Accepted Manuscript Title: High-Level Expression of a Novel Protease-Resistant ␣-Galactosidase from Thielavia terrestris Authors: Yu Liu, Shaoqing Yang, Qiaojuan Yan, Jun Liu, Zhengqiang Jiang PII: DOI: Reference:
S1359-5113(18)30169-7 https://doi.org/10.1016/j.procbio.2018.05.025 PRBI 11361
To appear in:
Process Biochemistry
Received date: Revised date: Accepted date:
1-2-2018 24-5-2018 31-5-2018
Please cite this article as: Liu Y, Yang S, Yan Q, Liu J, Jiang Z, High-Level Expression of a Novel Protease-Resistant ␣-Galactosidase from Thielavia terrestris, Process Biochemistry (2018), https://doi.org/10.1016/j.procbio.2018.05.025 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
High-Level Expression of a Novel Protease-Resistant α-Galactosidase from Thielavia terrestris
Yu Liua, Shaoqing Yanga, Qiaojuan Yanb, Jun Liua, Zhengqiang Jianga, * a
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Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food
Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China College of Engineering, China Agricultural University, Beijing 100083, China
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b
*
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Corresponding author:
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Zhengqiang Jiang
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Tel.: +86 10 62737689; Fax: +86 10 82388508; E-mail address:
[email protected] (Z.
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Graphical abstract
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Jiang)
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Research highlights A novel α-galactosidase gene (TtGal27A) was firstly cloned from Thielavia species. TtGal27A was highly expressed in Pichia pastoris.
TtGal27A exhibited strong resistance to various proteases
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TtGal27A displayed good thermal and pH stability.
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TtGal27A could hydrolyze raffinose family oligosaccharides and galactomannans.
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Abstract
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A novel α-galactosidase gene from Thielavia terrestris (designated TtGal27A) was cloned and expressed at high level in Pichia pastoris. The enzyme shared highest identity (40%) with
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glycoside hydrolase family 27 α-galactosidases from Penicillium purpurogenum and
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Penicillium simplicissimum among the characterized proteins. Extracellular expression of TtGal27A was up to 4402.1 U/mL. TtGal27A was purified and had a specific activity of 752.0 U/mg towards p-nitrophenyl-α-galactopyranoside. It showed optimal pH and temperature of
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4.5 and 60°C, respectively, and exhibited 36.0% of its maximum activity at pH 2.5. The enzyme was stable within pH 3.0–9.0 and at temperatures of up to 50°C after 30 min incubation. TtGal27A hydrolyzed raffinose family oligosaccharides and various galactomannans. In addition, the enzyme showed good protease resistance (>80% of initial activity retained)
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towards α-chymotrypsin, papain, proteinase K and trypsin. These properties of TtGal27A make it potentially applicable in the food and feed industries.
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Keywords: α-Galactosidase · Thielavia terrestris · High-level expression · Protease resistance
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1. Introduction
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α-Galactosidase (EC 3.2.1.22, α-D-galactoside galactohydrolase) is a glycosidase that removes the α-linked D-galactose residue from the non-reducing end of galactose
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oligosaccharides, galactomannans, galactolipids and glycoproteins. Usually, α-galactosidases
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are classified into two groups according to whether they can hydrolyze galactomannan [1]. Based on sequence similarity, the α-galactosidases are divided into glycoside hydrolase (GH) families 4, 27, 36, 57, 97 and 110 [2].
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α-Galactosidases have many applications in galactomannan modification [1], biomass
treatment [3,4], α-galacto-oligosaccharide synthesis [1,5] and other industrial processes. Legume products are important ingredients in food and feed. However, the raffinose family oligosaccharides (RFOs), such as raffinose and stachyose, can cause flatulence in humans and
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other monogastric animals [1]. α-Galactosidases can hydrolyze RFOs in foods and feeds. Protease-resistant α-galactosidases are more suitable because they can tolerate the proteases that are added exogenously or secreted endogenously by the stomach and intestines [6–8]. Moreover, α-galactosidases which have good acid stability and activity can perform well in
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acidic environments such as that in human and animal stomachs [7,8], providing an extra benefit for their application in food and feed industries. Therefore, α-galactosidases with both
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protease resistance and adaptability to acidic pH are in high demand. Many α-galactosidases
have been characterized in recent years, including those from Bacillus megaterium [9],
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Flavobacterium sp. TN17 [10], Pleurotus citrinopileatus [11], Pleurotus djamor [12],
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Pontibacter sp. HJ8 [13], Rhizomucor miehei [14], Sphingomonas sp. JB13 [15], and
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Streptomyces sp. S27 [16]. However, only a very few have both strong protease resistance and
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acid adaptability.
