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

Nyyssölä, Antti; Pihlajaniemi, Ville; Järvinen, Riikka; Mikander, Saara; Kontkanen, Hanna; Kruus, Kristiina; Kallio, Heikki; Büchert, Johanna

doi:10.1016/j.enzmictec.2013.01.005

Cited by 30 publications

(26 citation statements)

References 26 publications

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“…Martinez et al (47) suggested that the low levels of activity of cutinases toward pNP esters of long-chain fatty acids are related to the lack of a large hydrophobic area around the active site, which is present in lipases. To determine if the potential cutinases from A. adeninivorans are true cutinases, PCL was used as a model polyester substrate, because it has been used in previous studies (4,6,16,48,49). The test for a clear zone revealed PCL degradation by Acut1-6hp, Acut2-6hp, and Acut3-6hp and by FsCut-6hp, which served as a positive control.…”

Section: Discussionmentioning

confidence: 99%

Three New Cutinases from the Yeast Arxula adeninivorans That Are Suitable for Biotechnological Applications

Bischoff

Litwińska

Cordes³

et al. 2015

Appl Environ Microbiol

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The genes ACUT1, ACUT2, and ACUT3, encoding cutinases, were selected from the genomic DNA of Arxula adeninivorans LS3. The alignment of the amino acid sequences of these cutinases with those of other cutinases or cutinase-like enzymes from different fungi showed that they all had a catalytic S-D-H triad with a conserved G-Y-S-Q-G domain. All three genes were overexpressed in A. adeninivorans using the strong constitutive TEF1 promoter. Recombinant 6؋ His (6h)-tagged cutinase 1 protein (p) from A. adeninivorans LS3 (Acut1-6hp), Acut2-6hp, and Acut3-6hp were produced and purified by immobilized-metal ion affinity chromatography and biochemically characterized using p-nitrophenyl butyrate as the substrate for standard activity tests. All three enzymes from A. adeninivorans were active from pH 4.5 to 6.5 and from 20 to 30°C. They were shown to be unstable under optimal reaction conditions but could be stabilized using organic solvents, such as polyethylene glycol 200 (PEG 200), isopropanol, ethanol, or acetone. PEG 200 (50%, vol/vol) was found to be the best stabilizing agent for all of the cutinases, and acetone greatly increased the half-life and enzyme activity (up to 300% for Acut3-6hp). The substrate spectra for Acut1-6hp, Acut2-6hp, and Acut3-6hp were quite similar, with the highest activity being for short-chain fatty acid esters of p-nitrophenol and glycerol. Additionally, they were found to have polycaprolactone degradation activity and cutinolytic activity against cutin from apple peel. The activity was compared with that of the 6؋ His-tagged cutinase from Fusarium solani f. sp. pisi (FsCut-6hp), also expressed in A. adeninivorans, as a positive control. A fed-batch cultivation of the best Acut2-6hp-producing strain, A. adeninivorans G1212/YRC102-ACUT2-6H, was performed and showed that very high activities of 1,064 U ml ؊1 could be achieved even with a nonoptimized cultivation procedure.

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Section: Discussionmentioning

confidence: 99%

Three New Cutinases from the Yeast Arxula adeninivorans That Are Suitable for Biotechnological Applications

Bischoff

Litwińska

Cordes³

et al. 2015

Appl Environ Microbiol

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“…Expression of AnCUT2 using P. pastoris resulted in production of the active protein with an enzyme activity of 3.3 U/mL. The advantage of using P. pastoris for foreign protein expression [3,25] over E. coli expression host is the low expression of its native proteins compared to the yield of the recombinant protein. P. pastoris carries out posttranslational modifications that may contribute to enzyme stability and activity [26].…”

Section: Discussionmentioning

confidence: 99%

“…Homology search of the A. niger ATCC 1015 (Al-Tammar et al, unpublished work) and CBS 513.88 revealed the presence of five cutinase encoding genes and one of them, anig5 encoding AnCut5 of A. niger CBS 513.88, was previously reported as being active in acidic pH [3]. In this A. niger were first grown in Potato Dextrose Broth (PDB) in an incubator shaker for two days at 180 to 200 rpm and 28°C.…”

