Atom transfer radical polymerization to fabricate monodisperse poly[glycidyl methacrylate- co -poly (ethylene glycol) methacrylate] microspheres and its application for protein affinity purification

Yu, Ling; Shi, Zhuan Zhuan; Li, Chang Ming

doi:10.1016/j.jcis.2015.03.068

Cited by 4 publications

(2 citation statements)

References 31 publications

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“…coli BL21(DE3) with pET28a- fcs . Extraction and purification of FCS enzymes were conducted following a published method. , The bacteria were collected and resuspended for ultrasonic crushing in a 12.5 mL column buffer containing: 300 mM NaCl, 50 mM NaH 2 PO 4 , and 10 mM imidazole. The ProteinSafe Protease Inhibitor Cocktail (Transgen, Beijing) consisting of AEBSF, aprotinin, bestatin, E-64, leupeptin, and pepstatin A, with a final concentration of 0.1 mM was added to the suspension bacteria before ultrasonic crushing.…”

Section: Methodsmentioning

confidence: 99%

“…The pure enzymes were eluted with 2 mL protein eluent (200 mM imidazole, pH 7.0). Moreover, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting were performed as described previously with 10% polyacrylamide gels. − The protein concentration was measured using the UV method with a Nanodrop Ultra-Micro Reflectometer (Implen Gmbh, Germany).…”

Section: Methodsmentioning

confidence: 99%

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Engineering a Feruloyl–Coenzyme A Synthase for Bioconversion of Phenylpropanoid Acids into High-Value Aromatic Aldehydes

Chen

Jiang

Kang³

et al. 2022

J. Agric. Food Chem.

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Aromatic aldehydes find extensive applications in food, perfume, pharmaceutical, and chemical industries. However, a limited natural enzyme selectivity has become the bottleneck of bioconversion of aromatic aldehydes from natural phenylpropanoid acids. Here, based on the original structure of feruloyl–coenzyme A (CoA) synthetase (FCS) from Streptomyces sp. V-1, we engineered five substrate-binding domains to match specific phenylpropanoid acids. FcsCIAE407A/K483L, FcsMAE407R/I481R/K483R, FcsHAE407K/I481K/K483I, FcsCAE407R/I481R/K483T, and FcsFAE407R/I481K/K483R showed 9.96-, 10.58-, 4.25-, 6.49-, and 8.71-fold enhanced catalytic efficiency for degrading CoA thioesters of cinnamic acid, 4-methoxycinnamic acid, 4-hydroxycinnamic acid, caffeic acid, and ferulic acid, respectively. Molecular dynamics simulation illustrated that novel substrate-binding domains formed strong interaction forces with substrates’ methoxy/hydroxyl group and provided hydrophobic/alkaline catalytic surfaces. Five recombinant E. coli with FCS mutants were constructed with the maximum benzaldehyde, p-anisaldehyde, p-hydroxybenzaldehyde, protocatechualdehyde, and vanillin productivity of 6.2 ± 0.3, 5.1 ± 0.23, 4.1 ± 0.25, 7.1 ± 0.3, and 8.7 ± 0.2 mM/h, respectively. Hence, our study provided novel and efficient enzymes for the bioconversion of phenylpropanoid acids into aromatic aldehydes.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%