- Open Access
Pectinase secreted by psychrotolerant fungi: identification, molecular characterization and heterologous expression of a cold-active polygalacturonase from Tetracladium sp.
© The Author(s) 2019
- Received: 8 September 2018
- Accepted: 22 February 2019
- Published: 7 March 2019
Pectinolytic enzymes, which are used in several industries, especially in the clarification process during wine and fruit juice production, represent approximately 10% of the global enzyme market. To prevent the proliferation of undesired microorganisms, to retain labile and volatile flavor compounds, and to save energy, the current trend is to perform this process at low temperatures. However, the commercially available pectinases are highly active at temperatures approximately 50 °C and poorly active at temperatures below 35 °C, which is the reason why there is a constant search for cold-active pectinases. In preliminary studies, pectinolytic activity was detected in cold-adapted yeasts and yeast-like microorganisms isolated from Antarctica. The aim of the present work was to characterize pectinases secreted by these microorganisms and to express the best candidate in Pichia pastoris.
Degradation of pectin by extracellular protein extracellular extracts obtained from 12 yeast cultures were assayed in plates at 4 °C to 37 °C and pH from 5.4 to 7.0, obtaining positive results in samples obtained from Dioszegia sp., Phenoliferia glacialis and Tetracladium sp. An enzyme was purified from Tetracladium sp., analyzed by peptide mass fingerprinting and compared to genome and transcriptome data from the same microorganism. Thus, the encoding gene was identified corresponding to a polygalacturonase-encoding gene. The enzyme was expressed in Pichia pastoris, and the recombinant polygalacturonase displayed higher activity at 15 °C than a mesophilic counterpart.
Extracellular pectinase activity was found in three yeast and yeast-like microorganisms from which the highest activity was displayed by Tetracladium sp., and the enzyme was identified as a polygalacturonase. The recombinant polygalacturonase produced in P. pastoris showed high activity at 15 °C, representing an attractive candidate to be applied in clarification processes in the production of fermented beverages and fruit juices.
Currently, there is a high demand for enzymes used in several industrial applications for food, detergent, paper, textile and synthesis of organic compounds because they are highly efficient and environmentally friendly [1–3]. Additionally, these enzymes constitute a well-established global market projected to reach US$6.3 billion in 2021 [1, 4]. The current trend is to use cold-adapted or cold-active enzymes to decrease the temperature of the industrial processes, allowing energy savings and diminishing their carbon footprint and to manufacture products with better performance at ambient or lower temperatures [5–7]. Approximately 10% of the enzyme market is represented by pectinolytic enzymes [8, 9], which are used in the wine, food, paper and textile industries [9, 10]. The reduction of cloudiness and bitterness of fruit juices and grape must in the juice and wine industries is performed at low temperatures (≤ 15 °C) to prevent the proliferation of undesired microorganisms, to retain the labile and volatile flavor compounds, and to save energy [7, 9]. These requirements have led to the search for pectinases with high performance at lower temperatures but also at low pH, as the pH of fruit juices and grape must be in the range from 2.5 to 3.5 . Currently, the industrially available pectinases are obtained from mesophilic filamentous fungi, mainly from Aspergillus species; however, they work poorly at temperatures ≤ 35 °C . Thus, cold-active enzymes have higher enzymatic activities at lower temperatures than their mesophilic counterparts .
Microorganisms that thrive in cold environments have evolved several adaptations to live under this condition, including the synthesis of cold-active enzymes [14–17]. In particular, cold-adapted fungi secrete cold-active enzymes that hydrolyze the complex compounds available in the environment to use as nutrients [18–21]. The production of pectinases has been reported in cold-adapted bacteria and fungi [11, 22–36]; however, in most of these studies no purification or biochemical characterization of the enzyme were performed. Pectinolytic enzymes, or pectinases, are classified according to their mode of action and to their substrate: polygalacturonases, which are subclassified as endo-polygalacturonases (E.C. 22.214.171.124) and exo-polygalacturonases (E.C. 126.96.36.199); lyases, which are classified into pectate lyases (E.C. 188.8.131.52 and EC. 184.108.40.206) or pectin lyases (E.C. 220.127.116.11); and pectin methylesterases (E.C. 18.104.22.168). It is recommended the use of a combination of different kind of pectinases that degrade different parts of the polymer, to achieve a maximal degradation of pectin in various raw materials . Bacteria produce alkaline pectinases, most frequently polygalacturonases and pectate lyases . In fungi, the production of acidic pectinases has been described, mainly exo- and endo- polygalacturonases . The most frequently used pectinases in industry are the polygalacturonases, which belong to glycosyl hydrolase family 28 (GH28). Although there have been attempts to isolate cold-active pectinases from different sources, to the best of our knowledge, there are no commercially available cold-active pectinases.