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The high cost of α-galactosidases, due to low production levels, is another essential obstacle for their application in industry. Heterologous expression has been used to improve
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enzyme production levels [17]. Among the various protein-expression systems, Pichia
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pastoris is excellent due to high cell density fermentation, the strong and tightly controlled methanol-inducible alcohol oxidase 1 promoter, and the ability to secrete large amounts of protein into the medium [17]. Many industrial enzymes have been expressed to high levels in
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P. pastoris.15 The yeast has also been employed to express α-galactosidases [4,17–24]. Although in recent years, high expression levels of 1299 and 1953.9 U/mL have been achieved for α-galactosidases from Aspergillus niger [18] and R. miehei CAU432 [22], respectively, these levels need to be further improved to lower the enzyme's cost.
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Thermophilic fungi are important sources of industrial enzymes due to their good thermostability [25,26]. Thielavia terrestris is an important thermophilic fungus that secretes many cellulases and hemicellulases [25]. However, there are no reports on cloning or heterologous expression of α-galactosidase from T. terrestris. In this study, the
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recombinant enzyme was characterized and assessed for protease resistance.
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α-galactosidase of T. terrestris was cloned and expressed at high level in P. pastoris. The
2. Materials and methods
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2.1. Strain and reagents
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T. terrestris CAU709 was preserved at the China General Microbiological Culture
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Collection Center under the number 6233. The Pichia expression kit was purchased from
Japan).
Guar
gum,
locust
bean
gum,
synthetic
substrates
including
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(Tokyo,
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Invitrogen (Carlsbad, CA). Melibiose, raffinose and stachyose were purchased from TCI
p-nitrophenyl-α-galactopyranoside (pNPαGal) and other nitrophenyl glycosides were from
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Sigma-Aldrich (St. Louis, MO). Tara gum was kindly provided by Kang Ben Biological
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Technology (Zhengzhou, China). Fenugreek gum and cassia gum were purchased from Mahesh Agro Food Industries (Barmer, Rajasthan, India). All other chemicals in this work were of analytical grade unless otherwise stated.
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2.2. Cloning of an α-galactosidase gene from T. terrestris The cultivation of T. terrestris CAU709 was carried out as described by Xu et al [27]. Total RNA was prepared from mycelia with Trizol (Invitrogen). First-strand cDNA synthesis was performed with the Prime-Script First Strand cDNA Synthesis Kit (Takara Bio Inc., Otsu,
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Shiga, Japan). The coding sequence was amplified by LA Taq DNA polymerase (Takara Bio Inc.) with GC Buffer II using cDNA as the template. Primers for polymerase chain reaction (PCR)
amplification
of
TtGal27AF
(5′-ATGAAGTCTGCACTGAGCACT-3′)
and
TtGal27AR (5′-TCACGCCTTGGTGACCTTG-3′) were designed according to the glycoside
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hydrolase family 27 protein (THITE_2124844 of GenBank No CP003014.1) of genome sequence of T. terrestris NRRL 8126. The following protocol was used for PCR:
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pre-denaturation at 94°C for 5 min; 35 cycles of 94°C for 30 s, 51°C for 30 s, and 72°C for 1
min; post-extension for 5 min at 72°C followed by holding at 4°C. The product with the
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expected size was purified, cloned into pMD19-T vector (Takara Bio Inc.) and verified by
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2.3. Sequence analysis of the α-galactosidase
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sequencing.
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The putative amino acid sequence of TtGal27A was deduced by the Translate Tool of
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ExPASy [28]. The SignalP server [29] was used to predict the signal peptide. Homology search of the putative protein was performed at NCBI BLAST [30], and multiple-sequence alignment
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of the translated sequence was performed with Clustal Omega [31]. NCBI CD-search [32] was
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employed to find conserved domains. Theoretical molecular weight and pI were predicted by ExPASy ProtParam tool [28]. NetNGlyc 1.0 [33] and NetOGlyc 4.0 [34] were used to predict N- and O-linked glycosylation, respectively.
