Characterization of an acidic cold-adapted cutinase from Thielavia terrestris and its application in flavor ester synthesis
Introduction
Cutinase (EC 3.1.1.74) is an inducible lipolytic/estrolytic enzyme, capable of catalyzing the cleavage of not only the ester bonds of cutin, the major aliphatic polymer of the plant cuticle, but also other water-soluble esters and insoluble triglycerides (Chen et al., 2013, Sulaiman et al., 2012). Cutinases are considered to be intermediate between esterases and lipases as they can efficiently hydrolyze soluble esters and emulsified triacylglycerols. However, they do not exhibit interfacial activation, which is a typical characteristic of lipase (Egmond & Van Bemmel, 1997). Recently, cutinases have received much attention due to their potential application in various industrial fields such as foods, fine chemicals, pharmaceuticals and environmental (Chen et al., 2013). In the food industry, they can be used to hydrolyze excess fat in fat-rich foods such as milk, with the aim of reducing food energy, or used to synthesize flavor esters as a biocatalyst in clean green bioprocess, which has been considered as an alternative route to traditional synthetic chemical industry (De Barros, Azevedo, Cabral, & Fonseca, 2012). In the chemical industry, they are used to produce detergents for fat removal in household laundry (Castro-Ochoa et al., 2012). In the pharmaceutical industry, an enzymatic preparation containing cutinase has been developed to increase the pharmacological effect of agricultural agents (Pio & Macedo, 2007). In the environmental industry, cutinases can be used for the degradation of biodegradable plastics (Watanabe et al., 2014, Yang et al., 2013).
The availability of cutinases possessing desirable functional characteristics for specific purposes is a limiting factor in their application. 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 et al., 2012, Nyyssölä et al., 2014, Pio and Macedo, 2007, Roussel et al., 2014, Speranza and Macedo, 2013), although several bacterial cutinolytic enzymes have also been reported (Dutta et al., 2013, Hegde and 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 and Macedo, 2013), Fusarium solani (Kwon, Kim, Yang, Song, & Song, 2009), Monilinia fructicola (Wang, Michailides, Hammock, Lee, & Bostock, 2002), Sirococcus conigenus (Nyyssölä et al., 2014), Trichoderma harzianum (Rubio, Cardoza, Hermosa, Gutierrez, & Monte, 2008) and Trichoderma reesei (Roussel et al., 2014). Most of these are mesophilic fungi; there are no reports on cutinases from thermophilic fungi, except for Humicola insolens (Nielsen, Borch, & Westh, 2007). Thielavia terrestris is a type of soilborne thermophilic ascomycete which can grow normally at relatively low pH (e.g. 4.5) and elevated temperature (40–45 °C) (Yang, Xu et al., 2013). There is no report on the production or characterization of cutinases from any of the species of Thielavia, except a low molecular mass cutinase (TtcutA), which was reported in our laboratory recently (Yang, Xu et al., 2013). Here, we describe the purification and biochemical characterization of another acidic cutinase from T. terrestris CAU709. We then investigate this cutinase’s application on the synthesis of flavor esters.
Section snippets
Materials, microorganism and enzyme production
p-Nitrophenol (pNP) and pNP esters including p-nitrophenyl acetate (pNPA), p-nitrophenyl butyrate (pNPB), p-nitrophenyl caproate (pNPC), p-nitrophenyl dodecanoate (pNPD), p-nitrophenyl myristate (pNPM) and p-nitrophenyl palmitate (pNPP) were the products of Sigma Chemical Co. (St. Louis, MO, USA). p-Nitrophenyl hexacaprate (pNPH) was purchased from HEOWNS Company (Tianjing, China). Triacetin, tributyrin, tricaproin, tricaprylin and tricaprin were purchased from TCI Co. (Tokyo, Japan). Fast Red
Purification and identification of a cutinase from T. terrestris
An extracellular cutinase was purified to homogeneity from the culture supernatant of T. terrestris CAU709 with a purification fold of 16 and a recovery yield of 10% (Table 1). The specific activity of the enzyme was increased from 62 U mg−1 to 983 U mg−1. The purified cutinase was electrophoretically homogeneous as observed by SDS–PAGE with a molecular mass of approx. 27.3 kDa (Fig. 1), while the native molecular mass of the cutinase was found to be about 27.9 kDa by gel filtration, indicating that
Discussion
Cutinases have been purified and characterized from various fungi (Fraga et al., 2012, Koschorreck et al., 2010, Kwon et al., 2009, Roussel et al., 2014, Speranza and Macedo, 2013, Wang et al., 2002), especially phytopathogenic fungi such as Fusarium solani pisi, whose cutinase has been extensively studied with respect to its structure and function. However there are no reports on cutinase from Thielavia spp, except a recently reported low molecular mass cutinase (TtcutA) in our lab (Yang, Xu
Conclusions
A novel acidic and cold adapted cutinase (TtcutB) from T. terrestris was purified and characterized. The optimal pH and temperature of the enzyme are pH 4.0 and 55 °C, respectively. The enzyme is stable under a wide range of pH values and temperatures, and it also shows relatively high activity at low temperatures. The enzyme exhibits broad substrate specificity. In addition, the enzyme synthesized flavor ester butyl butyrate using 1-butanol and butyric acid with high esterification efficiency.
Acknowledgements
We thank Dr. Priti Katrolia for critical reading of the manuscript. This work was supported by the National Science Fund for Distinguished Young Scholars (No. 31325021) and the National Natural Science Foundation of China (No. 31371718).
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2019, Studies in MycologyCitation Excerpt :terrestris is the most eminent species. Genome analyses and enzymological studies showed that this thermophilic species is able to produce enzymes with biotechnological applications, including the hydrolysis of all major polysaccharides found in biomass (Berka et al. 2011, de Vries et al. 2011, Syed et al. 2014, Xu et al. 2015, Woon et al. 2016, Gao et al. 2017, García-Huante et al. 2017). The capacity of Th.