10.1002/cbic.202000196
ChemBioChem
FULL PAPER
lysed through ultrasonication with an ultrasonic oscillator (JY92- Ⅱ ,
and the pH was maintained at 6.0 with 500 mM Na2CO3. After the
reaction, the mixture was extracted with ethyl acetate (containing 0.5
mM n-dodecane as the internal standard), followed by drying with
anhydrous sodium sulfate. The conversion was determined using the
Shimadzu GC-2014 gas chromatography with a CP-Chirasil-Dex CB
column (25 m × 0.25 mm × 0.39 mm, Varian) and a flame ionization
detector (FID). Analytic conditions/retention time: Inj. temp., 280°C; Det.
temp, 280°C; Col. temp., 110°C; Rtacetophenone = 5.0 min; Rtn-Dodecane = 6.0
min; Rt(R)-1-phenylethanol = 10.8 min ; Rt(S)-1-phenylethanol = 11.8 min.
Scientz Biotech Co.) in an ice water bath for 15 min. Insoluble material
was removed by centrifugation (13000
× g, 40 min, 4°C). The
supernatant was loaded onto a 5 mL Ni2+ chelating His-Trap affinity
column equilibrated with buffer A. The fractions were collected by
gradient elution with an increasing concentration (20 mM, 50 mM, 100
mM, 150 mM and 500 mM) of imidazole for 8–10 column volumes,
respectively. The fractions containing target protein was verified by
SDS-PAGE analysis and concentrated by Millipore (10 KDa). The
concentrated solution was replaced by storing buffer (20 mM PBS, pH
8.0, 1 mM DTT) for three times and the purified enzyme was stored at -
80°C for further use. The protein concentration of purified enzyme was
determined by Nanodrop 2000c spectrophotometer (Thermo Scientific)
at 280 nm and the extinction coefficients from the Expayprot Param
Tool was taken into consideration. The purity of target protein was
checked by SDS-PAGE (Supporting Information, Figure S1).
Acknowledgements
This work was financially supported by the National Natural
Science Foundation of China (Nos. 21536004, 21672063,
21776085, 21871085
Foundation of Shanghai China (No. 19ZR1472900), the
National Key Research and Development Program of China
(Nos. 2019YFA09005000
Fundamental Research Funds for the Central Universities (No.
22221818014).
& 31971380), the Natural Science
Enzyme activity assay. The activities of BmGDHM0 and its mutants
were assayed at 30°C by monitoring the increase in the absorbance of
NADPH at 340 nm using a UV-spectrophotometer (Beckman DU730).
The standard assay mixture (1 mL) was composed of 870 μL PBS (100
mM, pH 7.0), 100 μL substrate D-glucose (2 M), 20 μL NADP+ (50 mM)
and 10 μL enzyme solution with an appropriate concentration. The
concentration of NADPH was calculated based on the extinction
coefficient of 6220 M-1 cm-1. One unit of enzyme activity was defined as
the amount of GDH that catalyzing the formation of 1 μM NADPH in
one minute. All these reactions were conducted in triplicate to avoid
artificial errors.
& 2018YFC1706200), and the
Keywords: biocatalysis • chemical stability • cofactor
regeneration • directed evolution • glucose dehydrogenase
[1]
a) U. T. Bornscheuer, G. W. Huisman, R. J. Kazlauskas, S. Lutz, J. C.
Moore, K. Robins, Nature 2012, 485, 185–194. b) G. W. Zheng, J. H.
Xu, Curr. Opin. Biotechnol. 2011, 22, 784–792. c) M. T. Reetz, J. Am.
Chem. Soc. 2013, 135, 12480–12496. d) J. M. Woodley, Curr. Opin.
Chem. Biol. 2013, 17, 310–316. e) J. Lalonde, Curr. Opin. Biotechnol.
2016, 42, 152–158. f) R. A. Sheldon, J. M. Woodley, Chem. Rev.
2018, 118, 801–838. g) F. H. Arnold, Angew. Chem. Int. Ed. 2018, 57,
4143–4148. h) M. Hönig, P. Sondermann, N. J. Turner, E. M. Carreira,
Angew. Chem. Int. Ed. 2017, 56, 8942–8973. i) C. A. Denard, H. Ren,
H. Zhao, Curr. Opin. Chem. Biol. 2015, 25, 55−64. j) S. Shoda, H.
Uyama, J. Kadokawa, S. Kimura, S. Kobayashi, Chem. Rev. 2016,
116, 2307−2413.
Determination of Kinetic Parameters. The D-glucose concentrations
for Km determinations were varied from 20 mM to 400 mM in 100 mM
PBS buffer (pH 7.0, 30°C) containing 1 mM NADP+. The initial-velocity
data obtained were fitted to the equation ν = νmax [S]/([S] + Km) (where ν
is the initial velocity, νmax is the maximum velocity, [S] is the D-glucose
concentration, and Km is the Michaelis constant) by using Origin 8.6
(nonlinear fitting). The kcat was calculated from the ratio of νmax and
BmGDH concentration.
