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

  • Khadijah Ahmed Al-Tammar , Othman Omar , Abdul Munir Abdul Murad and Farah Diba Abu Bakar EMAIL logo
Published/Copyright: March 15, 2016
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Open Life Sciences
From the journal Open Life Sciences Volume 11 Issue 1

Abstract

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.

Keywords:: Serine esterase; heterologous expression; Pichia pastoris; p-nitrophenyl esters; polycaprolactone; polyethylene terephthalate; scanning electron microscopy

1 Introduction

The filamentous Aspergillus niger is a member of the black aspergilla that secrete a massive amount of numerous enzymes in their natural habitat to liberate nutrients. This high secretory capability is extensively used by the fermentation industry for the production of organic acids and enzymes [1]. 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]. Cutinase belongs to the α/β hydroloase fold and serine esterase super-family with the classical Ser, His, and Asp triad [5,6].

Cutinase has been extensively studied due to its potential application in various industrial processes. 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. Cutinases can also be used as fat-based stain removers in detergent and laundry industry [9]. However, their instability at high temperatures and the limitation of their enzymatic activity towards one or two substrates have hindered the enzymes from being commercialized. Thus, continuous screening for efficient cutinases from microorganisms with new potential properties may lead to the practical application of cutinases in such industries [4].

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 paper, we report the isolation, cloning and expression of Ancut2 encoding cutinase from A. niger strain ATCC 10574 in Pichia pastoris. It is thereafter characterized and the gene sequence was analyzed in silico in an effort to determine unique features of the encoded enzyme.

2 Materials and Methods

2.1 Microorganisms, Vectors and Culture Media

The A. niger ATCC 10574 was obtained from the American Type Culture Collections (ATCC), 10801, University Boulevard, Manassas, VA 20110 USA. Escherichia colistrain DH5α (Promega, Madison, WI, USA) was used as the host for plasmid construction and propagation. The pGEM-T Easy vector (Promega) and pPICZαC vector (Invitrogen/Life Technologies, Carlsbad, CA, USA) were used for cloning inE. coli and expression in P. pastoris strain X33, respectively. A low salt Luria-Bertani medium (LB) containing 25 μg/mL Zeocin was used to cultureE. coli transformants. Yeast extract peptone dextrose (YPD) agar supplemented with different concentrations of Zeocin (from 100 μg/mL to 2,000 μg/mL) was used to grow P. pastoris and to screen for multiple integrants. Buffered complex glycerol medium (BMGY), buffered complex methanol medium (BMMY) and YPD medium were prepared following the manufacturer’s manual of the Easy Select™ Pichia Expression Kit (Invitrogen). Polymerase Chain Reaction (PCR) reagent, restriction endonucleases, Wizard® Plus SV minipreps DNA purification system and DNA ligase were from Promega (USA). MEGAquick-spin™ PCR and agarose gel DNA extraction kit were purchased from iNtRON Biotechnology, Jungwon-gu, Seongnam-si Gyeonggi-do, South Korea. Nucleotide sequencing was performed using BigDye terminator version 3.3 cycle sequencing kit (PE Applied Biosystems, Foster City, CA, USA). Prestained protein marker was purchased from New England Biolabs, Hitchin, UK. DNA ladders were purchased from Vivantis (Subang Jaya, Selangor DE, Malaysia). All primers used for DNA amplification and sequencing were purchased from First BASE Laboratories (Seri Kembangan, Selangor, Malaysia). All of the chemicals used were of analytical and molecular grade.

2.2 Growth Conditions, RNA Isolation and cDNA Synthesis

Biomass was obtained following the method of Rubio et al. [10] with small modifications. Briefly, 106 spores/mL of 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. Afterwards, the mycelia were harvested aseptically via filtration, washed with water and transferred into fresh minimal medium (MM) containing 0.05% 16-hydroxyhexadecanoic acid (16-hha) (Sigma-Aldrich, USA). After 24 h of incubation, mycelia were collected using muslin cloth, washed with water and used for RNA isolation. Total RNA was extracted with the Trizol® reagent according to the manufacturer’s instruction (Invitrogen). The cDNA was synthesized using SuperScriptTM III first strand synthesis system using RT-PCR and oligodT primer (Invitrogen). The Ancut2 was amplified using Go Taq polymerase kit. The PCR reaction mixture (20 μL) contained a final concentration of 1x Go Taq® Green Master Mix, 300 ng of template cDNA and 0.5 μM each of forward (Ancut2-F 5′-ATG AAG CTT CCT TAC TTT CTG CTC G-3′) and reverse (Ancut2-R 5′-TTA GAA AAG TGA TGC CAG AGA AGG G-3′) specific primers. Specific primers were synthesized based on the hypothetical protein sequence of A. niger strain ATCC 1015 (GenBank accession number: EHA19281.1). The PCR amplification protocol included an initial de-naturation at 94°C for 3 min followed by 30 cycles of 94°C for 20s, 59°C for 30s and 72°C for 1 min, followed by a final extension at 72°C for 10 min. The amplified cDNA was purified then ligated into pGEMT-Easy vector. This ligation mixture was then used to transform cells ofE. coli DH5α. The positive plasmid clone was confirmed by nucleotide sequencing using T7 and SP6 universal primers.

