Cutinase: Characteristics, Preparation, and Application
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Biotechnology Advances 31 (2013) 1754–1767 Contents lists available at ScienceDirect Biotechnology Advances journal homepage: www.elsevier.com/locate/biotechadv Research review paper Cutinase: Characteristics, preparation, and application Sheng Chen, Lingqia Su, Jian Chen, Jing Wu ⁎ State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Ave., Wuxi, Jiangsu 214122, China School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Ave., Wuxi, Jiangsu 214122, China article info abstract Article history: Cutinases (E.C. 3.1.1.74) belong to the α/β-hydrolase superfamily. They were initially discovered because they Received 15 May 2013 are secreted by fungi to hydrolyze the ester bonds of the plant polymer cutin. Since then, they have been Received in revised form 4 August 2013 shown to catalyze the hydrolysis of a variety of polymers, insoluble triacylglycerols, and low-molecular-weight Accepted 11 September 2013 soluble esters. Cutinases are also capable of catalyzing esterification and transesterification reactions. These Available online 19 September 2013 relatively small, versatile, secreted catalysts have shown promise in a number of industrial applications. This re- view begins by describing the characteristics of cutinases, pointing out key differences among cutinases, esterases Keywords: Cutinase and lipases, and reviewing recent progress in engineering improved cutinases. It continues with a review of the Identification methods used to produce cutinases, with the goal of obtaining sufficient quantities of material for use in indus- Crystal structure trial processes. Finally, the uses of cutinases in the textile industry are described. The studies presented here dem- Molecular modification onstrate that the cutinases are poised to become important industrial catalysts, replacing older technologies with Preparation more environmentally friendly processes. Textile industry © 2013 Elsevier Inc. All rights reserved. Contents 1. Introduction............................................................. 1754 2. Cutinase identification, structure and modification............................................ 1755 2.1. Identificationoftruecutinases.................................................. 1755 2.2. Cutinaseassays......................................................... 1756 2.3. Simplerassaysappliedtocutinases................................................ 1757 2.4. Cutinasestructureandfunction................................................. 1758 2.5. Modificationofcutinases.................................................... 1760 3. Preparationofcutinase........................................................ 1761 3.1. Preparationofcutinasesfromwild-typestrains.......................................... 1761 3.2. Preparationofcutinaseusingengineeredstrains.......................................... 1762 4. Applicationofcutinaseinindustry................................................... 1763 4.1. Cotton fibers.......................................................... 1763 4.2. Synthetic fibers......................................................... 1763 4.3. Woolfabrics.......................................................... 1764 5. Conclusions.............................................................. 1764 Acknowledgments............................................................. 1764 References................................................................. 1764 1. Introduction Abbreviations: CBM, carbohydrate-binding module; DHA, docosahexanoic acid; EPA, eicosapentanoic acid; PBM, polyhydroxyalkanoate-binding module; PET, polyethylene The plant cuticle is a protective layer that coats the epidermis of terephthalate; RBB, Remazol Brilliant Blue R. leaves, shoots, and other tender, aboveground portions of terrestrial ⁎ Corresponding author at: State Key Laboratory of Food Science and Technology, plants. Composed of waxes and lipid polymers, the cuticle protects the Jiangnan University, 1800 Lihu Ave., Wuxi, Jiangsu 214122, China. Tel.: +86 510 85327802; fax: +86 510 85326653. plant from dehydration, and is a barrier to infection by pathogen. The E-mail address: [email protected] (J. Wu). major constituent of the cuticle is insoluble lipid polyester called cutin, 0734-9750/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.biotechadv.2013.09.005 S. Chen et al. / Biotechnology Advances 31 (2013) 1754–1767 1755 which consists primarily of hydroxylated 16- and 18-carbon fatty acids 2. Cutinase identification, structure and modification that are linked together via ester bonds. Microorgaisms, principally fungi and bacteria, can hydrolyze this polymer by secreting enzymes 2.1. Identification