cells were cultured in lipase medium w10x at
unidentified ORF is coded in the same direc- 308C for 18 h. The polypeptides in the supernatant of the cultured
tion. We considered that the protein expressed media Ž0.2 OD equivalent units. were subjected to 12.5% SDS-
from this unidentified gene might be a real PAGE. The gels were stained by Coomassie brilliant blue G-250
ŽA. and analysed by immunoblotting with antisera against SlaA
substrate for Lip system and analyzed the pro- ŽB., LipA ŽC. and PrtA ŽD.. Molecular mass standardsŽsizes in
tein expressed from this gene. As a result, this kDa. are shown on the left. Positions of SlaA, LipA, PrtA and
gene coded a protein composed of 1004 amino flagellin are shown as S, L, P and F, respectively. Lane 1: SM176;
lane 2: SL2; lane 3: SL2 ŽpMWlipC12.; lane 4: SL13; lane 5:
acids. Analysis of the amino acid sequence of
SL13 ŽpMWlipB15.. SM176: wild type strain of S. marcescens;
this protein revealed that it has high homology SL2: LipC-deletion mutant of SM176; SL13: LipB-deletion mu-
with paracrystalline surface layer protein from tant of SM176; pMWlipC12: vector with LipC; pMWlipB15:
Caulobacter crescentus w15x and it was named vector with LipB.

SlaA as a homolog of cell surface protein. In

fact, SlaA is secreted abundantly in E. coli,
which is reconstructed from the vector plasmid from Has system and Prt system, Binet and
with Lip system. In addition, in S. marcescens Wandersman w14x demonstrated from analyses
or also in E. coli lipase, metalloprotease and of secretion profile of HasA and PrtC that secre-
SlaA are not secreted in the deletion mutants of tory proteins are recognized by the protein,
either lipB, lipC or lipD ŽFig. 4.. As a conclu- which locates nearest to cytoplasm and called as
sion, lipBCD functions as genes of transporter ATP-binding cassette. Furthermore, using the
system for the three proteins, lipase, metallopro- hybrid of Lip, Has and Prt system, we found
tease and cell surface protein. that ABC-protein recognizes not only secretory
proteins but also membrane fusionŽ MF. -protein
2.3. Recognition of secretory protein by ABC- w10x.
transporter

Each ABC-transporter has its own substrate 3. Overproduction of S. marcescens lipase by

specificity for secretory proteins. In S. cloning of lipA gene and its secretion device
marcescens, SlaA is secreted only through Lip genes
system and HasA is secreted only through Has
system. Lipase and metalloprotease are secreted The extracellular lipase from S. marcescens
through Lip system or Prt system from E. Žtriacylglycerol acylhydrolase wEC 3.1.1.3x. is
chrysanthemi and also partly secreted through an industrially important enzyme w3–5x. It is
Has system in the reconstructed E. coli. Using stable in some organic solvents and is applica-
hybrid transporter, which was reconstructed ble to the asymmetric synthesis of chiral com-
 T. Shibatani et al.r Journal of Molecular Catalysis B: Enzymatic 10 (2000) 141–149 145