2.3 Analysis ofAncut2 Sequence

Homology searches were carried out using the NCBI BLAST server [11]. The ExPASy translate tools were used to compute the theoretical molecular weight and to predict the amino acid sequence [12]. The signal peptide was predicted with PSORT II program [13]. The conserved domain and the catalytic active sites contained within the deduced AnCUT2 were predicted with InterPro Scan [14] and Pfam scan (http://www.ebi.ac.uk/Tools/pfa/pfamscan/) [15]. Putative N- and O-glycosylation sites were predicted using NetNGlyc 1.0 [16] and NetOGlyc 4.0 servers [17], respectively. The disulphide bonds of the deduced enzyme were predicted using DiANNA 1.1 web server [18,19]. ClustalW (http://www.ch.embnet.org/software/ClustalW.html) and BoxShade version 3.21 servers were used to compare the sequence with other homologous protein sequences from different fungal sequences obtained from the BLAST results.

2.4 Construction of AnCUT2-pPICZαC and Integration into P. pastoris Genome

The plasmid pGEMT-easy containing the complete Ancut2 cDNA (762 bp) was used as a template for PCR. Forward (ClaI-F 5′-ATC GAT GGA ACG TCA ACT TTC C-3′) and reverse (XbaI-F 5′-TCT AGA TTG AAA AGT GAT GCC AGA-3′) primers containing restriction site sequence of ClaI and XbaI (underlined), respectively, were used to amplify Ancut2 without its signal secretion sequence and stop codon. The PCR product was digested with XbaI and ClaI restriction endonucleases and ligated into pPICZαC vector at the same restriction sites resulting in AnCUT2-pPICZαC (4,362 bp) construct. Transformation of P. pastoris X-33 cells was achieved using 10 μg of PmeI-linearized plasmid recombinant construct as described by the manufacturer (Invitrogen). The transformants were then plated on YPDS agar supplemented with various concentrations of Zeocin® (500, 1,000 and 2,000 μg/mL) to select colonies with different copy numbers. Colony PCR [20] using 5′-AOX1 and 3′-AOX1 universal primers and P. pastoris transformed cells as the source of DNA template was conducted to verify the single crossover recombination at the AOX1 locus of the P. pastoris genome.

2.5 Recombinant AnCUT2 Expression

Colonies from each concentration of Zeocin were picked for enzyme production and that with the maximum secretion levels of AnCUT2 were chosen for further analyses. The expression of Ancut2 in P. pastoris was directed by the inducible strong AOX1 promoter. The transformant was grown overnight in 100 mL BMGY in 1 L baffled flasks at 28°C and 240 rpm until the culture reached an OD600 of 2 to 6. The cells were transferred into BMMY production medium and grown for three days. A final concentration of 2% (v/v) absolute methanol was added every 24 h to maintain induction of cutinase expression in P. pastoris. Next, the culture was centrifuged for 5 min at 3,077 x g and the supernatant was collected. This supernatant was partially purified and concentrated using Vivaspin™ centrifugal device with a molecular weight cut-off of 10 kDa (Vivascience, Germany). AnCUT2 expression was verified by SDS-PAGE (12% polyacrylamide) [21] and western blot analyses using mouse anti His-tag monoclonal antibodies (Novagen, USA) and HRP-conjugated anti-mouse antibodies (Promega).

2.6 Enzyme Assay

Protein concentration was measured according to the Bradford [22] method using bovine serum albumin (BSA) as the standard. Cutinase activity was assayed according to Kumar et al. [23] using colorimetric methods with p-nitrophenyl caprate (pNPC) as the substrate with some modifications. The enzymatic analysis was carried out in 50 mM citrate buffer pH 5.0 and the total reaction mixture of 100 μL contained 1 μg enzyme and 1 mM pNPC. This was incubated at 25°C for 10 min. A volume of 100 μL of 0.1 M Na2CO3 was added and the liberation of p-nitrophenol was measured at 405 nm. Controls containing heat-inactivated enzyme and/or buffer with substrate were also assayed. One unit of enzyme is defined as 1 μmol of p-nitrophenol liberated from the substrate per min under standard assay conditions.

2.7 Characterization of AnCUT2

2.7.1 Effect of pH and temperature

The optimum pH of cutinase was determined using 50 mM glycine-HCl buffer (pH 2.0 to 3.0), citrate buffer (3.0 to 6.0), phosphate buffer (6.0 to 7.0), Tris (7.0 to 9.0) and glycine-NaOH buffer (9.0 to 10.0). In order to determine pH stability, cutinase was pre-incubated separately in the above-mentioned buffers at 30°C without the substrate for 1 h. The residual cutinase activity was then assessed using the standard assay procedure. In order to assess the effect of temperature on cutinase activity, the cutinase activity was assayed at temperatures between 25 and 70°C. Cutinase was incubated in an optimal pH buffer at various temperatures (25-70°C) without the substrate for 1 h to determine the thermostability of the enzyme. After the incubation, the tubes were cooled for at least 15 min and the residual enzyme activity was estimated using standard procedures.

2.7.2 Effect of metal ions and reagents on cutinase activity

Metal ions and reagents were each added separately to the reaction mixture containing 1 μg enzyme in the optimum buffer, at a final concentration of 1.0 and 10.0 mM. After 1 h of incubation at the optimal temperature, residual enzyme activity was assayed. The activity of the control (without additives) was taken as 100%.