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2.4. Transformation of P. pastoris and expression in shake-flask The coding sequence of TtGal27A without the signal peptide was subcloned into P. pastoris expression vector pPIC9K using the EcoRI and NotI sites. Electroporation of P. pastoris GS115 cells was conducted according to Invitrogen's manual using 10 μg of
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SacI-linearized recombinant expression vector. Putative multiple-copy transformants were screened on YPD [1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) glucose] plates containing 1.0, 2.0, 4.0 and 6.0 mg/mL G418 (also known as Geneticin) as per Invitrogen’s protocol, since the Tn903 kanr gene in integration
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cassette of pPIC9K plasmid can endow P. pastoris with dose-independence G418-resistance [35]. The colonies on G418 plates were picked and initially inoculated into 10 mL buffered
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minimal glycerol complex medium BMGY and then transferred into 10 mL buffered minimal methanol complex medium BMMY in shake flasks; 50 μL of methanol was added at 24-h
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intervals to maintain induction. Both the BMGY and BMMY medium were prepared
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according to the Invitrogen’s guidelines. After 72 h, the culture broth was centrifuged at
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12,000 × g for 10 min. The supernatant was collected and the enzyme activity was determined.
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The strain showing the highest enzyme activity was selected for high cell-density
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fermentation. 2.5. High cell-density fermentation
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High cell-density fermentation of the recombinant yeast was carried out in 5-L fermentor
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(BIOTECH-5BG, BXBIO, Shanghai, China) with an initial medium volume of 1.5 L. Fermentation medium preparation and basic process control were performed according to Invitrogen’s manual unless otherwise stated. During the whole process, the temperature, air
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flow and pressure were kept at 30°C, 4 L/min and 0.04 MPa, respectively. After the cell wet weight reached 300 g/L, agitation rate was changed to 800 rpm and the induction was initiated according to Invitrogen’s guidelines. Methanol feed rate was set at 5.5 mL/h per liter of initial fermentation volume after 4 h induction, and finally increased to 6.6 mL/h per liter of initial
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fermentation volume after 72 h induction with dissolved oxygen fluctuating between 10% and 30%. Culture broth samples were drawn every 24 h and centrifuged at 10,000 × g for 3 min. The supernatants were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, and determinations of enzyme activity and protein content. Cell wet
2.6. Purification and deglycosylation of the recombinant α-galactosidase
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weight was measured by weighing the sediments after centrifugation.
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The culture broth was centrifuged at 12,000 × g at 4°C for 10 min to remove yeast cells.
The supernatant was dialyzed against 50 mM diethanolamine (DEA)-HCl buffer pH 9.0
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overnight. For anion-exchange chromatography, a Q-Sepharose Fast Flow (GE Healthcare,
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Uppsala, Sweden) column (1.0 × 10 cm) was pre-equilibrated with the same buffer and loaded
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with the supernatant. Unbound proteins were washed with equilibration buffer (50 mM
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DEA-HCl buffer pH 9.0) to optical absorbance at 280 nm below 0.05. The column was eluted
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with a linear gradient, from 0 to 0.5 M, of NaCl in the equilibration buffer at a flow rate of 1 mL/min. The fractions with α-galactosidase activity were collected and purity was checked by
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SDS-PAGE [36]. Endo H (New England Biolabs, Ipswich, MA) was used to remove N-linked
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glycan of purified TtGal27A. De-O-glycosylation was performed with O-glycosidase and neuraminidase (New England Biolabs, Ipswich, MA) according to the reagent manufacture’s manual. After deglycosylation, the reaction mixtures were applied to SDS-PAGE analysis.
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2.7. Molecular weight determination by gel filtration Molecular weight of the native protein was determined by loading the sample on a Sephacryl S200-HR (GE Healthcare) gel-filtration column (1 × 40 cm). The column was pre-equilibrated with 50 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer pH 6.5
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containing 150 mM NaCl at a flow rate of 0.8 mL/min. The protein markers were α-chymotrypsinogen A from bovine pancreas (25.6 kDa), albumin from chicken egg white (44.3 kDa), albumin bovine V (68.0 kDa), phosphorylase b from rabbit muscle (97.2 kDa) and alcohol dehydrogenase from Saccharomyces cerevisiae (141.0 kDa).