of true cutinases called cutinases. Early work focused on the role of cutinase in the infection of plants Cutinase genes were originally identified by observing the effect of by fungi. Fungal spores landing on the plant cuticle were shown to their deletion upon the virulence of the expressing organism. Although respond to cutin monomers by expressing cutinase, and specificinhibi- originally found in pathogenic fungi, cutinases have subsequently been tion of this enzyme blocked infectivity in several pathogen/host systems isolated and characterized from bacteria, such as Thermobifida fusca (Kolattukudy et al., 1989). Subsequent work with Alternaria brassicicola, (Chen et al., 2008), and from a variety of both pathogenic and non- demonstrated that cutinases are a diverse group of enzymes, in which pathogenic fungi, including the yeast Cryptococcus (Masaki et al., some cutinases are essential for pathogenicity, while others are 2005). The microbial strains that have been shown to express cutinases expressed during saprophytic growth on cutin as a carbon source (Fan are identified in Table 1. As the number of sequenced microbial and Köller, 1998; St Leger et al., 1997). More recent research has genomes has increased, so has the number of open reading frames demonstrated that both pathogenic and saprophytic microorganisms annotated as cutinases. In several instances, these putative cutinase express cutinases, that the genomes of individual species may harbor enzymes have been isolated and assayed using simple substrates. Un- several different putative cutinases, and that these enzymes are fortunately, since many of these enzymes have not been assayed using expressed at different times during the microbial life cycle (Skamnioti cutin as a substrate, it is not possible to distinguish them from similar et al., 2008a, 2008b). There is also evidence suggesting that cutinase- esterases or lipases that can also hydrolyze these simple substrates. To containing saprophytic bacteria, such as Pseudomonas putida, may pro- appreciate the number and diversity of the open reading frames that vide a carbon source to a wider microbial community (Sebastian et al., have been annotated as cutinases, and appreciate how varied the activ- 1987). ities of the proteins expressed from these genes are likely to be, studies Cutinases (EC 3.1.1.74) are serine esterases that belong to the α/β hy- of the molecular taxonomy of cutinases must be reviewed. drolase superfamily. They possess a classical Ser–His–Asp catalytic triad, Relatively recent work on the molecular taxonomy of fungal in which the catalytic serine is exposed to solvent. Because cutinases lack cutinases (Skamnioti et al., 2008a, 2008b) has suggested that the fungal the hydrophobic lid that covers the active site serine in true lipases, the cutinases can be divided into two ancient subfamilies that have been in cutinase active site is large enough to accommodate the high- existence since before the Ascomycota and the Basidobycota diverged molecular-weight substrate cutin, and some of them can also hydrolyse nearly a billion years ago. Interestingly, no cutinase sequences were high-molecular-weight synthetic polyesters. Besides, they are able to found in the “true yeasts” (Saccharomycotina). A similar study of the hydrolyse a greater variety of substrates, including low-molecular- cutinase sequences from Phytophthora, a genus of phytopathogenic weight soluble esters, short- and long-chain triacylglycerols. Cutinases oomycetes (water molds) (Belbahri et al., 2008) identified related are also capable of catalyzing esterification and transesterification. cutinase sequences in three genera of Actinobacteria, and suggested First identified in the 1960's and characterized in the early 1970's, lateral gene transfer between ancient bacteria and oomycetes. the cutinase from the filamentous fungus Fusarium solani pisi rapidly be- Skamnioti et al. (Skamnioti et al., 2008a) recognized the existence of came a model system for the study of cutinase structure, function and mycobacterial cutinases, but rejected the idea of lateral gene transfer reactivity. This early work has been described in concise reviews to fungi, placing the bacterial cutinase sequences in a separate group. (Carvalho et al., 1998; Egmond and de Vlieg, 2000; Longhi and Both research groups recognized that a single species may contain Cambillau, 1999). Cutinase was found to be distinct among the α/β several putative cutinases, sometimes more than a dozen. Skamnioti hydrolases because, unlike the majority of esterases (Panda and et al. (Skamnioti et al., 2008a) also performed a transcriptional analysis Gowrishankar, 2005), cutinase is able to hydrolyse lipid substrates ofthe14putativecutinasesfromMagnaporthe grisea strain Guy11.