Modification of Galactose Oxidase to Introduce Glucose 6-Oxidase Activity

Sun, Lianhong; Bülter, Thomas; Alcalde, Miguel; Petrounia, Ioanna P.; Arnold, Frances H.

doi:10.1002/1439-7633(20020802)3:8<781::aid-cbic781>3.0.co;2-8

Cited by 101 publications

(97 citation statements)

References 8 publications

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“…on May 10, 2018 by guest http://aem.asm.org/ stability and substrate specificity (9,(27)(28). To immobilize the enzyme on the surface of the PhaC protein, the protein was endowed with a coiled-coil sequence containing glutamic acid (Ecoil) or lysine residues (Kcoil), which are known to form tight heterodimers with estimated K D (equilibrium dissociation constant) values of 1 nM (6).…”

Section: Vol 76 2010 Enzyme Immobilization By Inclusion Body Displamentioning

confidence: 99%

In Vivo Enzyme Immobilization by Inclusion Body Display

Steinmann

Christmann

Heiseler

et al. 2010

Appl Environ Microbiol

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A novel strategy for in vivo immobilization of enzymes on the surfaces of inclusion bodies has been established. It relies on expression in Escherichia coli of the polyhydroxybutyrate synthase PhaC from Cupriavidus necator, which carries at its amino terminus an engineered negatively charged ␣-helical coil (Ecoil) and forms inclusion bodies upon high-level expression. Coexpression in the same cell of galactose oxidase (GOase) from Fusarium spp. carrying a carboxy-terminal positively charged coil (lysine-rich coil [Kcoil]) sequence results in heterodimeric coiled-coil formation in vivo and in the capture of the enzyme in active form on the surface of the inclusion body particle. These round-shaped enzyme-decorated microparticles, with sizes of approximately 0.7 m, can be isolated from lysed cells simply by centrifugation. The cost-effective one-step generation and isolation of enzymes immobilized on inclusion body particles may become useful for various applications in bioprocessing and biotransformation.

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Section: Vol 76 2010 Enzyme Immobilization By Inclusion Body Displamentioning

confidence: 99%

In Vivo Enzyme Immobilization by Inclusion Body Display

Steinmann

Christmann

Heiseler

et al. 2010

Appl Environ Microbiol

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“…One suitable strategy to study this function in more detail could be carried out through the construction of a small library by saturation mutagenesis. 32 Other clones with ~2-fold better ac- A B tivities were characterized. Among them, the 3G10 variant showed just 1 substitution, GAG/AAG, located at the C-terminal processing site of the protein introducing the change E615K.…”

Section: Figmentioning

confidence: 99%

“…This method can be valid not only for screening for laccase activity in organic media but also for other redox systems (e.g., peroxidases, galactose oxidases) in complex biological mixtures. 32,33 …”

Section: Figmentioning

confidence: 99%

Screening Mutant Libraries of Fungal Laccases in the Presence of Organic Solvents

Alcalde¹,

Bülter²,

Zumárraga³

et al. 2005

SLAS Discovery

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Reliable screening methods are being demanded by biocatalysts'engineers, especially when some features such as activity or stability are targets to improve under nonnatural conditions (i.e., in the presence of organic solvents). The current work describes a protocol for the design of a fungal laccase-expressed in Saccharomyces cerevisiae-highly active in organic cosolvents. A high-throughput screening assay based on ABTS (2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)) oxidation was validated. The stability of the ABTS radical cation was not significantly altered in the presence of acetonitrile, ethanol, or DMSO. With a coefficient of variance below 10% and a sensitivity limit of 15 pg laccase/µL, the assay was reproducible and sensitive. The expression system of Myceliophthora thermophila laccase variant T2 in S. cerevisiae was highly dependent on the presence of Cu 2+. Copper concentration was limited up to 10 µM CuSO 4 where expression levels (~14-18 mg/L) were acceptable without compromising the reliability of the assay. A mutant library was created by error-prone PCR with 1.1 to 3.5 mutations per kb. After only 1 generation of directed evolution, mutant 6C9 displayed about 3.5-fold higher activities than parent type in the presence of 20% acetonitrile or 30% ethanol. The method provided here should be generally useful to improve the activity of other redox enzymes in mixtures of water/cosolvents. (Journal of Biomolecular Screening 2005:624-631)

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“…The selective functionalization of the glucose C-6 18 position would be a synthetically useful reaction facilitating further chemical modifications as described above for GAO treated galactose containing polysaccharides. Accordingly, after generating the GAO variant M 1 , Sun et al (2002) used a structure guided approach to identify four amino acids predicted to interact with galactose or stabilize the radical form of GAO[94]. Whereas wild-type GAO is 10 6 times more active on galactose than glucose, a combination of three substitutions (R330K, Q406T and W290F) generated variant M 3 , which demonstrated 100 times more activity on D-glucose than M 1 .…”

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confidence: 99%