Characterization of a cutinase from Myceliophthora thermophila and its application in polyester hydrolysis and deinking process

Highlights

• A cutinase, MtCUT, from Myceliophthora thermophila ATCC 42464 was expressed in Pichia pastoris.

• MtCUT showed the highest specific activity for p-nitrophenyl butyrate.

• MtCUT hydrolyzed and broke down apple cutin into C16 and C18 family fatty acids.

• MtCUT could efficiently degrade poly caprolactone.

• MtCUT showed more efficient performance in the deinking of laser-printed paper and newspaper than commercial lipase.

Abstract

A gene encoding cutinase (MtCUT) from Myceliophthora thermophila was expressed in Pichia pastoris. The optimal temperature and pH for MtCUT were 30 °C and 8.5, respectively. MtCUT showed high activities toward a broad range of p-nitrophenyl esters, and the highest specific activity was recorded for p-nitrophenyl butyrate. MtCUT hydrolyzed and broke down apple cutin into C16 and C18 family fatty acids. MtCUT could efficiently degrade polycaprolactone (PCL), the weight loss of which was 78.5% in 36 h. In addition, MtCUT could degrade PVAc, a kind of adhesive that is frequently used in papermaking and in synthetic toner or ink. Its performance in the deinking of waste papers was investigated and compared with that of commercial lipase in this study. The results showed that an ink removal efficiency of 78.4% on laser-printed paper and 81.3% on newspaper was obtained using MtCUT at 30 °C, which was better than that of lipase from Candida rugosa (CrLIP) (77.0% and 76.0%, respectively). The properties of the MtCUT-treated samples were similar to that of the control samples. This revealed that the cutinase MtCUT from M. thermophila has a great potential in PCL hydrolysis and the deinking process.

Introduction

Cutinases (3.1.1.74) are the smallest members of the α/β hydrolase family, which can hydrolyze the ester bonds of cutin, the insoluble biopolyester matrix in plant surfaces [1]. Cutinases were identified and characterized mainly from fungi and several bacteria [2], [3]. Among them, the cutinase from Fusarium solani pisi was the most studied and widely used in industrial application [4]. The biochemical properties of cutinases from other fungi such as Alternaria brassicicola [5], Aspergillus niger [6], Fusarium oxysporum [7], Monilinia fructicola [8], Sirococcus conigenus [9], Trichoderma reesei [10], and Thielavia terrestris [11] have been studied in recent years, and it is believed that the cutinase-catalyzed hydrolysis of the cuticular layer is essential for fungal initial attack on plants [12].

Structural analyses of cutinase crystals show that their catalytic triad is composed of Ser-His-Asp, in which the catalytic serine is exposed to solvent [7], [10], [13], [14]. Contrary to lipases, cutinases do not display or display little interfacial activation. Cutinases can act on water-soluble esters such as p-nitrophenyl esters and insoluble triglycerides, with a preference for short-chain substrates.

Cutinases have received much attention because of their potential applications in the degradation of aliphatic and aromatic polyesters [3]. Cutinase 1 from Thermobifida cellulosilytica was reported to hydrolyze several polyethylene terephthalate (PET) substrates [15] and the environmentally friendly polyester polyethylene furanoate (PEF) [16]. The cutinase from Thielavia terrestris was able to successfully hydrolyze polycaprolactone (PCL) and polybutylene succinate (PBS) [17]. Cutinases from other fungi, such as F. solani, A. oryzae, A. brassicicola, and Humicola insolens, were also reported to hydrolyze PBS [18]. The latter was also demonstrated to be active in the hydrolysis of polylactic acid (PLA) [19] or deacetylation of polyvinyl acetate (PVAc) [20].

Waste papers are now the major fiber resources for paper manufactures in the world. In the waste paper recycling process, the printed ink in the paper needs to be removed by a deinking process. Previous reports had demonstrated the successful use of various enzymes for waste paper deinking, such as cellulases, xylanases, pectinases, amylases, lipase, and laccases [21]. Although enzymatic treatment has shown some advantages over chemical deinking, further efforts are required to discover more effective enzymes for commercial applications [22]. Various polymers, such as polyvinyl acetate and polyacrylate with ester bonds, have been widely used as adhesives in papermaking or as binders in synthetic toner or ink [23]. The deinking ability of the lipases is due to the hydrolysis of triglycerides in vegetable oil-based inks into di- and monoglycerides and glycerol, thereby dislodging the ink particles from waste paper [24]. Thus, we speculate that cutinases could be used effectively in the deinking process because of their multiple capacities in the degradation of polyester binders, water-insoluble triglycerides, and soluble esters.

In this paper, a cutinase from Myceliophthora (Thermothelomyces) thermophila ATCC 42464 was heterologously expressed in Pichia pastoris (Komagataella phaffii), and its recombinant protein was efficiently purified in a single step using Ni-NTA affinity chromatography. The properties of this enzyme and its potential in the deinking of laser-printed paper and newspaper were investigated. To the best of our knowledge, it is the first report on the characterization and deinking application of cutinase from the species M. thermophila.