In previous work, pectinolytic activity was detected in 12 yeasts and yeast-like microorganisms isolated from soils of King George Island in the sub-Antarctic region . In this work, these microorganisms were further studied to characterize the pectinases secreted by them. Secretion of pectinase was confirmed in three of them, and among these, Tetracladium sp. showed the highest pectinase activity. The enzyme from Tetracladium sp. was purified and analyzed by peptide mass fingerprinting. The peptide sequences were compared to genome and transcriptome data, and the gene, which encodes a polygalacturonase, was identified. The gene was expressed in Pichia pastoris, and the recombinant polygalacturonase displayed higher activity at 15 °C than a mesophilic counterpart.
Strains, plasmid and growth conditions
Microorganism, plasmids and oligonucleotides used in this work
Rhodotorula glacialis (Phenoliferia glacialis) (T8Rg)
Leuconeurospora sp. T17Cd1
Leucosporidiella fragaria (Leucosporidium fragarium)
Rhodotorula glacialis (Phenoliferia glacialis) (T11Rs)
Rhodotorula (Cystobasidium) laryngis
PichiaPink strain 4
P. pastoris strain: ade2, prb1, pep4
PichiaPink strain 2
P. pastoris strain: ade2, pep4
Escherichia coli Top10
F-mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 Δ lacX74 recA1 araD139 Δ(araleu)7697 galU galK rpsL (StrR) endA1 nupG
P. pastoris integrating vector for high-copy expression of a secreted protein
Molecular and biochemical methods
Standard molecular and biochemical procedures, such as plasmid DNA purification, digestion with restriction enzymes, cloning procedures, PCR assays, SDS-PAGE, protein quantification and electrotransformation, were performed according to standard protocols . Protein quantification was made using the BCA Kit Assay (Thermo Scientific, IL, USA). Plasmid and genomic DNAs were purified using the GeneJet Plasmid Miniprep Kit (Thermo Scientific, Waltham, MA, USA) and the Wizard Genomic DNA Purification kit (Promega, WI, USA) according to manufacturer’s instructions.
Extraction and purification of extracellular proteins
Yeast cultures (100–300 mL) at the initial stationary phase of growth were centrifuged at 7000×g for 10 min at 4 °C and filtered through sterile 0.45-μm pore size polyvinylidene fluoride membrane filters (Millipore, Billerica, MA, USA). Ammonium sulfate was added to the cell-free supernatants to reach 80% of saturation to obtain total proteins or in the case of protein fractioning, to reach increasing saturation from 20 to 80%. Samples were incubated at 4 °C for 2 h and centrifuged at 10,000×g for 15 min at 4 °C. The protein pellets were suspended in 2–4 mL of 20 mM potassium phosphate and 150 mM NaCl pH 7.0, and samples were desalted using a HiTrap Desalting column (GE, Schenectady, Nueva York, USA) in AKTA Prime purification equipment (GE). For ion exchange protein purification, the protein sample was loaded onto a DEAE-Sephadex column equilibrated with 50 mM Tris–HCl pH 7.0, proteins were eluted using a NaCl gradient from 0 to 200 mM as the mobile phase at 0.5 mL min−1, and 1 mL fractions were collected. For gel filtration purification, 0.2 mL of concentrated protein extracellular extracts were loaded onto a Superdex 200 Increase 10/300 GL column (GE Healthcare, IL, USA) equilibrated with 20 mM sodium phosphate buffer, using the same buffer as the mobile phase at flow rate of 0.5 mL min−1. In both cases, proteins of the fractions were monitored at an absorbance of 280 nm. The relative molecular weight (rMW) of proteins were calculated from SDS-PAGE by comparison its relative mobilities to that of the proteins standard.
