Green synthesis of (R)-3-TBDMSO glutaric acid methyl monoester using Novozym 435 in non-aqueous media

Article abstract of DOI:10.1039/c5ra14348a

An efficient biocatalytic synthesis of (R)-3-TBDMSO glutaric acid methyl monoester (R-J₆), an important intermediate in the synthesis of rosuvastatin, has been developed using a green catalytic route in the presence of lipase, conducted under mild conditions without additional chiral reagents. Enzyme screening indicated Novozym 435 to be the most efficient biocatalyst for R-J₆ synthesis. Methanol, which was the most effective alcohol for synthesis of R-monoester, was identified as the best acyl acceptor by molecular docking. The optimal conditions for synthesis of R-J₆ were as follows: 50 g L^-1 catalyst, 3 sp;:sp;1 molar ratio of methanol sp;:sp;substrate, 200 g L^-1 substrate, iso-octane as solvent, orbital shaking at 200 rpm, and an incubation time of 24 h at 35°C. The key factor affecting the yield of R-J₆ was the molar ratio of methanol to substrate found by an orthogonal array experimental design. Consequently, the desired product, R-J₆, was afforded with a titer of 117.2 g L^-1, a yield of 58.6%, and productivity of 4.88 g L^-1 h^-1. This green method holds promise for the preparation of kilogram quantities of (R)-3-substituted glutaric acid monoesters.

Full text of DOI:10.1039/c5ra14348a

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PAPER
Green synthesis of (R)-3-TBDMSO glutaric acid
methyl monoester using Novozym 435 in non-
aqueous media†
Cite this: RSC Adv., 2015, 5, 75160
Hongjiang Wang,abc Zebiao Li,d Xiaoxia Yu,abc Ruidong Chen,abc Xiulai Chenabc
abc
*
and Liming Liu
An efficient biocatalytic synthesis of (R)-3-TBDMSO glutaric acid methyl monoester (R-J6), an important
intermediate in the synthesis of rosuvastatin, has been developed using a green catalytic route in the
presence of lipase, conducted under mild conditions without additional chiral reagents. Enzyme
screening indicated Novozym 435 to be the most efficient biocatalyst for R-J6 synthesis. Methanol,
which was the most effective alcohol for synthesis of R-monoester, was identified as the best acyl
acceptor by molecular docking. The optimal conditions for synthesis of R-J6 were as follows: 50 g Lꢀ1
ꢀ1
This article can be cited before page numbers have been issued, to do this please use: H. Wang, Z. Li, X.
catalyst, 3 : 1 molar ratio of methanol : substrate, 200 g L substrate, iso-octane as solvent, orbital
shaking at 200 rpm, and an incubation time of 24 h at 35 ꢁC. The key factor affecting the yield of R-J6
Yu, X. Chen and L. Liu, RSC Adv., 2015, DOI: 10.1039/C5RA14348A.
Received 20th July 2015
Accepted 13th August 2015
was the molar ratio of methanol to substrate found by an orthogonal array experimental design.
Consequently, the desired product, R-J6, was afforded with a titer of 117.2 g Lꢀ1, a yield of 58.6%, and
DOI: 10.1039/c5ra14348a
productivity of 4.88 g Lꢀ1
h
ꢀ1. This green method holds promise for the preparation of kilogram
www.rsc.org/advances
quantities of (R)-3-substituted glutaric acid monoesters.