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2.8. Enzyme activity and protein content Enzyme activity was assayed according to Katrolia et al [14]. Briefly, the activity was
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measured by adding 50 μL suitably diluted enzyme solution to 200 μL of 5 mM pNPαGal. The assay was performed in 50 mM citrate buffer pH 4.5 at 60°C for 10 min; 750 μL of 2 M
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Na2CO3 was added to terminate the reaction. The released p-nitrophenol was quantified by
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spectrometric method at 410 nm. One unit of enzyme activity was defined as the amount of
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enzyme liberating l μmol p-nitrophenol per minute under the given conditions. The Lowry
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method [37] was employed to determine the protein content.
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2.9. Effects of pH and temperature on enzyme activity and stability The optimal pH of α-galactosidase was investigated at 50°C in 50 mM various buffers in
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the pH range of 2.0–10.0. pH stability of the enzyme was determined by incubating it in
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different buffers at 50°C for 30 min, followed by cooling in ice-water bath. The residual activity was determined by standard assay and relative activity was calculated using untreated samples as control (100%).
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The optimal temperature of the enzyme was determined by standard assay at various
temperatures from 20°C to 85°C. Thermostability was measured by assessing the residual activity after incubating the enzyme over the same temperature range for 30 min. To further investigate the half-lives at optimal pH, the enzyme was incubated at 50, 55, 60 and 65°C.
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Samples were withdrawn after incubation for 15, 30, 60, 120 and 240 min. Residual activity was determined and the relative activity was calculated as above. 2.10. Effects of metal ions, sugars and various chemicals on enzyme activity The enzyme was incubated with 1 mM of metal ions or various chemicals at 50°C for 30
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min. Then the mixture was immediately cooled in ice-water bath and residual activity was determined by standard assay. For the control, the enzyme was incubated without metal ions
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or chemicals and the activity was set at 100%. To assess the effects of sugars on α-galactosidase activity, the activity of 1 mg/mL enzyme solution was determined by standard
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method in the presence of galactose, glucose, mannose, melibiose, raffinose, stachyose, and
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sucrose. The final concentrations of the sugars in the reaction mixture were 5 mM, 10 mM, 20
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mM, 50 mM and 100 mM, and the reaction mixture without sugars was used as control
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(100%).
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2.11. Substrate specificity, kinetic and inhibition constants of the recombinant α-galactosidase To test specificity of the enzyme towards synthetic chromogenic substrates such as p-nitrophenyl-β-galactopyranoside
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pNPαGal,
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o-nitrophenyl-β-D-galactopyranoside (pNPαGlu),
(oNPβGal),
(pNPβGal),
p-nitrophenyl-α-D-glucopyranoside
p-nitrophenyl-α-D-mannopyranoside
(pNPαMan)
and
p-nitrophenyl-α-D-xylopyranoside (pNPαXyl) were used in the standard activity assay. In the
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case of oligosaccharides (raffinose and stachyose) and galactomannans (locust bean gum, cassia gum, tara gum, guar gum and fenugreek gum), 100 μL enzyme solution was added to 400 μL prewarmed 50 mM sodium citrate buffer pH 4.5 containing 5 mM oligosaccharide or 0.5% (w/v) galactomannan and incubated for 10 min. The reducing sugar content was measured by
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3,5-dinitrosalicylic acid method [38] using galactose as the standard. One unit of enzyme activity was defined as the amount of enzyme that liberates 1 μmol reducing sugar equivalents to galactose per minute under the given conditions. Specific activity towards melibiose was determined by measuring the glucose released using glucose oxidase–peroxidase kit (Biosino,
forming 1 μmol glucose per minute under the given conditions.
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Beijing, China). One unit of α-galactosidase activity was defined as the amount of enzyme
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For the determination of the kinetic parameters, the enzymatic reactions were carried out
at 60°C in citrate buffer pH 4.5 for 5 min. The substrate concentrations ranged from 0.7-2.7
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mM for pNPαGal, 20–80 mM for melibiose and 135–535 mM for raffinose. The Ki value of
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galactose was determined using 5 mM galactose and 3.2–12.8 mM pNPαGal. To obtain the Ki
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value of melibiose, the reaction mixture contained 50 mM melibiose and 1.6–6.6 mM
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pNPαGal. Nonlinear curve fitting was used to calculate the kinetics parameters and inhibition
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constants using “GraFit” software (Erithacus Software Limited, UK). 2.12. Protease resistance of the α-galactosidase
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The reaction cocktail contained 1 mg/mL of different proteases and 0.1 mg/mL
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α-galactosidase; the control group contained only 0.1 mg/mL α-galactosidase. After incubation at 37°C for 30 and 60 min, samples were withdrawn and the residual activities were determined. For α-chymotrypsin (Sigma-Aldrich C4129), proteinase K (Calbiochem 539480), subtilisin A
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(Sigma-Aldrich P5380) and trypsin (Sigma-Aldrich P5380), the incubation pH was 7.5 (100 mM Tris-HCl buffer). For papain (Sigma-Aldrich P3250), the incubation pH was 7.0 (100 mM Tris-HCl buffer); pH 2.0 (100 mM glycine-HCl buffer) was adopted for pepsin (Sigma-Aldrich P7000) due to its optimum at acidic pH.
