10.1002/cssc.201900185
ChemSusChem
FULL PAPER
(10 ml 37% HCl, 40 mg orcinol and 5.4 mg ferric trichloride). One volume
of supernatant was mixed with two volumes orcinol reagent and boiled
together for 30 min, then cooled to room temperature. Glucuronic acid
was quantified by measuring absorbance at 670 nm. The reaction
mixture without substrate was used as a control to account for the
background. The activity of UDH was measured at 30˚C in a reaction
system which contained 200 mM Tris-HCl (pH 7.5), 20 mM glucuronic
acid and 2 mM NAD+. The activity was assayed by the reduction rate of
NAD+ at 340 nm using a spectrophotometer (UV-2550, Shimadzu, Japan).
The activity of NOX was measured as previously with some
modifications[32]. NOX activity was analyzed in a reaction system which
contained 200 mM Tris-HCl and 2 mM NADH at 30˚C. The activity was
a flow rate of 0.5 mL/min. Retention times of G1P/G6P (overlap), fructose,
inositol and GA was 8.352, 12.205, 11.270 and 10.250 min, respectively
(Figure S4). The concentration of G6P in the supernatants was assayed
by G6P dehydrogenase reaction.
The qualitative of glucaric acid was performed by Ultra high pressure
liquid chromatography high resolution mass spectrometry (LC-MS)
Agilent 1290/maXis impact (Bruker, German) (Figure S5). The LC
separation was determined using an Agilent SB-C18 RRHD (1.8 um,
2.1*50mm; Bruker, Germany). The mobile phases were formic acid
(0.1%) and acetonitrile. And a 0-15 min linear gradient with a flow rate
(0.2 ml/min) was used.
measured by the oxidation rate of NADH at 340 nm using
a
Statistical analysis
spectropotometer (UV-2550). One unit of enzyme activity was defined as
Data were obtained at least in triple and expressed as mean ± standard
deviation (SD).
the amount of enzyme needed to produce 1 μmol product per min.
Optimization of reaction conditions for multi-enzyme synthesis of
glucaric acid
The synthesis of GA from sucrose by multi-enzyme catalysis was carried
out in a 2.0 mL reaction system with seven enzymes, substrate,
coenzymes and metal ions. In this study, we optimized the reaction
conditions (substrate concentration, buffer type and concentration,
temperature, pH value, coenzyme and metal ions concentration) to
enhance flux through the multi-enzyme reaction system. A one-pot
biosynthesis strategy was employed in 2.0 mL 200 mM Tris-HCl buffer
(pH 7.5), initially including 30-100 mM sucrose, 50 mM disodium
hydrogen phosphate, 2 mM MgCl2, 2 mM NAD+, 2 mM ammonium
ferrous sulfate, 0.1 mg/mL SP, 0.1 mg/mL PGM, 0.15 mg/mL MIPS, 0.15
mg/mL IMPase, 0.2 mg/mL MIOX, 0.1 mg/mL UDH and 0.1 mg/mL NOX
at 30˚C for 12 h. After centrifugation, concentration of GA in supernatant
was mesure by reverse phase high pressure liquid chromatography
(HPLC).
Acknowledgements
The authors gratefully acknowledged the National Natural
Science Foundation of China (No. 21676104 and 21878105),
the National Key Research and Development Program of China
(2018YFC1603403), the Science and Technology Program of
Guangzhou for partially funding this work.
Conflict of interest
All authors declare no conflict of interest.
Enzyme concentrations were optimized, first, by evaluating the effect of
doubling the concentration of certain enzymes. Subsequently, some
enzymes were selected for second-round optimization as detailed in the
Results. The enzyme concentrations resulting from this second-round
optimization are listed in Table 3. Firstly, the improvement GA yield was
evaluated by doubling the concentration of the tested enzyme.
Subsequently, the appropriate enzyme composition was selected for the
second-round optimization. The second round optimized enzyme
concentrations are was listed in Table 3.
Keywords: glucaric acid · in vitro · multi-enzyme system
·sucrose · value-added chemicals
[1]a) J.Singh, K. Gupta, Biomed. Environ. Sci. Bes. 2003, 16, 9-16; b) R.
Zółtaszek, M. Hanausek, Z. M. Kiliańska, Z. Walaszek, Postepy. Hig. Med.
Dosw. 2008, 62, 451-462.
[2]a) Z. Walaszek, J. Szemraj, M. Hanausek, A. K. Adams, U. Sherman, Nutr.
Res. 1996, 16, 673-681; b) S. Jaya, G. Krishna P, J. Environ. Pathol.
Toxicol. Oncol. 2007, 26, 63-73; c) T. Polen, M. Spelberg, M. Bott, J.
Biotechnol. 2013, 167, 75-84; d) C. Dwivedi, W. J. Heck, A. A. Downie, S.
Larroya, T. E. Webb, Biochem.Med. Metab. Biol. 1990, 43, 83-92.
[3]a) R. A. Sheldon, Cheminform.Eve. 2014, 16, 950-963; b) T. A. Werpy, J. E.
Holladay, J. F. White, Synthetic Fuels 2004; c) D. W. Morton, D. E. Kiely, J.
Appl. Polym. Sci. 2015, 77, 3085-3092.
Analytical methods
Cell density (OD600) was determined using the optical density with a
spectrophotometer (UV-2550). Protein mass concentration was
measured using the Bradford method[33] with bovine serum albumin as
the standard protein. The purities of recombinant proteins were analyzed
by SDS-PAGE (Bio-Rad Hercules, CA, USA) (Figure S3). The
concentration of G1P/G6P, phosphate, fructose, inositol and GA were
determined by HPLC which was equipped with Waters 1525 refractive
index and diode array detectors (Waters, USA), and Aminex HPX-87H
column (300 mm × 7.8mm, Bio-Rad Laboratories, Hercules, CA). The
mobile phase was 5 mM H2SO4 and samples were separated at 65˚C at
[4]C. Dwivedi, W. J. Heck, A. A. Downie, S. Larroya, T. E. Webb, Biochem.
Med. Metab. Biol. 1990, 43, 83-92.
[5]H. Kochkar, L. Lassalle, M. Morawietz, W. F. Hölderich, J. Catal. 2000, 194,
343-351.
[6]C. L. Mehltretter, C. E. Rist, J. Agric. Food Chem. 1953, 1, 779-783.
[7]T. N. Smith, K. Hash, C. L. Davey, H. Mills, H. Williams, D. E. Kiely,
Carbohydr. Res. 2012, 350, 6-13.
[8]a) M. T. Seok, Y. Sang-Hwal, A. M. Lanza, J. D. Roy-Mayhew, K. L. J.
Prather, Appl. Environ. Microbiol. 2009, 75, 589-595; b) Y. N. Qu, H. J. Yan,
Q. Guo, J. L. Li, Y. C. Ruan, X. Z. Yue, W. X. Zheng, T. W. Tan, L. H. Fan,
Metab. Eng. 2018, 47, 393-400; c) A. Gupta, M. A. Hicks, S. P. Manchester,
K. L. Prather, Biotechnol. J. 2016, 11, 1201-1208; d) Z. Shuang, H. Jin, Z.
Yi, F. Hao, W. Ting-Ting, L. Fei, W. Feng-Shan, S. Ju-Zheng, Metab. Eng.
2018, 49,212-219.
[9]C. Jaturapaktrarak, S. C. Napathorn, M. Cheng, K. Okano, H. Ohtake, K.
Honda, Biores. Biopro. 2014, 1, 18.
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