world's top ten, with annual sales of $5.3 billion recorded
in 2013.8
Current industrial production of rosuvastatin is mainly by
1. Introduction
Statins are a class of pharmaceuticals that inhibit the enzyme
hydroxymethylglutaryl-CoA reductase (HMGR) and are widely
used as hypolipidemic agents to lower the level of cholesterol in
the blood.1 Clinical trials have conrmed that statins can adjust
blood lipid levels2,3 and reduce the risk of fatal and nonfatal
cardiovascular disease.4 In particular, rosuvastatin, the so-
called “super statin”, has high efficacy, few side-effects, low
toxicity, and outstanding selectivity. Dose-for-dose, rosuvastatin
is by far the most efficacious statin for reducing plasma low-
density lipoprotein (LDL) cholesterol, reducing total choles-
terol signicantly, and the duration of inhibition is longer than
for other statins such as atorvastatin, simvastatin, and pravas-
tatin.5,6 The market for cholesterol-lowering drugs is the largest
in the pharmaceutical sector,7 and industrial production of
rosuvastatin is signicant. Sales of rosuvastatin remain in the
chemical synthesis,9,10 in which the pyrimidine nucleus
and chiral side chain are condensed using the Wittig reaction.11
The chiral side chain acts as the functional group, presenting
the pharmacophore for HMGR recognition.12 (R)-3-Substituted
glutaric acid monoesters are important intermediates for the
assembly of the chiral side chain. In recent years, several
effective methods have been used for the preparation of (R)-3-
substituted glutaric acid monoesters, including chemical
synthesis,12 enzymatic methods13 and chiral resolution.14
Industrial production of (R)-3-substituted glutaric acid mono-
esters is mostly by chemical synthesis.12,15,16 Green synthetic
routes involving renewable raw materials and the replacement
of environmentally “unfriendly” syntheses are receiving
increasing attention. However, chemical syntheses of 3-
substituted glutarates require extreme conditions, such as low
temperature (ꢀ78 ꢁC)12 and expensive reagents (benzyl (R)-
(ꢀ)-mandelate and Pd(OH)2–C)15,17 containing heavy-metals,
which affects the quality of the chiral end-product, and high
energy consumption; and the poor extraction and expensive
additional purication steps made the whole process costly,
which made the method unsuitable for large-scale R-J6
preparation.
aState Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu
Road, Wuxi, Jiangsu 214122, China. E-mail: mingll@jiangnan.edu.cn; Fax: +86-0510-
85197875; Tel: +86-0510-85197875
bThe Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan
University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China
cLaboratory of Food Microbial-Manufacturing Engineering, Jiangnan University, 1800
Lihu Road, Wuxi, Jiangsu 214122, China
dNantong Chanyoo Pharmatech Co., Ltd., Coastal Economic Development Zone, No. 2
Tonghaisi Road, Rudong county, Nantong, Jiangsu 226407, China
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c5ra14348a
With high catalytic efficiency, mild reaction conditions,
fewer side reactions, and environmental friendliness, biological
catalysts have been widely applied to industrial production.18–21
75160 | RSC Adv., 2015, 5, 75160–75166
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Enzymatic methods provide an alternative to traditional
complex chemical synthesis.22 Kinetic resolution by biological
catalysts can remove one enantiomer from the racemate selec-
tively and mildly, and the unwanted enantiomer can be sepa-
rated. However, since the maximum theoretical yield is only
50%,23 application of kinetic resolution on a large scale has
been hampered. Hydrolysis of 3-substituted glutaric acid dies-
ters using hydrolases or esterases has been applied to the
preparation of (R)-3-substituted glutaric acid monoesters,24 but,
because the substrates are poorly soluble in water, the reaction
is slow. To achieve higher yields, the hydrolysis has been con-
ducted in a two-phase aqueous–organic system,25 but yields are
still limited because the reaction only occurs at the solvent
interface.
The use of organic solvents in biocatalytic reactions has
addressed the problem of low aqueous solubility of the
substrate. A possible alternative for green production of (R)-3-
substituted glutaric acid monoesters is acylation of alcohols
with 3-substituted glutaric anhydride using biocatalysts
(Scheme 1), which has a theoretical yield of 100%. Since
suitable organic solvents26 can enhance the “rigid” confor-
mation of lipases,27–29 improve heat resistance18,30 and main-
tain high catalytic activity,27 mono-esterication using lipases
is more promising for industrial application. Unfortunately, a
lot of work on preparation of 3-substituted glutaric acid
monoesters has revealed that natural lipases favor the
product with S-conguration (ESI data Table S1†).22,23 The
yield of R-J6 was low and could not meet industrial demand,
therefore enzymatic synthesis of (R)-3-substituted glutaric
acid monoesters (R-monoester) by lipases has rarely been
reported.
In this study, we have extended the scope of the synthesis
of (R)-3-TBDMSO glutaric acid methyl monoester (R-J6) using
organic solvent by alcoholysis of 3-substituted glutaric
anhydride with an S-selective lipase. The lipase from Candida
antarctica (CALB) has been screened and employed for
R-J6 production with high catalytic efficiency.31 Following
selection of the best performing enzyme, the co-substrate
(alcohol) that would be favored for synthesis of the R-mono-
ester was selected as the acyl acceptor by molecular docking.
The nal objective was to optimize the reaction conditions.