2.7.3 Substrate specificity

Substrate specificity of the partially purified enzyme was determined using p-nitrophenyl palmitate (pNPP C16), p-nitrophenyl myristate (pNPM C14), p-nitrophenyl laurate (pNPL C12), p-nitrophenyl caprate (pNPC C10), p-nitrophenyl valerate (pNPV C5) and p-nitrophenyl butyrate (pNPB C4) at a final concentration of 1 mM. The enzyme activity was measured under standard assay condition.

2.7.4 Enzymatic treatment of polyesters

Enzymatic degradation of PCL and PET pellets (Sigma-Aldrich, St. Louis, MO, USA) was performed according to Sulaiman et al. [7] and Donelli et al. [24] with slight modifications. Both PCL and PET pellets were incubated with 1% (w/v) SDS for 30 min at 50°C and then washed with distilled water. PCL and PET pellets with initial weights of 30 and 20 mg respectively, were added into 2 mL microfuge tubes that contained 1 mL of 50 mM sodium phosphate buffer at pH 6. Partially purified enzyme (50 μg) was added and incubated at 25°C for 7 days with shaking at 100 rpm. After incubation, the pellets were rinsed with water and ethanol, then dried in a fume hood overnight. Surface morphologies of PCL and PET pellets were examined using Scanning Electron Microscopy (SEM) (VPSEM LEO 1450, LEO Co. Ltd, England). PCL and PET pellets treated with supernatant of untransformed P. pastoris were used as negative controls.

3 Results

3.1 Cloning and Sequence Analysis

The Ancut2 cutinase encoding gene from A. niger ATCC 10574 was amplified by PCR and this yielded a distinct single band of approximately 800 bp. The resulting fragment was then cloned into the pGEM-T Easy cloning vector and its sequence was verified. The sequenced Ancut2 cDNA showed that it comprises of an open reading frame (ORF) of 816 nucleotides encoding a protein of 271 amino acid residues with a theoretical molecular weight (Mw) of 27.67 kDa and a deduced isoelectric point (pI) of 4.73. PSORT II program revealed possible cleavage sites between 16-17, hence, it is deduced that the mature proteins contained 255 residues with a calculated Mw and pIof 26.04 and 4.64, respectively. The deduced enzyme was predicted to contain 33 sites for O-glycosylation while there were no sites for N-glycosylation. The protein is predicted to have three disulphide bonds between cysteine residues (Cys): 37-115, 63-76 and 177-184. BlastP searches showed that AnCUT2 has 99%, 99% and 97% identity to a hypothetical protein of A. niger ATCC 1015 (GenBank accession number: EHA19281.1), cutinase2 from A. niger CBS 513.88 (NCBI Reference Sequence: XP_001394015.1) and A. kawachii cutinase (GenBank accession number: GAA84164.1), respectively. Multiple sequence alignment (Figure 1) shows the active-site residues Ser126, Asp181 and His194, and that the Gly-X-Ser-X-Gly motif was conserved in all homologous proteins. A serine-rich region at the C-terminal was also observed in AnCUT2. The nucleotide sequence of Ancut2 was deposited into the GenBank database under accession number KR064617.

Figure 1 Amino acid sequence alignment of AnCUT2 cutinase from A. niger (this study) and other fungal cutinases. Af: A. flavus (GenBank accession no. EED52785.1); An: A. nidulans (GenBank accession no. ABF50887.1); Ao: A. oryzae (GenBank accession no. XP_001817153.1): Cg: Colletotrichum gloeosporioides (GenBank accession no. AAL38030.1); Fs: Fusarium solani (GenBank accession no. AAA33335.1). Black and grey backgrounds indicate identical and similar residues, respectively. (*): Asterisks indicate conserved identical residues; (.): Full stops indicate residues with weak similarity; (#): indicates amino acids involved in the catalytic triad; (=): indicates four conserved cysteines in all cutinases; (+): indicates two cysteines that are conserved in cutinases from the genus Aspergillus. The penta-peptide motif (G-Y-SQ-G) is shown in bold and green. The serine-rich region comprises of residues shown in bold and lowercase letters (s).
Figure 1

Amino acid sequence alignment of AnCUT2 cutinase from A. niger (this study) and other fungal cutinases. Af: A. flavus (GenBank accession no. EED52785.1); An: A. nidulans (GenBank accession no. ABF50887.1); Ao: A. oryzae (GenBank accession no. XP_001817153.1): Cg: Colletotrichum gloeosporioides (GenBank accession no. AAL38030.1); Fs: Fusarium solani (GenBank accession no. AAA33335.1). Black and grey backgrounds indicate identical and similar residues, respectively. (*): Asterisks indicate conserved identical residues; (.): Full stops indicate residues with weak similarity; (#): indicates amino acids involved in the catalytic triad; (=): indicates four conserved cysteines in all cutinases; (+): indicates two cysteines that are conserved in cutinases from the genus Aspergillus. The penta-peptide motif (G-Y-SQ-G) is shown in bold and green. The serine-rich region comprises of residues shown in bold and lowercase letters (s).

