A practical high-throughput screening system for feruloyl esterases: Substrate design and evaluation

Expression of feruloyl esterase A from Aspergillus terreus and its application in biomass degradation

“…Methyl esters of ferulic (MFA), p-coumaric (MpCA), caffeic (MCA) and sinapic (MSA) acids were purchased from Apin Chemicals Ltd. (Abingdon, UK). The substrate 2-chloro-4-nitrophenyl ferulate (CNPF) was synthesized in our laboratory as previously described [16]. Corn stalk and corncob were collected in the local farm and were smashed into particles of small size.…”

“…Bound activity was eluted with the same buffer containing 1 M NaCl. The purified AtFaeA was confirmed through enzymatic activity assay with 1 mM substrate CNPF as reported [16].The molecular weight of AtFaeA was determined with SDS-PAGE. The glycosylation of AtFaeA was confirmed with 0.5 mU of endo-b-Nacetylgluco saminidase H (New England Biolabs, MA, USA) at 37°C for 2 h following the manufacture's protocol.…”

Multiple amino acid substitutions significantly improve the thermostability of feruloyl esterase A from Aspergillus niger

“…The substrate CNPF was synthesized as previously described with >99% purity (Zhang et al, 2012). Methyl feulate (MFA) was purchased from Alfa Aesar (Tianjin, China) with 98% purity.…”

“…The expression of AnFaeA mutants were induced by adding 150 ll buffered methanol-complex medium (BMMY) (same as BMGY except using 0.5% (v/v) methanol instead of 1% (v/v) glycerol as an inducer) to the cell pellets and the cultivation was continued for 36 h. After centrifugation at 1800g for 10 min, 20 ll supernatant in each well was transferred into two duplicate 96-well plates, and one plate was pretreated at 63°C for 30 min in a BINDER oven (BINDER, Germany). Then the reaction mixture in each well was prepared by adding 170 ll of sodium phosphate buffer (100 mM, pH 6.4) containing 2.5% (v/v) Triton X-100 and 10 ll of DMSO solution containing the substrate CNPF (20 mM) (Zhang et al, 2012). After 15 min incubation at 40°C with shaking at 100 rpm, the release of 2-chloro-4-nitrophenol was measured at 425 nm in a Thermo Scientific Varioskan Flash microplate reader (Thermo Scientific, USA).…”

“…To the best of our knowledge, no previous work has been reported on the directed evolution of FAEs. Only recently, a high-throughput screening system for FAEs has been evaluated based on a chromogenic substrate, 2-chloro-4-nitrophenyl ferulate (CNPF) with the coefficient of variance of 10.3% as determined for the single sequence library of the AnFaeA-D93G/S187F mutant (Zhang et al, 2012). This screening system was adapted in this work to test a random library constructed in Pichia pastoris using the same double mutant as the parental enzyme.…”