An efficient process for large-scale production of (R)-3-
substituted glutaric acid monoesters by reaction of
3-substituted glutaric anhydride with alcohols has been
developed.
2. Materials and methods
2.1. Materials
Novozym 435 (CALB, lipase from C. antarctica immobilized on a
macroporous anionic resin) was purchased from Novozymes
(Beijing, China). 3-TBDMSO glutaric anhydride (TBDMSO: t-
butyl-dimethyl-silyloxy) was purchased from Yuchen Fine Co.,
Ltd (Henan, China). a-Chymotrypsin was purchased from San-
gon Biological Engineering Technology & Services Co., Ltd
(Shanghai, China). Isopropanol and n-hexane (HPLC grade)
were purchased from Sigma (St Louis, USA). Other chemicals
and solvents (analytical grade) were from local suppliers (Wuxi,
China). Standards of (R)-3-TBDMSO glutaric acid methyl
monoester and racemic 3-TBDMSO glutaric acid methyl
monoester were obtained as a gi from Chanyoo Pharmatech
Co., Ltd (Nantong, China). The Chiralpak AD-H column (4.6 ꢂ
250 mm) was purchased from Daicel Chiral Technologies
(Shanghai, China).
2.2. Analytical procedure
Methyl esters in the reaction mixture were analyzed by high
performance liquid chromatography (HPLC) using a Daicel
Chiralpak AD-H column (4.6 ꢂ 250 mm) and an ultraviolet
detector supplied by Agilent. The mobile phase consisted of
96% hexane and 4% isopropanol with 0.02% (v/v) acetic acid,
which was ltered through a 0.45 mm membrane. A 10 mL
sample was injected into the column with a detection temper-
ature of 25 ꢁC and a ow rate of 1 mL minꢀ1. The run time was
15 min.
R-J6 and racemic 3-TBDMSO glutaric acid methyl monoester
(racemic J6) were the internal standards. Under these conditions
the retention times were as follows: R-J6, 7.7 min; (S)-3-TBDMSO
glutaric acid methyl monoester (S-J6), 8.3 min. Aliquots (50 mL)
of the reaction mixture were taken and solvent removed in an
oven at 70 ꢁC. Each sample was diluted with 1 mL of mobile
phase then ltered through a 0.22 mm membrane.
2.3. Molecular docking of alcohols
The crystal structure of CALB32 [PDB: 1TCA] was taken from
the Protein Data Bank (http://www.rcsb.org/pdb/explore/
explore.do?structureId¼1TCA). In the molecular docking, a
series of alcohols were used as acyl acceptors. Three-
dimensional structures of the ester products were obtained
from Chemoffice Ultra 11.0 then minimized using CHARMM.
Scheme 1 Enzymatic preparation of rosuvastatin side-chain intermediate (R-JR) from 3-TBDMSO glutaric anhydride using Novozym 435. When
R is CH3–, R-JR ¼ R-J6; alcohol can be methanol, ethanol, n-propanol, n-butanol, iso-butanol, tert-butanol, hexanol, benzyl alcohol, 1-phe-
nylethanol or 2-phenylethanol.
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The compounds were docked into the CALB binding site to
determine docking energies and hydrogen bonding. Only the
ligand molecules were considered exible during the docking
simulation, and only the free energy of the best pose was taken
for comparison.
2.4. Esterication reaction
A typical esterication reaction was conducted in 10 mL capped
asks using methyl tert-butyl ether (MTBE) as solvent, 3-
TBDMSO glutaric anhydride as substrate, and Novozym 435 as
ꢀ
catalyst (Scheme 1). Crushed 3 A molecular sieves were activated
by heating in an oven at 100 ꢁC for at least 3 days. The organic
ꢀ
solvent was dried over 3 A molecular sieves for 72 h before use.
The activated molecular sieves (2.5 g, Aldrich, 15–20 wt% based
on substrate) were added to the reaction mixture to absorb
water generated during the esterication. The mixture was
Fig. 1 Effect of Novozym 435 concentration on R-J6 production.
(
) R-J6 titer, (
) S-J6 titer, (
) yield of R-J6.