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3. Results 3.1. Cloning and sequence analysis of T. terrestris α-galactosidase A 1209-bp gene was successfully amplified from the cDNA of T. terrestris CAU709,
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encoding a protein of 402 amino acids with a signal peptide of 20 amino acids (Fig. 1). The molecular mass and pI of the deduced protein (TtGal27A) predicted by ExPASy ProtParam
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were 41.6 kDa and 6.12, respectively. In silico prediction revealed that TtGal27A has four presumed N- and O-linked glycosylation sites (Fig. 1).
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NCBI BLAST search showed that TtGal27A shared the highest identity of 40% with the
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characterized α-galactosidases from Penicillium purpurogenum (also known as Talaromyces
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purpureogenus) ATCC MYA 38 (GenBank accession no. AKH40275.1) and Penicillium
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simplicissimum VTT-D-78090 (GenBank accession no. CAA08915.1), followed by
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α-galactosidases from A. niger N400 (39%, GenBank accession no. CAB46229.1), Neosartorya fischeri P1 (39%, GenBank accession no. AGV79321.1) and Umbelopsis vinacea
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(39%, GenBank accession no. BAA33931.1) (Fig. 2). Moreover, NCBI CD-search showed a
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GH 27 family domain in TtGal27A. These results suggested that TtGal27A is a novel member of the GH 27 family α-galactosidase. Based on the multiple-sequence alignment, Glu-150 and Glu-220 are the presumed nucleophile and proton donor, respectively (Fig. 2) [39,40]. The
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gene (designated TtGal27A) was submitted to GenBank under accession no. KX909886.1. 3.2. High cell-density fermentation of recombinant P. pastoris One recombinant P. pastoris strain growing on a 6 mg/mL G418 plate showed the highest enzyme activity in the shake-flask. This strain was further subjected to high cell-density
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fermentation in a 5-L fermentor. After 216 h methanol induction, extracellular protein concentration, enzyme activity and cell wet weight gradually reached peaks of 8.3 g/L, 4402.1 U/mL and 522.2 g/L, respectively (Fig. 3A). SDS-PAGE showed broad and smeared bands of the extracellular proteins (Fig. 3B).
The
recombinant
protein
was
purified
through
single-step
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3.3. Purification and molecular weight determination of the recombinant α-galactosidase anion-exchange
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chromatography (Table 1). The molecular mass of TtGal27A determined by SDS-PAGE was
57.5 kDa, which is much higher than the predicted 41.6 kDa. After deglycosylation with Endo
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H, the molecular mass of the denatured protein decreased to 45.3 kDa (Fig. 4), demonstrating
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that the protein is highly N-glycosylated. No obvious band shift of TtGal27A was observed
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after de-O-glycosylation, indicating that no or slight O-glycosylation occurred in TtGal27A, or
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O-liked glycans was not detected. The native molecular mass, as determined by gel filtration,
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was 120.1 kDa, suggesting the homodimeric nature of TtGal27A. 3.4. Biochemical characterization of TtGal27A
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The optimal pH of TtGal27A was 4.5 in citrate buffer. At pH 2.5, TtGal27A still exhibited
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36.0% of its maximal activity (Fig. 5A). With respect to pH stability, the enzyme remained over 90.0% activity in the pH range of 3.0–9.0 (Fig. 5B) after incubation at 50°C for 30 min. TtGal27A showed maximal activity at 60°C (Fig. 5C), and over 90.0% activity remained after
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incubation at 50°C or lower for 30 min (Fig. 5D). The calculated half-lives of TtGal27A at 50°C, 55°C, 60°C and 65°C were 1214.3 min, 331.6 min, 231.8 min and 64.0 min, respectively (Fig. 5E). Most of the metal ions had little or no effect on TtGal27A activity (>90% activity
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remained), except for Ag+, Fe2+ and Hg2+ (data not shown). Both Ag+ and Fe2+ moderately inhibited TtGal27A activity (88.1% and 86.6% activity remained, respectively), and Hg2+ strongly inhibited the enzyme activity to 16.5%. After incubation with SDS, cetyltrimethyl ammonium bromide and thiol-reducing reagents such as β-mercaptoethanol and dithiothreitol,
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more than 95% of TtGal27A's original activity remained. Chelating agent ethylenediamine tetracetic acid (EDTA) had no effect on TtGal27A activity (data not shown). Among all of the
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chemicals tested, only Triton X-100 activated TtGal27A significantly to 114.6%.