Section: Introductionmentioning

confidence: 99%

“…In the food industry, acidic cutinases may be used as a facilitator to release bioactive compounds from acidic plant materials and to release cutin mono-and oligomers from acidic cutin-rich plant wastes [3]. A cutinase from leafbranch compost [7] and Thielavia terrestris [8] were able to degrade synthetic plastics such as polycaprolactone (PCL) and polyethylene terephthalate (PET) thus applicable in synthetic fiber modification.…”

Section: Introductionmentioning

confidence: 99%

“…Many of A. niger enzymes are generally recognized as safe (GRAS) and several enzymes such as cellulase, α-amylase, β-galactosidase, glucose oxidase, protease, lipase and pectinase are industrially important [2]. Cutinase is one of the extracellular enzymes produced by this fungus [3]. Interestingly, this enzyme is also implicated in the pathogenicity of plant pathogenic fungi to break down plant cell walls releasing cutin monomers [4].…”

Section: Introductionmentioning

confidence: 99%

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Expression and characterization of a cutinase (AnCUT2) from Aspergillus niger

et al. 2016

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Cutin hydrolase (EC 3.1.1.74), an extracellular polyesterase found in pollens, bacteria and fungi, is an efficient catalyst that exhibits hydrolytic activity on a variety of water-soluble esters, synthetic fibers, plastics and triglycerides. Thus, cutinase can be used in various applications such as ester synthesis, bio-scouring, food and detergent industries. Ancut2 is one of five genes encoding cutinases present in the Aspergillus niger ATCC 10574 genome. The cDNA of Ancut2 comprising of an open reading frame of 816 bp encoding a protein of 271 amino acid residues, was isolated and expressed in Pichia pastoris. The partially purified recombinant cutinase exhibited a molecular mass of approximately 40 kDa. The enzyme showed highest activity at 40°C with a preference for acidic pH (5.0-6.0). AnCUT2 showed hydrolytic activity towards various p-nitrophenyl esters with preference towards shorter chain esters such as p-nitrophenyl butyrate (C4). Scanning Electron Microscopy demonstrated that AnCUT2 was capable of modifying surfaces of synthetic polycaprolactone and polyethylene terephthalate plastics. The properties of this enzyme suggest that it may be applied in synthetic fiber modification and fruit processing industries.

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Toward rational thermostabilization of Aspergillus oryzae cutinase: Insights into catalytic and structural stability

et al. 2015

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Cutinases are powerful hydrolases that can cleave ester bonds of polyesters such as poly(ethyleneterephthalate) (PET), opening up new options for enzymatic routes for polymer recycling and surface modification reactions. Cutinase from Apsergillus oryzae (AoC) is promising owing to the presence of an extended groove near the catalytic triad which is important for the orientation of polymeric chains. However, the catalytic efficiency of AoC on rigid polymers like PET is limited by its low thermostability; as it is essential to work at or over the glass transition temperature (Tg) of PET i.e. 70 °C. Consequently, in this study we worked towards the thermostabilization of AoC. Use of Rosetta computational protein design software in conjunction with rational design led to a 6 °C improvement in the thermal unfolding temperature (Tm) and a 10-fold increase in the half-life of the enzyme activity at 60 °C. Surprisingly, thermostabilization did not improve the rate or temperature optimum of enzyme activity. Three notable findings are presented as steps toward designing more thermophilic cutinase: (a) surface salt bridge optimization produced enthalpic stabilization, (b) mutations to proline reduced the entropy loss upon folding and (c) the lack of a correlative increase in the temperature optimum of catalytic activity with thermodynamic stability suggests that the active site is locally denatured at a temperature below the Tm of the global structure.

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

Cited by 30 publications

References 26 publications

Three New Cutinases from the Yeast Arxula adeninivorans That Are Suitable for Biotechnological Applications

Three New Cutinases from the Yeast Arxula adeninivorans That Are Suitable for Biotechnological Applications

Expression and characterization of a cutinase (AnCUT2) from Aspergillus niger

Toward rational thermostabilization of Aspergillus oryzae cutinase: Insights into catalytic and structural stability

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