title: High efficiency preparation and characterization of intact poly(vinyl alcohol) dehydrogenase from Sphingopyxis sp.113P3 in Escherichia coli by inclusion bodies renaturation.
abstract: Poly(vinyl alcohol) dehydrogenase (PVADH, EC 1.1.99.23) is an enzyme which has potential application in textile industry to degrade the poly(vinyl alcohol) (PVA) in waste water. Previously, a 1,965-bp fragment encoding a PVADH from Sphingopyxis sp. 113P3 was synthesized based on the replacement of the rare codons in Escherichia coli (E. coli). In this work, the deduced mature PVADH (mPVADH) gene of 1,887 bp was amplified by polymerase chain reaction (PCR) and inserted into the site between NcoI and HindIII in pET-32a(+). The constructed recombinant plasmid was transformed into E. coli Rosetta (DE3). In shake flask, the fusion protein of thioredoxin (Trx)-mPVADH was expressed precisely; however, Trx-mPVADH was found to accumulate mainly as inclusion bodies. After isolating, dissolving in buffer containing urea, purification, dialysis renaturation, and digesting with recombinant enterokinase/His (rEK/His), the bioactive mPVADH fragments were obtained with protein concentration of 0.56 g/L and enzymatic activity of 194 U/mL. The K m and V max values for PVA 1799 were 2.33 mg/mL and 15.7 nmol/(min·mg protein), respectively. (1)H-NMR and infrared (IR) spectrum demonstrated that its biological function was oxidizing hydroxyl groups of PVA 1799 to form diketone, and PVA 1799 could be degraded completely by successive treatment with mPVADH and oxidized PVA hydrolase (OPH).
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China, jiadongxu@aliyun.com.
authors.forename: Dongxu
authors.initials: D
authors.lastname: Jia
authors.forename: Yu
authors.initials: Y
authors.lastname: Yang
authors.forename: Zhengcong
authors.initials: Z
authors.lastname: Peng
authors.forename: Dongxu
authors.initials: D
authors.lastname: Zhang
authors.forename: Jianghua
authors.initials: J
authors.lastname: Li
authors.forename: Long
authors.initials: L
authors.lastname: Liu
authors.forename: Guocheng
authors.initials: G
authors.lastname: Du
authors.forename: Jian
authors.initials: J
authors.lastname: Chen
mesh_terms: D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D001673:Biodegradation, Environmental; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry; D003062:Codon; D004785:Environmental Pollutants / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D002479:Inclusion Bodies / Q000737:chemistry; D007700:Kinetics; D011142:Polyvinyl Alcohol / Q000737:chemistry; D058849:Protein Refolding; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D042301:Sphingomonadaceae / Q000737:chemistry* / Q000201:enzymology; D013879:Thioredoxins / Q000737:chemistry / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D003062:Codon; D004785:Environmental Pollutants; D011993:Recombinant Fusion Proteins; D013879:Thioredoxins; D011142:Polyvinyl Alcohol; D000429:Alcohol Oxidoreductases; C048312:polyvinyl-alcohol dehydrogenase (acceptor); D002265:Carboxylic Ester Hydrolases; C409492:oxidized polyvinyl alcohol hydrolase, Pseudomonas sp.