3.2 Transformation of P. pastoris with AnCUT2

Integration of the expression cassette carrying Ancut2 into the P. pastoris genome has produced more than 40 transformants. Ten transformant colonies were randomly picked and re-streaked on YPDS plates containing different concentrations of Zeocin®. All transformants grew on 500 μg/mL Zeocin®, seven colonies withstood 1000 μg/mL Zeocin®, while the number of survivors declined to two upon transfer to 2000 μg/mL Zeocin®. The results reflected possible multi-copy integration of the expression cassette into P. pastoris genome (Pichia Expression Kit manual, Invitrogen). PCR analysis using 5′- and 3′-AOX1 primers of positive Pichia transformants supported that genomic integration event took place in the colonies. The positive clones produced two distinct bands, one at 1.2 kb, which confirms the expected size of the vector harboring the insert and the other at 2.2 kb, which is the size of the amplified indigenous AOX1 in the genome of P. pastoris (Figure 2).

Figure 2 Verification of the plasmid integration via colony PCR in P. pastoris X-33. M: 1 kb DNA ladder; Lane 1: Untransformed P. pastoris shows one band of 2.2 kb that corresponds to the size of the AOX1 in the P. pastoris genome; Lane 2: P. pastoris transformed with pPICZαC vector shows two bands with approximately 2.2 kb and 600 bp that correspond to the indigenous AOX1 in the host and the AOX1 in the vector, respectively; Lanes 3 to 7: P. pastoris transformed with pPICZαC vector carrying Ancut2 shows two bands with approximately 2.2 kb and 1.4 kb that correspond to the AOX1 in the host genome and Ancut2 within the AOX1 region, respectively
Figure 2

Verification of the plasmid integration via colony PCR in P. pastoris X-33. M: 1 kb DNA ladder; Lane 1: Untransformed P. pastoris shows one band of 2.2 kb that corresponds to the size of the AOX1 in the P. pastoris genome; Lane 2: P. pastoris transformed with pPICZαC vector shows two bands with approximately 2.2 kb and 600 bp that correspond to the indigenous AOX1 in the host and the AOX1 in the vector, respectively; Lanes 3 to 7: P. pastoris transformed with pPICZαC vector carrying Ancut2 shows two bands with approximately 2.2 kb and 1.4 kb that correspond to the AOX1 in the host genome and Ancut2 within the AOX1 region, respectively

3.3 Expression of AnCUT2

In order to produce the highest concentration of recombinant cutinase, the expression was optimized by determining the optimum day for harvesting, medium for biomass production and medium for methanol induction expression. Recombinant AnCUT2 was well expressed under methanol induced conditions and day 3 was optimal for protein production. The SDS-PAGE showed a protein band with a molecular mass of ~40 kDa, which was further verified by western-blot analysis (Figure 3). However, the molecular mass of the target protein was higher than expected at about 40 kDa (the theoretical molecular weight of AnCUT2 expressed in P. pastoris and taking into account the His-tag is 29 kDa).

Figure 3 SDS-PAGE (A) and Western blot (B) profiles of partially purified recombinant AnCUT2 in P. pastoris X-33. M: Protein molecular mass marker; Lane 1: Control, untransformed P. pastoris; Lane 2: Control, P. pastoris transformed with pPICZαC vector; Lane 3: P. pastoris transformed with pPICZαC vector carrying Ancut2.
Figure 3

SDS-PAGE (A) and Western blot (B) profiles of partially purified recombinant AnCUT2 in P. pastoris X-33. M: Protein molecular mass marker; Lane 1: Control, untransformed P. pastoris; Lane 2: Control, P. pastoris transformed with pPICZαC vector; Lane 3: P. pastoris transformed with pPICZαC vector carrying Ancut2.

3.4 Characterization of A. niger Recombinant Cutinase AnCUT2

The optimum pH of AnCUT2 was 5.0-6.0 (Figure 4A) where this enzyme displayed its highest activity (27 U/mL). Interestingly, this enzyme was stable across a wide pH range of 2-10 and retained more than 60% of its activity after 1 h of incubation at very acidic and basic pHs (Figure 4B). The enzyme exhibited its maximum activity (42 U/mL) at 40°C (Figure 4C). The enzyme is stable up to 40°C and retained more than 80% of its activity when incubated at 40oC for 60 min (Figure 4D). However, at 50°C, almost 60% of its activity was lost over the 60 min period.

Figure 4 Enzyme activity and stability profiles of AnCUT2 in varying pH and temperature. A: Activity profile to determine pH optimum; B: Stability profile in varying pH; C: Activity profile to determine temperature optimum; D: Stability profile in varying temperature. Each point represents the mean ± standard error from two independent experiments. The experiments were carried out at least twice and the average values are indicated with error bars. The highest cutinase activity was taken as 100% which is equivalent to 27.5 U/mg.
Figure 4

Enzyme activity and stability profiles of AnCUT2 in varying pH and temperature. A: Activity profile to determine pH optimum; B: Stability profile in varying pH; C: Activity profile to determine temperature optimum; D: Stability profile in varying temperature. Each point represents the mean ± standard error from two independent experiments. The experiments were carried out at least twice and the average values are indicated with error bars. The highest cutinase activity was taken as 100% which is equivalent to 27.5 U/mg.

The effects of metal ions showed that at 1 mM, most of the metal ions had negligible effect on the enzyme activity (Figure 5). However, denaturing agents sodium dodecyl sulphate (SDS) and phenylmethylsulfonyl fluoride (PMSF) at 1 mM strongly inhibited AnCUT2 activity. Ba2+, Zn2+, Ni2+ and Cu2+ ions had inhibitory effects (30% to 50% inhibition) at 10 mM towards AnCUT2. Dithiothreitol (DTT), EDTA and urea however, had no effect on AnCUT2 activity at 1 mM.