Analyse of chemical and thermal stabilities. Determination of half-
life in 1-phenylethanol (10%, v/v). The half-lives (t1/2
) of purified
[2]
a) J. Dong, E. Fernández-Fueyo, F. Hollmann, C. E. Paul, M. Pesic,
S. Schmidt, Y. Wang, S. Younes, W. Zhang, Angew. Chem. Int. Ed.
2018, 57, 9238–9261. b) G. Grogan, Curr. Opin. Chem. Biol. 2018, 43,
15–22. c) W. Hummel, Trends Biotechnol. 1999, 17, 487–492.
C. Rodriguez, I. Lavandera, V. Gotor, Curr. Org. Chem. 2012, 16,
2525–2541.
BmGDHM0 and its mutants were determined by incubating purified
protein (0.5 mg mL-1) in 1-phenylethanol (10%, v/v) at 30°C for a proper
time followed by measuring the residual activity. The half-life (t1/2) was
calculated by polynomial Fit using Origin 8.6. Set the fitted variable to
50% in the fitted equation to calculate the half-life (t1/2).
[3]
[4]
a) J. B. Jones, D. W. Sneddon, W. Higgins, A. J. Lewis, Chem.
Commun. 1972, 856–857. b) K. E. Taylor, J. B. Jones, J. Am. Chem.
Soc. 1976, 98, 5689–5694. c) T. Quinto, V. Köhler, T. Ward, Top.
Catal. 2014, 57, 321–331. d) F. Hollmann, I. W. C. E. Arends, K.
Buehler, ChemCatChem 2010, 2, 762–782. e) X. Wang, H. H. P. Yiu,
ACS Catal. 2016, 6, 1880–1886.
Determination of T5060. The temperature at which enzymes lose 50%
activity after incubation for 60 min, was determined by addition of 40 μL
purified enzyme (0.5 mg mL-1) into a row of a 96-well plate. The 96-well
plate was incubated at temperatures ranging from 60°C to 85°C for 60
min. The residual activities of BmGDHM0 and its mutants were assayed
after cooled down on ice.
[5]
a) F. Hollmann, A. Schmid, Biocatalysis 2004, 22, 63–88. b) X. Wang,
T. Saba, H. H. P. Yiu, R. F. Howe, J. A. Anderson, J. Shi, Chem 2017,
2, 621–654. c) C. S. Morrison, W. B. Armiger, D. R. Dodds, J. S.
Dordick, M. A. G. Koffas, Biotechnol. Adv. 2018, 36, 120–131.
a) K. Nakamura, R. Yamanaka, Chem. Commun. 2002, 1782–1783. b)
S. H. Lee, D. S. Choi, S. K. Kuk, C. B. Park, Angew. Chem. Int. Ed.
2018, 57, 7958–7985.
Asymmetric reduction of OPBE. The reaction mixture (10 mL)
contained 1 M OPBE, 0.5 mM NADP+, 50 U mL-1 CgKR2 (lyophilized
cells), 1.5
M D-glucose, 30 U
mL−1 of BmGDHM0 or BmGDHM6
[6]
[7]
(lyophilized powders), and 100 mM PBS buffer (pH 6.0). The reaction
was performed at 30°C, and the pH was maintained at 6.0 with 2 M
NaOH. After the reaction, the mixture was extracted with ethyl acetate,
followed by drying with anhydrous sodium sulfate. The conversion was
determined using the Shimadzu GC-2014 gas chromatography with a
CP-Chirasil-Dex CB column (25 m × 0.25 mm × 0.39 mm, Varian) and
a flame ionization detector (FID). Analytic conditions/retention time: Inj.
temp., 280°C; Det. temp, 280°C; Col. temp., 160°C; RtOPBE = 7.1 min;
Rt(R)-HPBE = 8.8 min.
a) H. Zhao, W. A. van der Donk, Curr. Opin. Biotechnol. 2003, 14,
583–589. b) W. A. van der Donk, H. Zhao, Curr. Opin. Biotechnol.
2003, 14, 421–426. c) H. Wu, C. Tian, X. Song, C. Liu, D. Yang, Z.
Jiang, Green Chem. 2013, 15, 1773–1789.
[8]
[9]
C. Nowak, A. Pick, P. Lommes, V. Sieber, ACS Catal. 2017, 7,
5202−5208.
a) V. I. Tishkov, V. O. Popov, Biomol. Eng. 2006, 23, 89–110. b) U.
Kragl, D. VasicRacki, C. Wandrey, Bioprocess Eng. 1996, 14,
291−297. c) F. F. Chen, S. C. Cosgrove, W. R. Birmingham, J.
Mangas-Sanchez, J. Citoler, M. P. Thompson, G. W. Zheng, J. H. Xu,
N. J. Turner, ACS Catal. 2019, 9, 11813−11818. d) F. F. Chen, Y. H.
Zhang, Z. J. Zhang, L. Liu, J. P. Wu, J. H. Xu, G. W. Zheng, J. Org.
Chem. 2019, 84, 14987−14993.
Asymmetric reduction of acetophenone. The reaction mixture (10
mL) contained 100 mM acetophenone, 0.5 mM NADP+, 3.4 U mL-1
SsCR, 150 mM D-glucose, and 0.34 U mL-1 of BmGDHM0 or BmGDHM6
,
and 100 mM PBS buffer (pH 6.0). The reaction was performed at 30°C,
7
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