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Catalysis Science & Technology
Page 5 of 7
DOI: 10.1039/C6CY00786D
ARTICLE
Journal Name
was defined as the amount of enzyme required to hydrolyse 1.0
µmol of Vince lactam per minute.
5
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Kinetic parameters of the purified SvGL towards Vince lactam
were determined by assaying the activity under various substrate
concentrations (10–392 mM) in Tris-HCl buffer (pH 8.0, 100 mM),
using the standard protocol. The maximal reaction rate (Vmax) and
Michaelis–Menten constant (KM) were calculated by fitting the
data to the Michaelis-Menten equation utilizing Origin 9.0
software (OriginLab, Northampton, USA).
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Effects of pH and temperature on activity and stability
The optimal pH of the SvGL was determined at 30 °C in different
buffers (100 mM) within a pH range of 6.0 to 12.0 (sodium citrate,
5.0–6.0; potassium phosphate, 6.0–8.5; Tris-HCl, 8.0–9.0; Gly-
NaOH, 9.0–12.0). The optimal temperature was determined under
standard conditions at different temperatures ranging from 20 °C
to 80 °C. The thermostability was determined by measuring the
residual activity against pNPA after incubating the purified enzyme
G. Zhu and S. Wu, Appl. Microbiol. Biotechnol., 2015, 99
,
3069–3080; (f) J. J. Wang, J. G. Zhu and S. Wu, Appl.
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Vitnik, W. M. F. Fabian, D. Ribitsch, G. M. Guebitz, K. Faber
(0.1 g L−1) at 70 °C. Aliquots were withdrawn periodically and the
and M. Hall, ChemCatChem, 2014, 6, 2517–2521.
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residual activities were assayed. Kinetic thermal stability (T ) of
50
enzymes is generally used to denote the temperature required to
15
reduce the initial enzyme activity by 50% in 15 min. The T was
50
examined by incubating the purified enzyme for 15 min in the
phosphate buffer (pH 7.0) at different temperatures, and then the
residual activity was assayed against pNPA.
Analytical method
The enantiomeric excess of substrate (ees) and conversion of Vince
lactam were determined by HPLC (SHIMADZU LC-2010AHT)
equipped with a Daicel Chiralpak AS-H column (Daicel, Tokyo,
Japan). The mobile phase consisted of 90% acetonitrile and 10%
isopropanol (v/v) at a flow rate of 0.8 mL min−1. The UV
absorbance was determined at 230 nm.
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Acknowledgements
This work was financially supported by National Natural Science
Foundation of China (Nos. 21472045, 21506055 & 21536004),
Ministry of Science and Technology, P.R. China (Nos.
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Notes and references
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