Characterization of Thermobifida fusca Cutinase-Carbohydrate-Binding Module Fusion Proteins and Their Potential Application in Bioscouring

Abstract

Cutinase from Thermobifida fusca is thermally stable and has potential application in the bioscouring of cotton in the textile industry. In the present study, the carbohydrate-binding modules (CBMs) from T. fusca cellulase Cel6A (CBM Cel6A ) and Cellulomonas fimi cellulase CenA (CBM CenA ) were fused, separately, to the carboxyl terminus of T. fusca cutinase. Both fusion enzymes, cutinase-CBM Cel6A and cutinase-CBM CenA , were expressed in Escherichia coli and purified to homogeneity. Enzyme characterization showed that both displayed similar catalytic properties and pH stabilities in response to T. fusca cutinase. In addition, both fusion proteins displayed an activity half-life of 53 h at their optimal temperature of 50°C. Compared to T. fusca cutinase, in the absence of pectinase, the binding activity on cotton fiber was enhanced by 2% for cutinase-CBM Cel6A and by 28% for cutinase-CBM CenA , whereas in the presence of pectinase, the binding activity was enhanced by 40% for the former and 45% for the latter. Notably, a dramatic increase of up to 3-fold was observed in the amount of released fatty acids from cotton fiber by both cutinase-CBM fusion proteins when acting in concert with pectinase. This is the first report of improving the scouring efficiency of cutinase by fusing it with CBM. The improvement in activity and the strong synergistic effect between the fusion proteins and pectinase suggest that they may have better applications in textile bioscouring than the native cutinase.Cotton fiber has a multilayered structure, with its outermost surface being the cuticle that is cross-linked to the primary cell wall of cotton fiber by esterified pectin substances. The major component of the cuticle is cutin, an insoluble polyester composed mainly of saturated C 16 and C 18 hydroxy and epoxy fatty acids (14,16,27,38). During the process of scouring in the textile industry, the cuticle layer has to be removed in order to improve the wettability of cotton fiber, which then facilitates uniform dyeing and finishing. Traditionally, this process is performed by hot hydrolysis in alkaline medium, which not only consumes large quantities of water and energy but also causes severe pollution and fiber damage (20,21,33). Therefore, environment-friendly scouring methods based on biocatalysts have been actively sought (2,30,36).Cutinase is a multifunctional esterase capable of degrading the cutin component of the cuticle. Earlier reports showed that the fungal cutinase from Fusarium solani pisi has potential use for cotton cuticle degradation and exhibits a good synergistic effect with pectinase, an enzyme utilized to degrade pectin, in the scouring of cotton fiber (1,7,8,14). Moreover, site-directed mutagenesis has been performed to replace the specific amino acid residues near the active site of cutinase (3) to improve its hydrolytic activity toward polyesters. More recently, a cutinase from the thermophilic bacterium Thermobifida fusca has been identified and overexpressed in Escherichia coli in our laboratory (10). The...

Engineering of Family-5 Glycoside Hydrolase (Cel5A) from an Uncultured Bacterium for Efficient Hydrolysis of Cellulosic Substrates

“…CBM6 was fused to the C -terminus of Cel5A_2R2 using 3-step overlap PCR [19]. The construction procedure is shown in Figure S1.…”

Which properties of cutinases are important for applications?

“…In the presence of pectinase, binding activity of the Thermobifida fusca cellulase and Cellulomonas fimi carbohydrate binding domain fusions with the cutinase was 40 and 45 % higher than that of the native enzyme, respectively. In addition, a far higher amount, up to 3-fold higher than with native cutinase, of released fatty acids were obtained with the fusion proteins (Zhang et al 2010).…”

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

“…The pH stability was highest for the three chimeric enzymes after incubation for 24 hrs at 37ºC under optimum pH (Zhang et al 2010). Figure 4 shows the activities of CUT-N1 at optimum pH and after incubation at temperatures of 30 to 70ºC during 30, 60 and 90 min.…”

“…Zhang et al (2010) determined optimal temperature of 50ºC for recombinant cutinases and observed the half-life of 53 hrs after incubation at optimum temperature. Ronkvist et al (2009) characterized cutinases produced by different microorganisms and determined thermostability at 50ºC for cutinases produced by Pseudomonas mendocina and F. solani, while cutinases from Humilica insolens showed thermostability at 70-80ºC.…”