ꢁ
incubated for 24 h on an orbital shaker (200 rpm) at 35 C.
increased from 0 to 60 g Lꢀ1, the titer of R-J6 increased. The R-J6
titer reached 15.7 g Lꢀ1 when the concentration of Novozym 435
was 60 g Lꢀ1, with a yield of 26.13% and productivity of 0.65 g
Lꢀ1 hꢀ1. The R-J6 titer increased 11-fold compared with that
obtained when 10 g Lꢀ1 Novozym 435 was used. CALB crystal
structures suggest a catalytic mechanism somewhat similar to
the serine proteases, with the Ser105–His224–Asp187 triad as the
catalytic centre.32,35,36 The mechanism of (R)-3-TBDMSO glutaric
acid methyl monoester production by Novozym 435 has been
studied by molecular dynamics simulation (more information
showing in ESI Scheme S4†).
2.5. Statistical analysis
Three different factors (molar ratio of methanol to substrate,
Novozym 435 concentration and 3-TBDMSO glutaric anhydride
concentration) were explored using an L9-orthogonal array
design. The design was developed and analyzed using Design-
Expert 8.0 soware. All measurements were taken in triplicate
and experiments were repeated three times to evaluate the
standard deviation.
3. Results and discussion
3.1. Screening of the catalysts for R-J6 production
3.2. Effect of the acyl acceptor on R-monoester production
According to the literature33,34 there are four lipases (Lipozyme
TLIM from Thermomyces lanuginosus, porcine trypsin, Novozym
435 from C. antarctica, and a-chymotrypsin) that can be used as
biocatalysts for asymmetric alcoholysis of 3-substituted glutaric
anhydride. The performance of the four lipases in the synthesis
of R-J6 was investigated and the results are shown in Table 1.
Trypsin and a-chymotrypsin performed poorly, giving product
titers below 10 g Lꢀ1. Novozym 435 gave the best results, with
the titer of R-J6 reaching 12 g Lꢀ1 and productivity increased to
0.49 g Lꢀ1 hꢀ1. Based on these results, Novozym 435 was
selected as the catalyst for R-J6 production.
The ester bond between the alcohol and 3-hydroxy glutaric acid
is hydrolyzed in the nal step of rosuvastatin synthesis (Scheme
S2†). However, alcohols can affect the interactions between
product enantiomers and the enzyme. A series of alcohols were
used as acyl acceptors in the molecular docking (Scheme 1), and
the results are shown in Table 2. The larger the ER/ES ratio, the
more stable the predicted transition state of the R-isomer. Of
the alcohols studied, the best ER/ES ratios were for methanol
(1.072) and tert-butanol (1.043). Therefore, methanol and tert-
butanol were chosen as the acyl acceptors.
The effect of Novozym 435 concentration on R-J6 production
is shown in Fig. 1. As the concentration of Novozym 435
Table 2 Results of molecular docking
Energy of
R-isomer
Energy of
S-isomer
a
(kcal molꢀ1
)
(kcal molꢀ1
)
ER/ES
Table 1 Performance of different lipases in the synthesis of R-J6
Entry
R-OH
R-J6 titer
Productivity
1
2
3
4
5
6
7
8
9
10
Methanol
Ethanol
ꢀ97.74
ꢀ91.187
ꢀ109.65
ꢀ101.06
ꢀ107.47
ꢀ87.93
1.072
0.876
0.996
0.930
0.959
1.043
0.760
0.945
0.979
0.936
Entry
Enzyme
Time (h)
(g Lꢀ1
)
(g Lꢀ1 hꢀ1
)
ꢀ96.00
n-Propanol
n-Butanol
Iso-butanol
tert-Butanol
Hexanol
Benzyl alcohol
1-Phenylethanol
2-Phenylethanol
ꢀ100.70
ꢀ99.896
ꢀ84.362
ꢀ107.84
ꢀ85.98
1
2
3
4
Novozym 435
Lipozyme TLIM
a-Chymotrypsin
Trypsin
24
24
24
24
11.72
10.56
7.92
0.49
0.44
0.33
0.28
ꢀ103.37
ꢀ113.09
ꢀ102.17
ꢀ115.89
ꢀ115.27
6.64
ꢀ96.55
Reaction conditions: 100 g Lꢀ1 3-TBDMSO glutaric anhydride, 3 : 1
molar ratio of methanol to substrate, 30 g Lꢀ1 enzyme, MTBE as
solvent at 30 ꢁC with a shaking speed of 200 rpm.
a
ꢀ113.46
ꢀ107.87
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Fig. 2 Effect of methanol : substrate molar ratio on R-J6 production.
