title: Three New Cutinases from the Yeast Arxula adeninivorans That Are Suitable for Biotechnological Applications.
abstract: The genes ACUT1, ACUT2, and ACUT3, encoding cutinases, were selected from the genomic DNA of Arxula adeninivorans LS3. The alignment of the amino acid sequences of these cutinases with those of other cutinases or cutinase-like enzymes from different fungi showed that they all had a catalytic S-D-H triad with a conserved G-Y-S-Q-G domain. All three genes were overexpressed in A. adeninivorans using the strong constitutive TEF1 promoter. Recombinant 6× His (6h)-tagged cutinase 1 protein (p) from A. adeninivorans LS3 (Acut1-6hp), Acut2-6hp, and Acut3-6hp were produced and purified by immobilized-metal ion affinity chromatography and biochemically characterized using p-nitrophenyl butyrate as the substrate for standard activity tests. All three enzymes from A. adeninivorans were active from pH 4.5 to 6.5 and from 20 to 30°C. They were shown to be unstable under optimal reaction conditions but could be stabilized using organic solvents, such as polyethylene glycol 200 (PEG 200), isopropanol, ethanol, or acetone. PEG 200 (50%, vol/vol) was found to be the best stabilizing agent for all of the cutinases, and acetone greatly increased the half-life and enzyme activity (up to 300% for Acut3-6hp). The substrate spectra for Acut1-6hp, Acut2-6hp, and Acut3-6hp were quite similar, with the highest activity being for short-chain fatty acid esters of p-nitrophenol and glycerol. Additionally, they were found to have polycaprolactone degradation activity and cutinolytic activity against cutin from apple peel. The activity was compared with that of the 6× His-tagged cutinase from Fusarium solani f. sp. pisi (FsCut-6hp), also expressed in A. adeninivorans, as a positive control. A fed-batch cultivation of the best Acut2-6hp-producing strain, A. adeninivorans G1212/YRC102-ACUT2-6H, was performed and showed that very high activities of 1,064 U ml(-1) could be achieved even with a nonoptimized cultivation procedure.
authors.affiliation: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben, Germany.
authors.forename: Felix
authors.initials: F
authors.lastname: Bischoff
authors.affiliation: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben, Germany.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Litwińska
authors.affiliation: ASA Spezialenzyme GmbH, Wolfenbüttel, Germany.
authors.forename: Arno
authors.initials: A
authors.lastname: Cordes
authors.affiliation: School of Biological Sciences, University of Canterbury, Christchurch, New Zealand.
authors.forename: Keith
authors.initials: K
authors.lastname: Baronian
authors.affiliation: Institute of Microbiology, University of Greifswald, Greifswald, Germany.
authors.forename: Rüdiger
authors.initials: R
authors.lastname: Bode
authors.affiliation: Institute of Microbiology, University of Greifswald, Greifswald, Germany.
authors.forename: Frieder
authors.initials: F
authors.lastname: Schauer
authors.affiliation: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben, Germany kunzeg@ipk-gatersleben.de.
authors.forename: Gotthard
authors.initials: G
authors.lastname: Kunze
mesh_terms: D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D020134:Catalytic Domain; D002846:Chromatography, Affinity; D004795:Enzyme Stability / Q000187:drug effects / Q000528:radiation effects; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007220:Industrial Waste; D027845:Malus / Q000382:microbiology; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004718:Saccharomycetales / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D012997:Solvents; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D007220:Industrial Waste; D011994:Recombinant Proteins; D012997:Solvents; D002265:Carboxylic Ester Hydrolases; C019952:cutinase