Elsevier

Food Chemistry

Volume 188, 1 December 2015, Pages 439-445
Food Chemistry

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

https://doi.org/10.1016/j.foodchem.2015.05.026Get rights and content

Highlights

  • A novel acidic cutinase from Thielavia terristris was purified and characterized.

  • The enzyme exhibited good thermal and pH stability.

  • The enzyme displayed strong stability in the presence of organic solvents.

  • The enzyme efficiently synthesized flavor ester butyl butyrate under non-aqueous conditions.

  • The enzyme is a potential candidate in flavor esters-producing industries.

Abstract

An acidic cutinase (TtcutB) from Thielavia terrestris CAU709 was purified to apparent homogeneity with 983 U mg−1 specific activity. The molecular mass of the enzyme was estimated to be 27.3 and 27.9 kDa by SDS–PAGE and gel filtration, respectively. A peptide sequence homology search revealed no homologous cutinases from T. terrestris, except for one putative cutinase gene (XP003656017.1), indicating that TtcutB is a novel enzyme. TtcutB exhibited an acidic pH optimum of 4.0, and stability at pH 2.5–10.5. Optimal activity was at 55 °C, it was stable up to 65 °C, and retained over 30% activity at 0 °C. Km values toward p-nitrophenyl (pNP) acetate, pNP-butyrate and pNP-caproate were 8.3, 1.1 and 0.88 mM, respectively. The cutinase exhibited strong synthetic activity on flavor ester butyl butyrate under non-aqueous environment, and the highest esterification efficiency of 95% was observed under the optimized reaction conditions. The enzyme’s unique biochemical properties suggest great potential in flavor esters-producing industries.

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).

References (39)

  • J. Merz et al.

    Purification and identification of a novel cutinase from Coprinopsis cinerea by adsorptive bubble separation

    Separation and Purification Technology

    (2009)
  • A. Nyyssölä et al.

    Screening of microbes for novel acidic cutinases and cloning and expression of an acidic cutinase from Aspergillus niger CBS 513.88

    Enzyme and Microbial Technology

    (2013)
  • T.F. Pio et al.

    Optimizing the production of cutinase by Fusarium oxysporum using response surface methodology

    Enzyme and Microbial Technology

    (2007)
  • T. Raghavendra et al.

    Synthesis of the ‘green apple ester’ ethyl valerate in organic solvents by Candida rugosa lipase immobilized in MBGs in organic solvents: Effects of immobilization and reaction parameters

    Journal of Molecular Catalysis B: Enzymatic

    (2010)
  • A. Roussel et al.

    A Cutinase from Trichoderma reesei with a lid-covered active site and kinetic properties of true lipases

    Journal of Molecular Biology

    (2014)
  • P. Speranza et al.

    Biochemical characterization of highly organic solvent-tolerant cutinase from Fusarium oxysporum

    Biocatalysis and Agricultural Biotechnology

    (2013)
  • S. Torres et al.

    Enzymatic synthesis of banana flavour (isoamyl acetate) by Bacillus licheniformis S-86 esterase

    Food Research International

    (2009)
  • G.Y. Wang et al.

    Molecular cloning, characterization, and expression of a redox-responsive cutinase from Monilinia fructicola (Wint.) honey

    Fungal Genetics and Biology

    (2002)
  • T. Watanabe et al.

    Xylose induces the phyllosphere yeast Pseudozyma antarctica to produce a cutinase-like enzyme which efficiently degrades biodegradable plastics

    Journal of Bioscience and Bioengineering

    (2014)
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