Figure 5 Effect of reagents and metal ions on the AnCUT2 cutinase activity. The experiment was carried out at least twice and the average values are indicated with error bars. Sample without reagents or metals added to the enzyme solutions (None) was taken as 100% residual activity where 100% relative activity is equivalent to 27.5 U/mg.
Figure 5

Effect of reagents and metal ions on the AnCUT2 cutinase activity. The experiment was carried out at least twice and the average values are indicated with error bars. Sample without reagents or metals added to the enzyme solutions (None) was taken as 100% residual activity where 100% relative activity is equivalent to 27.5 U/mg.

The AnCUT2 exhibited variable hydrolytic activity towards various p-nitrophenyl esters (Figure 6). The short carbon chain length substrates pNPB (C4) and pNPV (C5) were more efficiently hydrolyzed than other esters (102.6 and 90.7 U/mg, respectively).

Figure 6 Relative activity profile of AnCUT2 towards various p-nitrophenyl substrates. The activity on each substrate was expressed relative to the activity of pNPB, which was taken as 100% (equivalent to 102.6 U/mg). The experiment was carried out at least twice and the average values are indicated with error bars
Figure 6

Relative activity profile of AnCUT2 towards various p-nitrophenyl substrates. The activity on each substrate was expressed relative to the activity of pNPB, which was taken as 100% (equivalent to 102.6 U/mg). The experiment was carried out at least twice and the average values are indicated with error bars

3.5 Enzymatic Treatment of Synthetic Polymers

We examined the ability and effect of the recombinant cutinase on degradation of PCL and PET under SEM by comparing micrographs of the samples (treated with AnCUT2) with the negative control (treated with protein expressed from untransformed P. pastoris). Pellets incubated with AnCUT2 were corroded and pitted (Figure 7C, D) as compared to the smooth surface of negative controls (Figure 7A, B).

Figure 7 SEM photograph (1,000 x magnification) of the surface of PCL (polycaprolactone) and PET (polyethylene terephthalate) pellets. (A) PCL in protein of untransformed P. pastoris (negative control). (B) PET in protein of untransformed P. pastoris (negative control). (C) PCL treated with AnCUT2. (D) PET treated with AnCUT2. The scale bar is 10.0 μm.
Figure 7

SEM photograph (1,000 x magnification) of the surface of PCL (polycaprolactone) and PET (polyethylene terephthalate) pellets. (A) PCL in protein of untransformed P. pastoris (negative control). (B) PET in protein of untransformed P. pastoris (negative control). (C) PCL treated with AnCUT2. (D) PET treated with AnCUT2. The scale bar is 10.0 μm.

4 Discussion

As far as we are aware, this is the first report on the heterologous expression and characterization of AnCUT2. 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]. One of the most common posttranslational modifications is glycosylation (N- and O-linked glycan). Glycosylation increases protein mass [27] and this may have resulted in the mass increase of AnCUT2 expressed in P. pastoris. Although the calculated pIof AnCUT2 is 4.73, its activity was maximum at near neutral pH and this enzyme was stable over a wide range of pH. Guo et al. [28] reported that glycosylation influenced the isoelectric point as well as optimal pH of phytase where it was shown that the maximum activity of glycosylated phytase was at pH 5.0, while the activity peak was shifted to pH 2.5 by deglycosylation. It is yet to be determined but glycosylation may be responsible for the increase in AnCUT2 molecular mass. AnCUT2 was shown to retain more than 60% of its relative activity after 1 h incubation at different pHs. Although it is a mesophilic enzyme, AnCUT2 exhibits remarkable stability after 1 h incubation at 40°C compared to G. cingulata cutinase [29] and F. solani [30]. Similar data have been previously observed in some fungal cutinases [30,31]. This thermostability observed may be due to serine (Ser) residues that represent the highest percentage (15.2%) of AnCUT2 composition. Serine is a polar residue that contains a hydroxyl group on its side chain that takes part in the formation of hydrogen bonds [27,32]. Lin et al. [33] reported that the replacement of Ala by Ser enhanced fumarase enzymatic activity and thermostability. Also, the substitution of Lysine (Lys) by Ser at position 347 was responsible for enhancing the thermostability of β-amylase [32]. A serine-rich region was also identified in the cutinase of A. nidulans (GenBank accession no. ABF50887.1), A. niger (GenBank accession no. CAK41954.1) and A. flavus (GenBank accession no. EED52785.1) at the C-terminal [5]. In addition, the thermotolerance of AnCUT2 might be due to the presence of three disulphide bonds in the protein (Figure 1). A. oryzae cutinase is more stable than F. solani cutinase and this enhanced thermotolerance might be attributed to the presence of three bonds while the latter possesses only two [30].