pounds by asymmetric hydrolysis. Previously, analogous to hlyBDr tolC and prtDEFEC have
we cloned the lipA gene encoding this enzyme been reported in several bacteria: the aprDEF
from S. marcescens and determined its nu- genes for Pseudomonas aeruginosa alkaline
cleotide sequence w6x. The S. marcescens lipase protease w26x; the cyaBD genes for cyclolysin of
has no conventional N-terminal signal sequence B. pertussis w21x; and the lktBD genes for the P.
and is not subjected to processing at the N haemolytica leukotoxin w27x. Recently, the
terminus. Thus, extracellular secretion of the S. hasDE genes encoding HasD and HasE, which
marcescens lipase is not promoted by the gen- are two inner membrane components of the
eral secretion pathway that require an N-termi- secretion mechanism for the S. marcescens met-
nal signal sequence w16x. S. marcescens, there- alloprotease and the heme-binding protein HasA
fore, possesses a specific secretion system for w28x, have been cloned from S. marcescens
extracellular lipase, which is a signal peptide-in- w29–31x.
dependent pathway w17x. As the characteristics of the genes, lipA and
The 50-kDa metalloprotease encoded by the lipBCD, became clear, we challenged to over-
prtA gene is known as one of the major secre- produce the lipase from S. marcescens for ap-
tory proteins of S. marcescens w17x. No overall plication to industrial production scale.
homology was observed between the S. The wild type strain of S. marcescens ex-
marcescens lipase and metalloprotease amino pressed 30 unitsrml of broth when cultured on
acid sequences except for a homologous region the lipase medium. A S. marcecsens strain,
consisting of multiple repeats of nine amino which has lipA gene in the vector plasmid
acid residues Ž GGXGXDXXX., which is glycine pUC19 and also in the chromosome gene ex-
and aspartic acid rich. This sequence is found in pressed 400 unitsrml of broth when cultured on
the following proteins: metalloproteases; the the lipase medium. A S. marcescens strain,
prtB and prtC gene products of E. chrysan- which has lipA gene and also lipBCD genes on
themi w18x; hemolysin, encoded by the hlyA the same vector cannot grow well on the lipase
gene of E. coli w19x; leukotoxin, encoded by the medium, since too much expression of many
lktA gene of Pasteurella haemolytica w20x; cy- genes of lipBCD affects the growth of the
clolysin, a multifunctional protein with adeny- microorganism. Therefore, we cloned lipA on a
late cyclase activity and hemolytic activity, en- multicopy plasmid, pUC 19 and lipBCD on
coded by the cyaA gene of B. pertussis w21x; another small copy plasmid, pMW 121.
and Ca2q-binding protein, encoded by the nodO Self-transformation of both lipA gene and
gene of Rhizobium leguminosarum w22x. Colicin lipBCD genes resulted in the production of li-
V, the cyaC gene product of E. coli w23x, pase protein at a level of 4200 unitsrml of
possesses a repeated glycine-rich sequence, broth in the culture medium, which is 140-fold
which is not homologous to the GGXGXDXXX compared to the wild type S. marcescens strain.
sequence but shares some characteristics with Details of these results will be reported in an-
this consensus sequence. Although the other paper, which is in preparation.
GGXGXDXXX sequence has been proposed to
be responsible for Ca-binding, the function of
this region is unclear. Most of the proteins 4. Industrial production of optically active
containing the above sequence have been re- diltiazem intermediate using hollow fiber type
ported to be secreted into culture medium by bioreactor
strains with a specialized secretion mechanism
encoded by the hlyBDrtolC genes of E. coli A lipase excreted from S. marcescens Sr41
w23x or the prtDEFEC genes w24x of E. chrysan- 8000 enantioselectively hydrolysed Žq.-MPGM
themi w25x. Genes encoding secretion machinery Ž E s 135, w1x.. The reaction proceeded effi-
146 T. Shibatani et al.r Journal of Molecular Catalysis B: Enzymatic 10 (2000) 141–149

