Production Optimization and Characterization of Recombinant Cutinases from Thermobifida fusca sp. NRRL B-8184

Abstract

Two genes, cut1 and cut2, of Thermobifida fusca NRRL B-8184 with cutin-hydrolyzing activity were cloned and expressed in Escherichia coli BL21 (DE3) separately. Enhanced expression was achieved after screening of six different media, optimization of the culture conditions and medium components. Among the screened media, modified Terrific Broth was found to be the best for maximum production of recombinant cutinases in E. coli BL21 (DE3). Under optimal conditions, the production of recombinant Cut1 and Cut2 (cutinases) were found to be 318±0.73 and 316±0.90 U/ml, respectively. The production of recombinant cutinases was increased by 11-fold as compared with T. fusca NRRL B-8184 wild-type strain. Both the recombinant cutinases were purified to homogeneity. They were found to be thermostable, organic solvent, and surfactant tolerant. Both the cutinase were active in a broad range of temperature (40-80 °C) and pH (6.8-9) with optimum activity at pH 8.0 and 55 °C.

Structural and functional studies of a Fusarium oxysporum cutinase with polyethylene terephthalate modification potential

“…Similarly, activity and efficiency of FoCut5a (i.e. k cat and k cat /K m constants) have similar values with cutinases already characterized [45,46], except the one from Pseudomonas cepacia, which appears to be highly active (k cat 22000 s -1 ) [59].…”

“…Expression of eukaryotic proteins that contain cysteine residues, in E. coli, can be [42][43][44] and from bacteria (three Thermobifida strains) [45][46][47].…”

“…On the other hand, bacterial thermophilic cutinases from T. fusca expressed in E. coli BL21, showed an optimal enzyme activity at 55-60 ο C [45,64].…”

Characterization of an acidic cold-adapted cutinase from Thielavia terrestris and its application in flavor ester synthesis

“…2c) is comparable to that of the enzyme from T. fusca sp. (Hegde & Veeranki, 2013), but higher than those of cutinases from A. brassicicola (40°C) (Koschorreck et al, 2010), F. solani (40°C) (Kwon et al, 2009) and T. terrestris CAU709 (50°C) , and a little lower than that of the cutinase from T. fusca (60°C) (Chen et al, 2008). TtcutB in the present study showed thermostability up to 65°C, retaining more than 95% of its activity after treatment at 65°C for 0.5 h (Fig.…”

“…Therefore, the isolation of novel cutinases that possess special properties is of great potential economic value and importance. Cutinases are mainly found in different species of fungi (Castro-Ochoa et al, 2012;Fraga, Carvalho, & Macedo, 2012;Nyyssölä et al, 2014;Pio & Macedo, 2007;Roussel et al, 2014;Speranza & Macedo, 2013), although several bacterial cutinolytic enzymes have also been reported (Dutta, Krishnamoorthy, & Dasu, 2013;Hegde & Veeranki, 2013;Kitadokoro et al, 2012). A number of cutinases have been purified and characterized from various fungi, including Alternaria brassicicola (Koschorreck, Liu, Kazenwadel, Schmid, & Hauer, 2010), Coprinopsis cinerea (Merz, Schembecker, Riemer, Nimtz, & Zorn, 2009), Fusarium oxysporum (Fraga et al, 2012;Speranza & Macedo, 2013), Fusarium solani (Kwon, Kim, Yang, Song, & Song, 2009), Monilinia fructicola (Wang, Michailides, Hammock, Lee, & Bostock, 2002), Sirococcus conigenus , Trichoderma harzianum (Rubio, Cardoza, Hermosa, Gutierrez, & Monte, 2008) and Trichoderma reesei (Roussel et al, 2014).…”