The activity of cutinase in the presence of anionic surfactants was diminished and this result is similar with previous studies [34-38]. However, enzyme activity is unaffected in the presence of the chelating agent EDTA at a concentration of 1 mM, suggesting that the enzyme does not require metal ions for its activity or stability. Interestingly, AnCUT2 showed a tolerance to urea and DTT as the activity neither significantly decreased nor enhanced although urea is known to alter water structure leading to disruption of the structure of protein, while DTT reduces Cys side chains leading to a reduction of intramolecular disulphide bonds [27]. PMSF almost completely inhibited the enzyme activity by 90% at a concentration of 1 mM, which supported that Ser is involved in the formation of the active site of AnCUT2 cutinase. This observation is similar to studies on cutinase from Pseudomonas cepacia [39] but contradicts to those on G. cingulata cutinase [40]. Cutinases from various sources showed a different degree of sensitivity to metals and chemical agents

The highest esterase activity of AnCUT2 is towards short (C4, C5), followed by long (C14, C16), then medium (C10, C12) carbon chain pNP esters. This result is similar to that observed for the cutinase from T. terrestris which has a preference towards short chain fatty acyl esters (C4) [8]. Activity towards short chain substrates may reflect structural features of the active site of the cutinase [30]. Fungal cutinases show different p-nitrophenyl substrate specificities; A. oryzae cutinase prefers pNPV [30], Sirococcus conigenus [41], T. harzianum [10] and F. solani [30] cutinases prefer pNPA (C2), while G. cingulata cutinase prefers pNPC (C8), followed by pNPM (C14), and pNPL (C12) [39].

AnCUT2 showed the capability to modify the surface of the PCL and PET synthetic polyesters. Synthetic polyesters (PET), most commonly used polymer in beverage containers, food packaging and electronic industry [42], represents a total of 50% of the global market for textile fibers [43]. An increased hydrophilicity of the fiber is required to facilitate the dyeing process of these fibers using enzymatic treatments as opposed to using harsh chemicals. Cutinase has been reported to free carboxylic acid and hydrophilic hydroxyl groups hence enhancing their wetting ability [44]. Thus, the partial hydrolyzing properties observed in AnCUT2 towards such synthetic polyesters may also be exploited in synthetic fiber modification.

5 Conclusion

A cutin hydrolase AnCUT2 from A. niger was cloned and expressed in P. pastoris. It has potential application in various industries due to its hydrolytic ability, stability across pH, relative thermostability, lack of cofactor requirements and resistance to denaturing agents. Thus, such properties make AnCUT2 a good candidate in the processes of esterification, fiber surface modification and food industries.

Acknowledgements

The authors would like to acknowledge the Ministry of Education, Malaysia, for providing funding for this research through the Grant, ERGS/1/2012/STG08/UKM/02/16 and the School of Biosciences and Biotechnology, UKM, for providing facilities to carry out the work published. The authors would also like to thank Shuhaila Mat Sharani and Suhaila Sulaiman from Malaysia Genome Institute for very helpful discussion in using bioinformatics tools. The first author also wishes to thank the Government of the Kingdom of Saudi Arabia for awarding the fellowship for doctoral research.

  1. Conflict of interest: The authors declare that there is no conflict of interests regarding the publication of this paper.

References

[1] Pel H.J., de Winde J.H., Archer D.B., Dyer P.S., Hofmann G., Schaap P.J., et al., Genome sequencing and analysis of the versatile cell factory Aspergillus niger CBS 513.88, Nat. Biotechnol., 2007, 25, 221-231.10.1038/nbt1282Search in Google Scholar

[2] Schuster E., Dunn-Coleman N., Frisvad J.C., Van Dijck P.W., On the safety of Aspergillus niger-a review, Appl. Microbiol. Biotechnol., 2002, 59, 426-435.10.1007/s00253-002-1032-6Search in Google Scholar

[3] Nyyssölä A., Pihlajaniemi V., Järvinen R., Mikander S., Kontkanen H., Kruus K., et al., Screening of microbes for novel acidic cutinases and cloning and expression of an acidic cutinase from Aspergillus niger CBS 513.88, Enzyme. Microb. Technol., 2013, 52, 272-278.10.1016/j.enzmictec.2013.01.005Search in Google Scholar

[4] Chen S., Su L., Chen J., Wu J., Cutinase: characteristics, preparation, and application, Biotechnol. Adv., 2013, 31, 1754-1767.10.1016/j.biotechadv.2013.09.005Search in Google Scholar

[5] Castro-Ochoa D., Peña-Montes C., González-Canto A., Alva-Gasca A., Esquivel-Bautista R., Navarro-Ocaña A., et al., AnCUT2, an extracellular cutinase from Aspergillus nidulans induced by olive oil, Appl. Biochem. Biotechnol., 2012, 166, 1275-1290.10.1007/s12010-011-9513-7Search in Google Scholar

[6] Egmond M.R., de Vlieg J., Fusarium solani pisi cutinase, Biochimie, 2000, 82, 1015-1021.10.1016/S0300-9084(00)01183-4Search in Google Scholar

[7] Sulaiman S., Yamato S., Kanaya E., Kim J.J., Koga Y., Takano K., et al., Isolation of a novel cutinase homolog with polyethylene terephthalate-degrading activity from leaf-branch compost by using a metagenomic approach, Appl. Environ. Microbiol., 2012, 78, 1556-1562.10.1128/AEM.06725-11Search in Google Scholar PubMed PubMed Central

[8] Yang S., Xu H., Yan Q., Liu Y., Zhou P., Jiang Z., A low molecular mass cutinase of Thielavia terrestris efficiently hydrolyzes poly (esters), Appl. Environ. Microbiol., 2013, 40, 217-226.10.1007/s10295-012-1222-xSearch in Google Scholar PubMed