Determination of pectinase activity
For semiquantitative determinations, 100 µL of the protein extracellular extracts was deposited into wells cut into agar plates containing 1% w/v pectin with pH of 5.4, 6.2 or 7.0, adjusted with phosphate-citrate buffer. The plates were incubated at 4 °C, 10 °C, 15 °C, 22 °C, 30 °C and 37 °C for 1 to 5 days, and the appearance of a clear halo around the well indicated pectinase activity. For quantitative determinations, the release of reducing sugars from pectin was quantified using the 3-amino-5-nitrosalicylic acid (DNS) method . Briefly, 50 µL of the protein sample was mixed with 50 µl of 10 mg mL−1 pectin, incubated at 30 °C for 1 h and then 100 µl of DNS solution was added. After incubation at 100 °C for 10 min, the samples were incubated on ice for 5 min, and the absorbance at 540 nm was measured. For comparative purposes, commercial polygalacturonase was used in activity assays.
Peptide mass fingerprinting
Protein extracellular extracts were separated by SDS-PAGE and stained with Coomassie Blue G-250. The protein band of interest was cut from the gel and analyzed using the protein analysis service of Alphalyse (Palo Alto, CA, USA). Briefly, the protein sample was reduced and alkylated with carbamidomethylation and subsequently digested with trypsin. The resulting peptides were spotted onto an anchorchip target for analysis on a Bruker Autoflex Speed MALDI-TOF/TOF instrument. The obtained data were analyzed by Mascot, and results having a score greater than 54 (P < 0.05) were considered statistically significant.
Next-generation Sequencing (NGS)
Cultures of Tetracladium sp. were centrifuged at 7000×g at 4 °C for 10 min. The cell pellets were used for DNA and RNA purification with the Wizard Genomic DNA Purification Kit (Promega, WI, USA) and RiboMinus Yeast Kit (Thermo Fisher, MA, USA), respectively. The quality and quantity of the samples were determined by absorbance at 260 and 280 nm, and those having a 260/280 ratio of 1.7 to 1.9 and a 260/230 ratio > 2 were used for whole genome sequencing by NGS at Macrogen Inc. (Seoul, South Korea) using the Hiseq 2000 platform.
Assemblies, ORFs and gene prediction, annotation and expression level analysis
Assemblies were done with ALLPATHS-LG . RNA-seq reads were mapped to the assembled genome with TopHat software  and STAR aligner . To capture all junctions, the RNA-seq reads were assembled according to Grabherr et al. . The gene model prediction was made with Augustus [45, 46] and Pasa . Functional annotation was performed using the standard BLAST and InterPro databases. CAZY annotation was made using dbCAN  and HMMER3 . ClustalW analysis and gene expression analysis were conducted using Geneious version 10.0.9  and the included plugins.
Alignment, modeling and bioinformatics analysis
Amino acid sequence alignments were conducted using MEGA7 software . The polygalacturonase sequences chosen for the amino acid sequence comparison had a minimum of 50% similarity and 50% coverage, and corresponded to: Venturia nashicola (BAG72101), Colletotrichum fioriniae (EXF76863), Colletotrichum lupini (ABL01533), Colletotrichum simmondsii (KXH46697), Achaetomium sp. (AGR51994), Colletotrichum higginsianum (XP_018155590), Venturia pyrina (BAG72133), Pestalotiopsis fici (XP0 07836731), Talaromyces cellulolyticus (GAM33350), Cadophora sp. (PVH80831), Pseudomassariellvae xata (ORY56346).