ciently when the enzymatic hydrolysis was car-

ried out using a conventional emulsion reactor
with toluene–aqueous biphasic system w1x.
However, to achieve industrial production of
Žy. -MPGM using the emulsion reactor, de-
struction of the emulsion and separation into
two phases were required for effective isolation
of the product. Enzyme stability and recovery
are also required for its repeated utilization. In
particular, the recovery of enzyme is very im-
portant for cost reduction in industrial processes
involving enzyme reactions. However, efficient
recovery is difficult in the case of a stable
Fig. 5. Flow diagram of membrane reactor. T:1.15 l of toluene
emulsion. We proposed an immobilized enzyme
phase containing 1.15 mol of Ž".-MPGM; W: 12.3 l of aqueous
system to solve these disadvantages of the phase containing 1.15 mol of sodium hydrogen sulfite; HM:
emulsion reactor. hydrophilic membrane; P: recycle pump; V: throttle valve.
With respect to the immobilization of lipase,
various methods have already been proposed in Fig. 6 w5x. The asymmetric hydrolysis of
w32–35x. When lipase was immobilized on vari- Ž". -MPGM by the lipase from S. marcescens
ous adsorbents, lipase could be efficiently im- immobilized on the membrane reactor is started
mobilized on these reactors. However, it is diffi- by circulating both Ž". -MPGM solution in
cult to achieve reaction and product separation toluene in the shell loop and sodium hydrogen
simultaneously. Furthermore, since the hydroly- sulfite solution ŽpH 8.5. in the lumen loop, and
sis of oil by lipase is an interfacial reaction Žq. -MPGM is hydolyzed to Žq.-Ž2 S,3 R .-3-
involving the adsorption of enzyme on the sur- Ž4-methoxyphenyl. glycidic acid and methanol.
face of the oil droplet w36x, the expressed activi- The glycidic acid is spontaneously decarboxyl-
ties of most of the immobilized lipases are low ated to p-methoxyphenylacetaldehyde Ž RCHO.
compared to those obtained with other immobi- and accumulate in the toluene phase as shown
lized enzyme w34x. We devised a membrane in Fig. 6. Though the aldehyde acted as an
reactor based on the principle of physical ad- inactivator of the lipase, it can be removed by
sorption of enzyme and the principle of contact transfer to the aqueous phase by formation of
between organic and aqueous phases for the water-soluble adduct with sodium hydrogen sul-
purposes of efficient asymmetric hydrolysis and fite added into the aqueous phase w5x. The stabil-
simplification of the product separation process. ity of the lipase is nominally influenced when
Since a pioneering work on the membrane sodium hydrogen sulfite concentration is below
bioreactor with liquid–liquid contact mode by 0.1 M at pH 8.5 and 228C. In practice, 0.0935
Hoq et al. w37x, Matson and Quinn w38x have M sodium hydrogen sulfite solution Ž pH 8.5. is
proposed a theory of hydrophilic membrane applied to the aqueous phase to prevent the
bioreactor in which organic and aqueous phases lowering of enzyme activity. Another by-prod-
are circulated along shell and lumen sides of the uct, methanol, is transferred from the toluene
membrane, respectively. In recent years, the phase to the aqueous phase. As the concentra-
membrane reactor has been commercialized by tion of methanol in the aqueous phase is below
Sepracor ŽS.L. Matson, US Pat. No. 4800162. . 0.2% Žwrv., the hydrolysis of Ž".-MPGM is
Flow diagram of membrane reactor is illus- not influenced.
trated in Fig. 5 and the behavior of substrate The stability of the lipase on the membrane
and products in membrane reactor is illustrated reactor is superior as compared to emulsion
 T. Shibatani et al.r Journal of Molecular Catalysis B: Enzymatic 10 (2000) 141–149 147

brane reactor is measured. After the regenera-

tion for eight subsequent batch runs is repeated
10 times over a period of 4 months according to
the Sepracor procedure Ž using a sodium hydro-
gen sulfite solution, a sodium hypochlorite one
and a sodium hydroxiderisopropanol one. , the
water flux rate of the membrane is 4.3 mlrmin
P kPa P m2 , which is the same as that of a new
membrane. From the result, it is thought that the
membrane reactor is appropriate to achieve in-
Fig. 6. Behavior of products in membrane reactor. Pressure 1 in dustrial utilization from the viewpoint of mem-
the shell side was kept at excess pressure compared with pressure brane regeneration.
2 in the lumen side. T: 1.15 l of toluene phase containing 1.15 mol
The velocity constant of the lipase immobi-
of sodium hydrogen sulfite; RCHO: p-methoxyphenylacetalde-
hyde; RPCHPOHPSO3 Na: an adduct formed between sodium lized on the membrane reactor is equivalent to
hydrogen sulfite and p-methoxyphenylacetaldehyde. about 8% of that of the free lipase in the
emulsion reactor. This value is higher than that
of lipase immobilized on stainless steel beads
w32x, urethane prepolymers, duolite or alky-
reaction; the half-life of enzyme activity is 127 lamine-CPG w34x, w36x. The value is approxi-
h in the membrane reactor, which is about 30 mately equivalent to that of lipase immobilized
times that in the emulsion reactor. But the ve- on photo-cross-linkable resin prepolymer w33x,
locity constant on the emulsion reactor is 3.1 polyvinylchloride, chitin, agarose, Sepharose
hy1 and that on the membrane reactor is 0.23 w34x or octyl-Sepharose w35x. The hydrolysis re-
hy1. In the case of emulsion reactor, a signifi- action using the membrane in a biphasic system
cant decrease of enzyme activity was observed. has to be carried out under excess pressure to
When the Sepracor membrane reactor, which avoid the penetration of the aqueous phase
immobilizes lipase on the toluene side of the through the hydrophilic membrane into organic
membrane is utilized, the lipase is allowed to phase. It is therefore presumed that not only the
localize on the interface of the toluene solution small area of the interface but also the enlarge-
and the aqueous solution, which permeated ment of diffusion resistance of molecule in the
through the hydrophilic membrane. The density 50-mm-thick membrane cause a decreased ex-
of protein at the interface of the two phases in pression of activity.
the membrane reactor is maintained at a high On the membrane reactor, p-methoxypheny-
value compared to that in the emulsion reactor. lacetaldehyde, which is a spontaneously de-
Therefore, by the use of the Sepracor membrane graded derivative from Ž q. -Ž 2 S,3 R . -3-Ž 4-
reactor, the hydrolyzing activity on Ž".-MPGM methoxyphenyl.glycidic acid, formed an adduct
is low but it is possible to stabilize the lipase. with sodium hydrogen sulfite, and the adduct is
From these results, it has become possible to transferred to the aqueous phase. When the
achieve repeated runs of the lipase. asymmetric hydrolysis of Ž".-MPGM is carried
Membrane deterioration is an important prob- out in eight subsequent batch runs using the
lem, which must be solved in the industrial membrane reactor with 0.80–1.6 = 10 5 units of
utilization of the membrane reactor. As the fil- lipase per m2 , Žq. -MPGM is completely de-
tration rate of the membrane is an important graded after 23 h of reaction at the initial reac-
factor in the clarification of membrane deterio- tion. As the toluene phase contained only Žy.-
ration, the water flux rate of the membrane MPGM, crystalline Žy.-MPGM with a high
before and after the regeneration of the mem- yield of 43% and optical purity of 99.9% e.e. is
148 T. Shibatani et al.r Journal of Molecular Catalysis B: Enzymatic 10 (2000) 141–149

