Purification and characterization of a novel cutinase from nasturtium (Tropaeolum majus) pollen

doi:10.1016/0003-9861(79)90292-3

Archives of Biochemistry and Biophysics

Volume 196, Issue 2, September 1979, Pages 412-423

https://doi.org/10.1016/0003-9861(79)90292-3 Get rights and content

Abstract

Cutinase from pollen grains of Tropaeolum majus was purified by Sephadex G-100 gel filtration, QAE-Sephadex chromatography, and isoelectric focusing. The purified enzyme was homogeneous as judged by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. The molecular weight of the enzyme was estimated to be 40,000 by both Sephadex G-100 gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This cutinase was found to be a glycoprotein containing about 7% carbohydrate and the isoelectric point of this enzyme was 5.45. It catalyzed hydrolysis of p-nitrophenyl esters of C₂ to C₁₈ fatty acids with similar K_m and V. The purified cutinase showed an optimum pH of 6.8 with cutin as the substrate, whereas with p-nitrophenyl esters of fatty acids the optimum pH was 8.0. This enzyme did not show any metal ion requirement. Unlike the previously studied fungal cutinases, the present pollen enzyme was strongly inhibited by thiol-directed reagents such as N-ethylmaleimide and p-hydroxymercuribenzoate whereas it was totally insensitive to the active serine-directed reagent, diisopropylfluorophosphate. The purified pollen cutinase showed preference for primary alcohol esters, but it did not catalyze hydrolysis of tripalmitoyl or trioleyl glycerol at significant rates. The properties of the pollen enzyme are, in general, in sharp contrast to those of the fungal cutinase, and the present results strongly suggest that the pollen enzyme belongs to a new class of cutinases. Another esterase which preferentially hydrolyzed p-nitrophenyl acetate was also found in the extracellular fluid. This enzyme, separated from cutinase, showed a pI of 5.6 and it was sensitive to diisopropylfluorophosphate, but not to SH-directed reagents.