[9] Dutta K., Sen S., Veeranki V.D., Production, characterization and applications of microbial cutinases, Process. Biochem., 2009, 44, 127-134.10.1016/j.procbio.2008.09.008Search in Google Scholar

[10] Rubio M.B., Cardoza R.E., Hermosa R., Gutiérrez S., Monte E., Cloning and characterization of the Thcut1 gene encoding a cutinase of Trichoderma harzianum T34, Curr. Genet., 2008, 54, 301-312.10.1007/s00294-008-0218-6Search in Google Scholar PubMed

[11] Altschul S.F., Gish W., Miller W., Myers E.W., Lipman D.J., Basic local alignment search tool, J. Mol. Biol., 1990, 215, 403-41010.1016/S0022-2836(05)80360-2Search in Google Scholar

[12] Gasteiger E., Hoogl and C., Gattiker A., Wilkins M.R., Appel R.D., Bairoch A., The proteomics protocols handbook, Walker J.M., Ed., Humana Press Inc, Totowa, New Jersey, 571-607, 200510.1385/1-59259-890-0:571Search in Google Scholar

[13] Nakai K., Horton P., PSORT: a program for detecting sorting signals in proteins and predicting their subcellular localization, Trends. Biochem. Sci., 1999, 24, 34-3510.1016/S0968-0004(98)01336-XSearch in Google Scholar

[14] Zdobnov E.M., Apweiler R., InterProScan-an integration platform for the signature-recognition methods in InterPro, Bioinformatics., 2001, 17, 847-84810.1093/bioinformatics/17.9.847Search in Google Scholar

[15] Mistry J., Bateman A., Finn R.D., Predicting active site residue annotations in the Pfam database. BMC. Bioinformatics., 2007, 8, 298.10.1186/1471-2105-8-298Search in Google Scholar

[16] Gupta R., Brunak S., Prediction of glycosylation across the human proteome and the correlation to protein function, Pac. Symp. Biocomput., 2002, 7, 310-322.Search in Google Scholar

[17] Steentoft C., Vakhrushev S.Y., Joshi H.J., Kong Y., Vester-Christensen M.B., Schjoldager K.T., et al., Precision mapping of the human O-GalNAc glycoproteome through SimpleCell technology, EMBO. J., 2013, 32, 1478-1488.10.1038/emboj.2013.79Search in Google Scholar

[18] Ferrè F., Clote P., DiANNA: A web server for disulfide connectivity prediction, Nucleic. Acids. Res., 2005, 33, 230-232.10.1093/nar/gki412Search in Google Scholar

[19] Ferrè F., Clote P., DiANNA 1.1: An extension of the DiANNA web server for ternary cysteine classification, Nucleic. Acids. Res., 2006, 34, 182-185.10.1093/nar/gkl189Search in Google Scholar

[20] Ayra-Pardo C., Martinez C.G., De La Riva G.A., A single-step screening procedure for Pichia pastoris clones, by PCR, Biotecnologia. Aplicada., 1998, 15, 173-175.Search in Google Scholar

[21] Laemmli U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature., 1970, 227, 680-685.10.1038/227680a0Search in Google Scholar

[22] Bradford M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 1976, 72, 248-254.10.1016/0003-2697(76)90527-3Search in Google Scholar

[23] Kumar S., Kikon K., Upadhyay A., Kanwar S.S., Gupta R., Production, purification, and characterization of lipase from thermophilic and alkaliphilic Bacillus coagulans BTS-3, Protein. Expr. Purif., 2005, 41, 38-44.10.1016/j.pep.2004.12.010Search in Google Scholar PubMed

[24] Donelli I., Freddi G., Nierstrasz V.A., Taddei P., Surface structure and properties of poly-(ethylene terephthalate) hydrolyzed by alkali and cutinase, Polym. Degrad. Stab., 2010, 95, 1542-1550.10.1016/j.polymdegradstab.2010.06.011Search in Google Scholar

[25] Kwon M.A., Kim H.S., Yang T.H., Song B.K., Song J.K., High-level expression and characterization of Fusarium solani cutinase in Pichia pastoris, Protein. Expr. Purif., 2009, 68, 104-109.10.1016/j.pep.2009.06.021Search in Google Scholar PubMed

[26] Cereghino J.L., Cregg J.M., Heterologous protein expression in the methylotrophic yeast Pichia pastoris, FEMS Microbiol. Rev., 2000, 24, 45-66.10.1111/j.1574-6976.2000.tb00532.xSearch in Google Scholar PubMed

[27] Price N.C., Nairn J., Exploring proteins: a student’s guide to experimental skills and methods, Oxford University Press Inc, New York, 2009.Search in Google Scholar

[28] Guo M., Hang H., Zhu T., Zhuang Y., Chu J., Zhang S., Effect of glycosylation on biochemical characterization of recombinant phytase expressed in Pichia pastoris, Enzyme. Microb. Technol., 2008, 42, 340-345.10.1016/j.enzmictec.2007.10.013Search in Google Scholar

[29] Chin I.S., Abdul Murad A.M., Mahadi N.M., Nathan S., Abu Bakar F.D., Thermal stability engineering of Glomerella cingulata cutinase, Protein. Eng. Des. Sel., 2013, 26, 369-375.10.1093/protein/gzt007Search in Google Scholar PubMed