Phialocephalasu balpina (CZR53206), Penicillium freii (KUM62405), Lepidopterella palustris (OCK84102), Thielavia arenaria (AIZ95162), Fusarium avenaceum (KIL90067), Neonectria ditissima (KPM736 08), Fusarium venenatum (CEl70336), Penicillium griseoroseum (MF06810), Colletotrichum gloeosporioides (ELA24368), Verticillium alfalfa (XP0 03002875), Verticillium dahlia (XP0 09653008), Diplodia corticola (XP_020130427), Pezoloma ericae (PMD23778), Penicillium camemberti (CRL23357), Fusarium langsethiae (KPA37956), Nectria haematococca (XP_003040641), Meliniomyces bicolor (XP_024742782), Ustilaginomycotina sp. (PWN47943), Talaromycesm arneffei (KFX46954), Bipolaris zeicola (XP_007711986), Penicillium subrubescens (OKO96599), Penicillium brasilianum (OOQ88122). The polygalacturonase model was constructed using the Swiss-model platform , using PDB: 2iq7.1 as the template, which has 91% coverage and 63% identity with the polygalacturonase from Tetracladium sp. For validation of the model structure the programs Verify 3D and ERRAT were used trough the AVES v5.0 server (http://servicesn.mbi.ucla.edu/SAVES/), and the values were 96.43% and 89.13%, respectively.
Heterologous expression of pectinase
The coding sequence of TPG1 identified in this work (see “Results and discussion” section) was analyzed bioinformatically and modified to generate a sequence of 1066 nt that lacks the first 57 nucleotides, which encode the signal peptide. Mly I and Kpn I restriction sites were added at the 5′ and 3′ end, respectively, and the codon usage was optimized to the one from P. pastoris. The modified gene was synthesized by Genescript company (New York, USA), and the cloning and expression of pectinase was performed using the PichiaPink™ Expression System (Invitrogen, CA, USA) according to manufacturers’ instructions. Briefly, synthetic CDS was ligated to a pPinkα-HC vector and transformed into E. coli Top10. The obtained transformants were selected on LB-ampicillin plates, and the presence of recombinant plasmid was confirmed by colony PCR using the primer pair Pectfw/Pectrev (Table 1). Plasmid DNA was purified from selected clones, digested with AflII and transformed into P. pastoris PichiaPink strain 2. Several transformants developed on SD plates were selected, and genomic DNA was extracted and checked by PCR using the primer pair Pectfw/Pectrev. Amplicon-positive clones were grown overnight in BCM supplemented with glycerol at 30 °C and then centrifuged at 1500 g for 5 min. Then, the yeast pellets were suspended in 10 ml of BMMY medium and incubated overnight at 30 °C, and methanol was added to reach 4% v/v final concentration. Ten 100 µL culture aliquots were collected at different times from 0 to 24 h and centrifuged at 1.500 g for 5 min, and cellular pellets were suspended in 50 mM sodium phosphate pH 7.4, 1 mM PMSF, 1 mM EDTA and 5% glycerol, and vortexed for 3 min. Samples were directly analyzed by SDS-PAGE.
Screening, selection and identification of secreted pectinases
Identification of the putative pectinase-encoding gene
Expression, at the transcript level, of putative pectate lyase- and polygalacturonase-encoding genes from Tetracladium sp
Heterologous expression and characterization of TPG1
The polygalacturonase TPG1 was identified in Tetracladium sp., and its expression, determined at the transcriptional and enzyme activity levels, is induced by pectin. The TPG1-encoding gene was successfully expressed in P. pastoris, and a recombinant polygalacturonase highly active at 15 °C was obtained. Furthermore, the feasibility to cultivate P. pastoris in high-cell density fermentations facilitates de the efficient production of pectinase at amount and purity required to be applied in industrial processes such as clarification processes of fermented beverage and fruit juice production. Therefore, the recombinant pectinase described in this work is economically attractive by both its efficient production and its activity at low temperatures allowing energy-saving in the processes.
MC carried out the experiments. MC, JMR and MB contributed to the design of the experiment and discussion of the results. MC, JA, VC and MB drafted the manuscript. All authors read and approved the final manuscript.
The authors declare a conflict of interest. Innovacold S.A. applied for a patent based on the results presented in this article.
Availability of data and materials
The datasets supporting this work are included in the manuscript and additional file.
Consent for publication
Ethics approval and consent to participate
The research was supported by the Fundación para la Innovación Agraria (Grant NoFIA PYT - 2014 – 0030).
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