Three New Cutinases from the Yeast Arxula adeninivorans That Are Suitable for Biotechnological Applications

The genesACUT1 ,ACUT2 , andACUT3 , encoding cutinases, were selected from the genomic DNA ofArxula 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 inA. adeninivorans using the strong constitutiveTEF1 promoter. Recombinant 6× His (6h)-tagged cutinase 1 protein (p) fromA. adeninivorans LS3 (Acut1-6hp), Acut2-6hp, and Acut3-6hp were produced and purified by immobilized-metal ion affinity chromatography and biochemically characterized usingp -nitrophenyl butyrate as the substrate for standard activity tests. All three enzymes fromA. 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 ofp -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 fromFusarium solani f. sp.pisi (FsCut-6hp), also expressed inA. 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.