(Below N is a link to NCBI taxonomic web page and E link to ESTHER at designed phylum.) > cellular organisms: NE > Eukaryota: NE > Opisthokonta: NE > Fungi: NE > Dikarya: NE > Ascomycota: NE > saccharomyceta: NE > Saccharomycotina: NE > Saccharomycetes: NE > Saccharomycetales: NE > Trichomonascaceae: NE > Blastobotrys: NE > Blastobotrys adeninivorans: NE
LegendThis sequence has been compared to family alignement (MSA) red => minority aminoacid blue => majority aminoacid color intensity => conservation rate title => sequence position(MSA position)aminoacid rate Catalytic site Catalytic site in the MSA MKTNFLIACASLIAGAAAAPLERRDGGCSKYTIIDTRGTGELQGPSAGFI TMNRNILSQVPGGVEYDTVYPADFSQISTQGTLDIVNKVQSTLESDPSHC FVLEGYSQGAAATVSALPKLTGASFDAVKAVFLIGDPMHKPGLACNVDTQ GGKSTADASGLEAYLGGIPDEWVSKTMDVCNFGDGVCDTLTGAGITPQHL DYPYDSNVQKMGTDFVVKALTGSS
References
Title: Cutinase ACut2 from Blastobotrysraffinosifermentans for the Selective Desymmetrization of the Symmetric Diester Diethyl Adipate to the Monoester Monoethyl Adipate Rauter M, Nietz D, Kunze G Ref: Microorganisms, 10:, 2022 : PubMed
Monoethyl adipate (MEA) is a highly valuable monoester for activating resistance mechanisms and improving protective effects in pathogen-attacked plants. The cutinase ACut2 from the non-conventional yeast Blastobotrys (Arxula) raffinosifermentans (adeninivorans) was used for its synthesis by the desymmetrization of dicarboxylic acid diester diethyl adipate (DEA). Up to 78% MEA with 19% diacid adipic acid (AA) as by-product could be synthesized by the unpurified ACut2 culture supernatant from the B. raffinosifermentans overexpression strain. By adjusting pH and enzyme concentration, the selectivity of the free ACut2 culture supernatant was increased, yielding 95% MEA with 5% AA. Selectivity of the carrier immobilized ACut2 culture supernatant was also improved by pH adjustment during immobilization, as well as carrier enzyme loading, ultimately yielding 93% MEA with an even lower AA concentration of 3-4%. Thus, optimizations enabled the selective hydrolysis of DEA into MEA with only a minor AA impurity. In the up-scaling, a maximum of 98% chemical and 87.8% isolated MEA yield were obtained by the adsorbed enzyme preparation with a space time yield of 2.6 g L(-1) h(-1). The high monoester yields establish the ACut2-catalyzed biosynthesis as an alternative to existing methods.
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 6x 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 degrees 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 6x 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.
