Purification and Properties of Secondary Alcohol Oxidase with an Acidic Isoelectric Point

Expression and fermentation optimization of oxidized polyvinyl alcohol hydrolase in E. coli

“…To enhance the expression of OPH in E. coli [10,14,18], we modified the published oph sequence (AB190288) from Sphingopyxis sp. 113P3: 16 relative low-usage codons encoding leucine and arginine were replaced by high-usage codons from E. coli.…”

“…are commonly researched species that can produce a variety of enzymes that degrade PVA [6,10,19,24,25,28]. The PVA biodegradation process is generally divided into two steps: (1) adjacent hydroxyl groups are oxidized to form a b-diketone structure by PQQ-dependent PVA dehydrogenase (PVA-DH) [6,8,21] or secondary alcohol oxidase [5,11,19,24,27]; (2) the b-diketone is further hydrolyzed to a methyl ketone and a carboxylic acid by b-diketone hydrolase [18] and oxidized PVA hydrolase (OPH). OPH has been found to exhibit activity only towards oxidized PVA, showing no activity towards monoketones and diketones [12,22].…”

Purification and Properties of Oxidized Poly(vinyl alcohol) Hydrolase with an Acidic Isoelectric Point

“…The enzyme was purified according to the procedures reported in our previous paper. 8 ) Ultracentrifugation. Sedimentation equilibrium analysis was at 12,000 rpm at 20°C with a Hitachi 282 ultracentrifuge.…”

Mechanistic and biocatalytic enzymology of flavoprotein oxidases

“…VAO/PCMH-type ChoXs are found in Gram-positive bacteria such as Brevibacterium sterolicum and various species of Rhodococcus and Gram-negative bacteria such as Chromobacterium sp. DS-1, Pseudomonas aeruginosa and Burkholderia cepacia (135)(136)(137)(138)(139)(140). Unlike most ChoXs, the VAO/PCMH-type enzymes from Gram-negative bacteria do not catalyse the isomerization of cholest-5-en-3-one to cholest-4-en-3-one and the main product of the reaction is 6-hydroperoxycholest-4-en-3-one (Scheme 11) (141).…”

“…Relatively little is known about the biochemical properties and substrate oxidation mechanism of SAO. The SAOs that have been purified display similar catalytic properties and contain a single non-heme iron atom per protein molecule, but differ in properties such as molecular weight (ranging from 26 to 75 kDa), pI (ranging from 4.5 to 10) and localisation (most are secreted, but a single membrane-associated SAO has been described) (137)(138)(139)(140)(141). In addition to polyvinyl alcohol, SAO oxidises various medium-chain secondary alcohols, with substrates with chain lengths of six to eight carbon atoms being oxidised most efficiently (though the observed decrease in activity for alcohols with longer chain lengths may be due to their poor solubility in water).…”