obtained by concentrating the toluene solution. w2x H. Abe, H. Inoue, T. Nagao, Jpn. J. Pharmacol. 108 Ž1988.
716.
From the second to the sixth reaction, 0.2% to 4 w3x H. Matsumae, M. Furui, T. Shibatani, J. Ferment. Bioeng. 75
% of Ž".-MPGM is unreactive after 23 h of Ž1993. 93.
each reaction. The solubility of optically pure w4x H. Matsumae, T. Shibatani, J. Ferment. Bioeng. 77 Ž1994.
152.
MPGM in toluene is nearly equal to half of the w5x H. Matsumae, M. Furui, T. Shibatani, T. Tosa, J. Ferment.
solubility of racemic Ž".-MPGM in toluene. Bioeng. 78 Ž1994. 59.
After the toluene solution containing MPGM is w6x H. Akatsuka, E. Kawai, K. Omori, S. Komatsubara, T.
concentrated from 1.15 to 0.12–015 l, pure Shibatani, T. Tosa, J. Bacteriol. 176 Ž1994. 1949.
w7x H. Akatsuka, E. Kawai, K. Omori, T. Shibatani, J. Bacteriol.
Žy. -MPGM with a high yield of 40–43% and 177 Ž1995. 6381.
optical purity of 99.9% e.e. is obtained by the w8x H. Akatsuka, E. Kawai, K. Omori, S. Komatsubara, T.
crystallization from the concentrated toluene so- Shibatani, J. Ferment. Bioeng. 81 Ž1996. 115.
w9x H. Akatsuka, R. Binet, E. Kawai, C. Wandersman, K. Omori,
lution at 48C. At the seventh and eighth reac- J. Bacteriol. 179 Ž1997. 4754.
tions, the yields of crystalline Žy. -MPGM Ž99.9 w10x E. Kawai, H. Akatsuka, A. Idei, T. Shibatani, K. Omori,
% e.e.. reduced to 38% and 36%, respectively. Mol. Microbiol. 27 Ž1998. 941.
w11x S. Letoffe, J.-M. Chigo, C. Wandersman, J. Bacteriol. 175
The productivity of Sepracor membrane biore- Ž1993. 7321.
actor is about 40 kg Žy. -MPGM per m2 per w12x S. Letoffe, J.-M. Chigo, C. Wandersman, J. Bacteriol. 176
year. Ž1994. 5372.
In conclusion, when the lipase having a high w13x S. Letoffe, P. Delepelaire, C. Wandersman, EMBO J. 9
Ž1990. 1375.
enantioselectivity is immobilized on the mem- w14x R. Binet, C. Wandersman, Mol. Microbiol. 22 Ž1996. 265.
brane reactor suitable for the asymmetric hydro- w15x A. Gilchrist, J.A. Fisher, J. Smit, Can. J. Microbiol. 38
lysis of the key intermediate in the synthesis of Ž1992. 193.
w16x A.P. Pugsley, C. d’Enfert, I. Reyss, M.G. Kornacker, Annu.
diltiazem hydrochloride, the production of Rev. Genet. 24 Ž1990. 67.
Žy. -MPGM is achieved efficiently by simulta- w17x C. Wandersman, Trends Genet. 8 Ž1992. 317.
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