[30] Liu Z., Gosser Y., Baker P.J., Ravee Y., Lu Z., Alemu G., et al., Structural and functional studies of Aspergillus oryzae cutinase: enhanced thermostability and hydrolytic activity of synthetic ester and polyester degradation, J. Am. Chem. Soc., 2009, 131, 15711-15716.10.1021/ja9046697Search in Google Scholar PubMed PubMed Central

[31] Baker P.J., Poultney C., Liu Z., Gross R., Montclare J.K., Identification and comparison of cutinases for synthetic polyester degradation, Appl. Microbiol. Biotechnol., 2012, 93, 229-240.10.1007/s00253-011-3402-4Search in Google Scholar PubMed

[32] Ma Y., Evans D., Logue S., Langridge P., Mutations of barley β-amylase that improve substrate-binding affinity and thermostability, Mol. Genet. Genomics., 2001, 266, 345-352.10.1007/s004380100566Search in Google Scholar PubMed

[33] Lin W., Chan M., Goh L.L., Sim T.S., Molecular basis for thermal properties of Streptomyces thermovulgaris fumarase C hinge at hydrophilic amino acids R163, E170 and S347, Appl. Microbiol. Biotechnol., 2007, 75, 329-335.10.1007/s00253-006-0822-7Search in Google Scholar PubMed

[34] Creveld L.D., Meijberg W., Berendsen H.J., Pepermans H.A., DSC studies of Fusarium solani pisi cutinase: consequences for stability in the presence of surfactants, Biophys. Chem., 2001, 92, 65-75.10.1016/S0301-4622(01)00187-9Search in Google Scholar

[35] Verripsab T., Duboc P., Visser C., Sagt C., From gene to product in yeast: production of fungal cutinase, Enzyme Microb. Technol., 2000, 26, 812-818.10.1016/S0141-0229(00)00176-9Search in Google Scholar

[36] Brissos V., Melo E.P., Martinho J.M., Cabral J.M., Biochemical and structural characterisation of cutinase mutants in the presence of the anionic surfactant AOT, Biochim. Biophys. Acta., 2008, 1784, 1326-1334.10.1016/j.bbapap.2008.04.017Search in Google Scholar

[37] Goncalves A.M.D., Aires-Barros M.R., Cabral J., Interaction of an anionic surfactant with a recombinant cutinase from Fusarium solani pisi: a spectroscopic study, Enzyme. Microb. Technol., 2003, 32, 868-879.10.1016/S0141-0229(03)00054-1Search in Google Scholar

[38] Pocalyko D.J., Tallman M., Effects of amphipaths on the activity and stability of Fusarium solani pisi cutinase, Enzyme. Microb. Technol., 1998, 22, 647-651.10.1016/S0141-0229(98)00013-1Search in Google Scholar

[39] Dutta K., Krishnamoorthy H., Venkata Dasu V., Novel cutinase from Pseudomonas cepacia NRRL B 2320: purification, characterization and identification of cutinase encoding genes, J. Gen. Appl. Microbiol., 2013, 59, 171-184.10.2323/jgam.59.171Search in Google Scholar PubMed

[40] Seman W.M.K., Bakar S.A., Bukhari N.A., Gaspar S.M., Othman R., Nathan S., et al., High level expression of Glomerella cingulata cutinase in dense cultures of Pichia pastoris grown under fed-batch conditions, J. Biotechnol., 2014, 184, 219-228.10.1016/j.jbiotec.2014.05.034Search in Google Scholar PubMed

[41] Nyyssölä A., Pihlajaniemi V., Häkkinen M., Kontkanen H., Saloheimo M., Nakari-Setälä T., Cloning and characterization of a novel acidic cutinase from Sirococcus conigenus, Appl. Microbiol. Biotechnol., 2014, 98, 3639-3650.10.1007/s00253-013-5293-zSearch in Google Scholar PubMed

[42] Zimmermann W., Billig S., Enzymes for the biofunctionalization of poly(ethylene terephthalate), Adv. Biochem. Eng. Biotechnol., 2011, 125, 97-120.10.1007/10_2010_87Search in Google Scholar PubMed

[43] Silva C.M., Carneiro F., O’Neill A., Fonseca Luis P., Cabral J.S.M., Guebitz G., et al., Cutinase-A new tool for biomodification of synthetic fibers, J. Polym. Sci. Pol. Chem., 2005, 43, 2448-2450.10.1002/pola.20684Search in Google Scholar

[44] Alisch-Mark M., Herrmann A., Zimmermann W., Increase of the hydrophilicity of polyethylene terephthalate fibres by hydrolases from Thermomonospora fusca and Fusarium solani f. sp. pisi, Biotechnol. Lett., 2006, 28, 681-685.10.1007/s10529-006-9041-7Search in Google Scholar PubMed

Received: 2015-8-5
Accepted: 2016-1-6
Published Online: 2016-3-15
Published in Print: 2016-1-1

© 2016 Khadijah Ahmed Al-Tammar et al., published by De Gruyter Open.

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

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https://doi.org/10.1515/biol-2016-0004
Keywords for this article
Serine esterase; heterologous expression; Pichia pastoris; p-nitrophenyl esters; polycaprolactone; polyethylene terephthalate; scanning electron microscopy
Creative Commons
BY-NC-ND 3.0

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