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Cutinases produced by fungi and bacteria have been investigated in detailed, for example, Humicola insolens (Kold et al., 2014), Fusarium solani pisi (Egmond and Vlieg, 2000), Aspergillus oryzae (Maeda et al., 2005), Fusarium oxysporum (Macedo and Pio, 2007), Monilinia fructicola (Wang et al., 2002), Thermobifida fusca (Chen et al., 2008), Pseudomonas putida (Sebastian and Kolattukudy, 1988). In addition, cutinase was also identified in plant pollen, such as Tropaeolum majus (Maiti et al., 1979). Since discovery, cutinase has been a research hotspot in different fields such as food processing and textile industry.
The paper industry is one of the most important basic raw material pillar industries. With the decrease of forest wood resources, the recycling of wastepaper has drawn increasingly attention. However, the stickies generated in the process of wastepaper recycling will flocculate and deposite in the pulp, resulting in production accidents and inferior product quality. The biological enzymatic method, with the advantages of high efficiency, specificity, and pollution-free, can prevent the flocculation of the stickies by enzymatically hydrolyzing the ester bond of the stickies components. Previous studies have demonstrated that cutinase (EC 3.1.1.74) had the ability to degrade polyester components of stickies. Meanwhile, relevant studies have shown that anchor peptides possessed the ability to bind polyester. Herein, the cutinase from Humicola insolens (HiC) was fused with Escherichia coli anchor peptide OMP25, the enzymatic properties of the fusion protein HiC-OMP25 and its degradation efficiency of the stickies model substrate, poly(ethyl acrylate) (PEA) and poly(vinyl acetate) (PVAc), as well as stickies sediment were determined. All of the results demonstrated that OMP25 efficiently enhanced the degradation ability of HiC.
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The nonmetallic compound boron executes a requisite role in the advancement of new meristematic cells in plants. Micronutrient boron is a very special element because it has a key aspect in the flowering and fertilization phenomenon. The most crucial performance of boron in plants is to enhance the pollination and fruit setting. It is reported that the reproductive organs such as flowers, anthers, ovary and stigma carry more boron content than the vegetative organs leaves, stems, and roots implying more requirement of boron during the reproductive phase. The role of boron has been locked up to pollen germination and growth of pollen tubes in vascular plants. Thus the present study is related to the impact of boron nutrition on pollen–pistil interaction and specific enzymes like peroxidase, acid phosphatases, and esterases which is found to be crucial for fertilization. The present study is also in the direction of the impact of boron nutrition on seed yield and quality.
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Cutinases (EC 3.1.1.74) are serine hydrolases and members of the α/β hydrolase superfamily [1–3].
The specific activity and enantioselectivity of immobilized cutinases from Aspergillus oryzae (AoC) and Humicola insolens (HiC) were compared with those of lipases from Thermomyces lanuginosus (TLL), Rhizomucor miehei (RML) and Lipase B from Candida antarctica (CALB) for menthol and its analogs that include isopulegol, trans-2-tert-butylcyclohexanol (2TBC), and dihydrocarveol (DHC). Common features of these alcohols are two bulky substituents: a cyclohexyl ring and an alkyl substituent. Dissimilarities are that the alkyl group reside at different positions or have dissimilar structures. The aim was to develop an understanding at a molecular level of similarities and differences in the catalytic behavior of the selected cutinases and lipases as a function of substrate structural elements. The experimental results reflect the (-)-enantioselectivity for AoC, HiC, TLL, and RML, while CALB is only active on DHC with (+)-enantioselectivity. In most cases, AoC has the highest activity while HiC is significantly more active than other enzymes on 2TBC. The E values of AoC, HiC, TLL, and RML for menthol are 27.8, 16.5, 155, and 125, respectively. HiC has a higher activity (>10-fold) on (-)-2TBC than AoC while they exhibit similar activities on menthol. Docking results reveal that the bulky group adjacent to the hydroxyl group determines the enantioselectivity of AoC, HiC, TLL, and RML. Amino acid residues that dominate the enantioselectivity of these enzymes are AoC’s Phe195 aromatic ring; HiC’s hydrophobic Leu 174 and Ile 169 groups; TLL’s ring structures of Trp89, His258 and Tyr21; and Trp88 for RML. Results of this study highlight that cutinases can provide important advantages relative to lipases for enantioselective transformation, most notably with bulky and sterically hindered substrates.
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Consequently, cutinase active sites can accommodate a variety of rigid and hindered substrates [10]. Cutinases (EC 3.1.1.74) are serine esterases that belong to the α/β hydrolase superfamily [11–13]. Their natural function is hydrolysis of ester bonds in the complex cutin bio-polyester network that lies within the plant cuticle [14,15].
Developing active immobilized enzymes and characterization of their use conditions is critically important prior to initiating studies of enzyme selectivity and substrate specificity in organic media. To this end, physical immobilization by hydrophobic interactions was performed with three well-characterized cutinases (Aspergillus oryzae Cutinase (AoC), Humicola insolens Cutinase (HiC), and Thielavia terrestris Cutinase (TtC)) using Lewatit VP OC 1600 as the macroporous support. We found that immobilization yields >98% were achieved for all three cutinases under the following immobilization conditions: 100 mg/g loading ratio, immobilization buffers of 100 mM phosphate pH 8 (AoC and HiC) and 100 mM acetate pH 5 (TtC), mixing at 150 rpm and 30 °C for 24 h. Among the three cutinases, HiC has the highest tolerance towards solvents of increased polarity while TtC has the highest thermal stability (up to 80 °C) in a bulk reaction system that consists of the reactants butanol and lauric acid. In nonane, these cutinases retain >64% of their activity at 90 °C. Furthermore, kinetic stability (residual activity as a function of time) analysis reveals that the cutinases retain >75% residual activity at 70 °C in 3 h. Moreover, at 80 °C, the kinetic stability of TtC is higher than that of HiC and AoC. Collectively, the results herein set the stage for the in-depth evaluation of these catalysts for selective transformations in organic media.
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Cutinase is an enzyme that catalyses the degradation of insoluble biopolyester cutin, a structural component of plants. This enzyme has some properties of lipase and esterase. Because of its unique nature, it has potential of being an industrially important enzyme. Some of the useful applications of cutinase include hydrolysis of fats and oils, esterification and transesterification reactions. This enzyme is mainly produced by phytopathogenic fungi, but there are several bacteria which are known to produce cutinase. In this article, the production, purification, characterizations, enhancement of activity and stability, immobilization of the enzyme and its applications in various industries have been discussed.

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^☆: Scientific paper No. 5301, project 2001, College of Agriculture Research Center, Washington State University, Pullman, Washington. This work was supported in part by National Science Foundation Grant PCM-74-09351 and by Washington State Tree Fruit Commission.

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Purification and characterization of a novel cutinase from nasturtium (Tropaeolum majus) pollen☆

Abstract

J. Biol. Chem

Arch. Biochem. Biophys

Arch. Biochem. Biophys

Planta

Plant Cell Physiol

Annu. Rev. Plant Physiol

Plant Physiol

Z. Bot

Nature (London)