Identification and Characterization of Bacterial Cutinase

Abstract

Cutinase, which exists in both fungi and bacteria, catalyzes the cleavage of the ester bonds of cutin. Fungal cutinases have been extensively studied, however, reports on bacterial cutinases have been limited due to the lack of knowledge concerning the identity of their open reading frames. In the present study, the cutinase from Thermobifida fusca was induced by cutin and purified to homogeneity by following p-nitrophenyl butyrate hydrolyzing activity. Peptide mass fingerprinting analysis of the wild-type enzyme matched two proteins, Tfu_0883 and Tfu_0882, which are 93% identical in sequence. Both proteins were cloned and overexpressed in their mature form. Recombinant Tfu_0883 and Tfu_0882 display very similar enzymatic properties and were confirmed to be cutinases by their capability to hydrolyze the ester bonds of cutin. Comparative characterization of Fusarium solani pisi and T. fusca cutinases indicated that they have similar substrate specificity and catalytic properties except that the T. fusca enzymes are thermally more stable. Homology modeling revealed that T. fusca cutinases adopt an ␣/␤-hydrolase fold that exhibits both similarities and variations from the fungal cutinase structure. A serine hydrolase catalytic mechanism involving a Ser 170 -His 248 -Asp 216 (Tfu_0883 numbering) catalytic triad was supported by active site-directed inhibition studies and mutational analyses. This is the first report of cutinase encoding genes from bacterial sources.Cutin is a major component of the plant cuticle. It is an insoluble lipid-polyester formed primarily from C 16 and C 18 hydroxy and epoxy fatty acids (Fig.

Production of heterologous cutinases by E. coli and improved enzyme formulation for application on plastic degradation

“…Moreover, cutinases produced by F. solani pisi and T. fusca, after isolation and purification, showed optimum pH 8.0 for the hydrolysis of triolein and pNPB. These enzymes were produced in specific media under orbital agitation of 200 rpm at 50ºC (Chen et al 2008). Speranza et al (2011) characterized cutinases produced by F. oxysporum and also determined maximal activity at pH 8.0 in different solid culture media in the presence of wheat bran, soybean rind and rice bran; and at pH 9.0 in the presence of Jatropha curcas seed cake.…”

“…The optimum temperature and the thermal stability ranged between 30 and 37ºC depending on the substrate. Chen et al (2008) characterized cutinases produced by F. solani pisi and T. fusca and determined optimum temperatures of 30 and 40ºC, respectively. Cutinases from T. fusca exhibited higher thermal stability; the residual activity was higher than 80% after 160 hrs, at 40ºC.…”

Substrate Inhibition Growth Kinetics for Cutinase Producing Pseudomonas cepacia Using Tomato-peel Extracted Cutin

“…Firstly, a non-specific yet simple spectrophotometric assay, which does not distinguish between esterase and cutinase, was used to measure the release of p-nitrophenol (pNP) from p-nitrophenylbutyrate (pNPB). 12 The production of cutinase was further confirmed by using cutinase-specific substrate p-nitrophenyl (16 methyl sulphone ester) hexadecanoate (p-NMSH).…”

“…Cutin as substrate for cutinase was prepared in the laboratory from fresh tomato peels using the method described by Chen et al 12 In brief, the tomato peels collected from fresh tomatoes were boiled in oxalic acid/ammonium oxalate buffer for 3-4 h. After cooling to room temperature, the peels were digested with enzymes (cellulase and pectinase) to remove pectin and cellulose, subjected to extensive solvent extraction with methanol-chloroform in soxhlet apparatus to remove the embedded waxes, and then dried in an oven at 40 ºC. These dried peels were ground to powder (< 20 mesh) to obtain cutin.…”

“…5 Extensive studies have been carried out on the production, purification and characterization of cutinase from phytopathogenic fungi. [6][7][8][9] On the other hand, there are only a few isolated studies on bacterial cutin degradation ability or bacterial cutinase [10][11][12] , which are even better in terms of thermal or pH stability when compared to their fungal counterpart. Unlike fungal cutinase, which loses its activity at 45 °C, bacterial cutinase could be stable at as high as 70 °C.…”