(
) R-J6 titer, (
) S-J6 titer, (
) the yield of R-J6. Reaction
conditions: 3-TBDMSO glutaric anhydride (50 g Lꢀ1), the solvent was
MTBE, 60 g Lꢀ1 Novozym 435. Methanol was added in one portion at
the beginning of the reaction.
However, the shorter the carbon chain of the alcohol, the
higher the rate of transesterication would be.37 For the
realization of large-scale production and reducing cost,
methanol (the cheaper and the shortest carbon chain) was
selected as the best acyl acceptor. To investigate the effect of
the methanol concentration on R-J6 production, the substrate
was alcoholyzed at 30 ꢁC for 24 h with various amounts of
methanol by Novozym 435. The results (Fig. 2) show that when
the molar ratio of methanol to substrate was 1 : 1, the R-J6
titer was below 5.0 g Lꢀ1. Of great interest, when the molar
ratio of methanol to substrate reached 2 : 1, the titer and yield
of R-J6 rose to 16.7 g Lꢀ1 and 33.4%, respectively, while
productivity reached 0.695 g Lꢀ1 hꢀ1
.
3.3. Effect of reaction conditions on R-J6 production
The effect of reaction temperature on R-J6 production was
investigated (Fig. 3A). The titer of R-J6 increased continually as
the temperature increased from 22 to 35 ꢁC, and the produc-
tivity of R-J6 reached 0.71 g Lꢀ1 hꢀ1 at 35 C.
ꢁ
The effect of solvents with different log P values on R-J6
production are shown in Fig. 3B. No R-J6 was detected in the
solvent-free reaction (only methanol, substrate and enzyme in
the reaction system). The R-J6 titer was 21.5 g Lꢀ1 when iso-
octane was used as the solvent; the productivity and the yield
were 0.895 g Lꢀ1 hꢀ1 and 35.7%, respectively.
Fig. 3 Effect of reaction conditions on R-J6 production. (
) R-J6
titer, (
) S-J6 titer, (
) the yield of R-J6. (A) Effect of temper-
ature. Reaction conditions: the solvent was MTBE, 3-TBDMSO glutaric
anhydride (60 g Lꢀ1), 60 g Lꢀ1 Novozym 435, 2 : 1 molar ratio of
methanol to substrate. (B) Effect of different organic solvents: MTBE
The effect of substrate concentration is presented in Fig. 3C.
The results show that the R-J6 titer increased as the substrate
concentration increased from 40 to 200 g Lꢀ1. At a substrate (log P ¼ 0.96), n-hexane (log P ¼ 2.50), cyclohexane (log P ¼ 3.00),
concentration of 200 g Lꢀ1, the R-J6 titer was up to the maximum
value, 67.1 g Lꢀ1, 3.7-fold higher than that at 40 g Lꢀ1 substrate.
However, the yield of R-J6 decreased as the substrate concen-
tration increased (40–200 g Lꢀ1). Excess substrate had a negative
iso-octane (log P ¼ 3.72), n-octane (log P ¼ 3.84); reaction conditions:
3-TBDMSO glutaric anhydride (60 g Lꢀ1), 60 g Lꢀ1 Novozym 435, 2 : 1
molar ratio of methanol to substrate, at 35 ꢁC with a shaking speed of
200 rpm. (C) Effect of substrate concentration. Reaction conditions:
the solvent was iso-octane, 60 g Lꢀ1 Novozym 435, 2 : 1 molar ratio of
effect on the yield of R-J6. When the substrate concentration was methanol to substrate, at 35 ꢁC with a shaking speed of 200 rpm.
above 200 g Lꢀ1, the yield of R-J6 dropped below 30%, therefore
Methanol was added in one portion at the beginning of the reaction.
the optimal concentration was 200 g Lꢀ1
.
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3.4. Statistical analysis
The optimum values of the molar ratio of methanol to substrate
(molar ratio), Novozym 435 concentration (catalyst), and 3-
TBDMSO glutaric anhydride concentration (substrate) were
examined using an orthogonal array design (Table 3). The order
of the effect of the factors on R-J6 production was molar ratio >
substrate > catalyst. The molar ratio of methanol to substrate
was the main factor for R-J6 production. In this study, the
optimal conditions for synthesis of R-J6 were as follows: 50 g Lꢀ1
catalyst, 3 : 1 molar ratio, and 200 g Lꢀ1 substrate. The R-J6 titer
was 117.2 g Lꢀ1, and the yield was 58.6%. Under the optimal
conditions, production of R-J6 with time is shown in Fig. 4,
indicating a maximum at 24 h. The biocatalytic process
described in this study achieved a highest synthesis rate of 4.84
g Lꢀ1 hꢀ1, and thus has great potential for large-scale produc-
tion of (R)-3-TBDMSO glutaric acid methyl ester, the purity of
the desire product was up to 98%. The R-J6 titer can be main-
tained at 90 g Lꢀ1, therefore, the biocatalyst (Novozym 435) can
be reused at least four times (Fig. 5).
Fig. 4 Time course of R-J6 production under optimized reaction
conditions. (
) R-J6 titer, (
) S-J6 titer, (
) the yield of R-
J6. Reaction conditions: 3-TBDMSO glutaric anhydride (200 g Lꢀ1), the
solvent was iso-octane, 50 g Lꢀ1 Novozym 435, 3 : 1 molar ratio of
methanol to substrate, at 35 ꢁC with a shaking speed of 200 rpm.
Methanol was added in one portion at the beginning of the reaction.
R-J6 has been prepared by using the lithium salt of benzyl (R)-
(ꢀ)-mandelate,12,17 three additional steps were required to
generate R-J6 (see ESI data Scheme S1†), the yield was only
42.3% from 3-TBDMSO glutaric anhydride, isolation and puri-
cation and waste treatment were difficult. a-Chymotrypsin
with R-selective has been used to prepare R-isomers by hydro-
lysis of diethyl-3-hydroxyglutarate, however, the maximum R-
recycled by vacuum distillation with no wastewater discharge.
Substrate concentration was up to 200 g Lꢀ1 and the reaction
time was 24 h, but the yield of R-J6 was only 58.6% and the ee
value was low. The S-isomer (S-J6) that was also produced can be
used to assemble other statins and their derivative products33
such as hapalosin,41 iostatine33 and dolastatin.42 In order to
obtain the statin skeleton of R-J6 in high optical purity, further
studies could explore isolation of R-J6 from the enzymatic
conversion solution containing racemic J6 by dynamic kinetic
resolution43–46 using vinyl acetate as acyl donor47 (Scheme S3†).
Novozym 435 can be reused directly for dynamic kinetic reso-
lution by ltering without any pretreatment, making the whole
process green. To enhance the yield and further reduce costs of
R-J6 production and simplify the process, we are doing our best
to change the enantioselectivity of CALB, and wanna to obtain a
catalyst with high R-selectivity on R-J6 preparation by directed
evolution.
isomer titer was 32.5 g Lꢀ1, and the productivity was only 0.68 g
Lꢀ1 hꢀ1
.
The 3-substituents group of the substrate signi-
38
cantly affect the enzyme activity and selectivity, the a-chymo-
trypsin performed a low catalytic efficiency on the substrate
whose 3-substituent was TBDMSO.39 The R-isomer productivity
was only 0.33 g Lꢀ1 hꢀ1 by a-chymotrypsin while the 3-substit-
uent was TBDMSO in our study.
Typical enzymatic process goals are a substrate loading > 100
g Lꢀ1, reaction time < 24 h, conversion > 98%, and enantiomeric
excess (ee) > 99%.40 In our study, the synthesis of R-J6 in non-
aqueous media requires only a single step, and the titer of R-
J6 was up to gram scale. The desired products can be isolated,
puried, and dried easily, and the organic solvents can be
Table 3 Orthogonal array design to improve R-J6 production
A
B
C
Factor
Run
A
B
C
Substrate (g Lꢀ1
)
Molar ratio
Catalyst (g Lꢀ1
)
R-J6 titer (g Lꢀ1
)
1
2
3
4
5
6
7
8
1
1
1
2
2
2
3
3
3
1
2
3
1
2
3
1
2
3
1
2
3
2
3
1
3
1
2
150
150
150
200
200
200
250
250
250
22.41
2
1
2
3
1
2
3
1
2
50
60
70
60
70
50
70
50
60
5.64
3
23.12 ꢃ 1.2
50.83 ꢃ 1.5
87.44 ꢃ 2.4
37.44 ꢃ 2.0
73.99 ꢃ 2.4
117.19 ꢃ 2.9
36.79 ꢃ 1.8
73.11 ꢃ 1.7
108.24 ꢃ 2.7
9
3
Range
Rank
Optimization
71.84
1
3
200
50
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