The activity of TtGal27A in the presence of various sugars was also investigated (data not
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shown). Galactose (5 mM) reduced TtGal27A to 54.8% of its original activity level. Only 6.6%
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activity was detected when 100 mM galactose was added to the reaction cocktail. Melibiose
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also inhibited the enzyme's activity. The residual activity of TtGal27A was 56.1% in the
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marginal (data not shown).
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presence of 100 mM melibiose. The impacts of other sugars on the activity of TtGal27A were
3.5. Substrate specificity, kinetic and inhibition constants of TtGal27A
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The specific activities of TtGal27A towards various substrates are given in Table 2. The
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enzyme was most active towards pNPαGal. TtGal27A did not attack the glyosidic bond in the β-linked chromogenic derivatives of galactose or α-linked p-nitrophenol derivatives of glucose, mannose or xylose. Among the oligosaccharides, TtGal27A favored hydrolysis of melibiose,
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followed by raffinose and stachyose (Table 2). The specific activity of TtGal27A towards galactomannans was positively related to their molar ratio of mannose to galactose (M/G ratio). The kinetic constants are presented in Table 3. TtGal2A showed the highest affinity (Km = 1.4 mM) and catalytic efficiency (kcat/Km = 0.719 mM-1 s-1) towards pNPαGal. The Km and
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kcat/Km value were 39.5 mM and 0.0013 mM-1 s-1 for melibiose, and 268.0 mM and 0.0005 mM-1 s-1 for raffinose. The Michaelis constant and Vmax value observed in presence of inhibitors, namely Km(obs) and Vmax(obs), were 6.52 mM and 1093.2 μmol min-1 mg-1 for galactose, and 3.27 mM and 1074.0 μmol min-1 mg-1 for melibiose. Both the Km(obs) values were higher than the Km
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value in the absence of inhibitors and the Vmax(obs) values were almost the same as the Vmax of 1050 μmol min-1 mg-1. These results demonstrated that galactose and melibose were the
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competitive inhibitors of TtGal27A. The calculated Ki of galactose and melibiose were 1.39 and 38.46 mM, respectively.
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3.6. Protease resistance of TtGal27A
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As shown in Fig. 6, TtGal27A was highly stable when it was co-incubated with
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α-chymotrypsin, proteinase K, subtilisin A and trypsin. Its activity remained above 90%, even
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after 60 min incubation. TtGal27A also exhibited good stability against papain, with residual
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activities of 92.8% and 83.3% after incubation for 30 min and 60 min, respectively (Fig. 6). For pepsin, although the respective residual activities were 29.4% and 14.1%, the corresponding
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activity ratios with and without pepsin were 91.0% and 89.9%, respectively (Fig. 6). This
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phenomenon indicated that loss of TtGal27A activity can be mainly attributed to the acidic pH, and that pepsin degradation has only slight effect on the enzyme activity.
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4. Discussion
Enzymes from thermophilic fungi have great potential in industrial applications because of their good thermostability [25,26]. Many α-galactosidases from thermophilic fungi have been heterologously expressed and characterized in recent years [4,14,19,22] In this study, we
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expressed and characterized a novel α-galactosidase from T. terrestris. The enzyme showed the highest identity with the α-galactosidases from P. purpurogenum and P. simplicissimum belonging to the GH 27 family among the characterized proteins [39,41]. Hence, TtGal27A is a novel member of the GH 27 family.
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Heterologous expression is an efficient method to produce enzymes on a large scale. Many hosts have been used for heterologous expression of α-galactosidases (Table 4), and P. Previous report indicates that
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pastoris is among the most successful (Table 4).
α-galactosidase is expressed by P. pastoris at higher levels and with higher specific activity
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than with other expression systems [1]. In this study, much higher extracellular enzyme
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activity and protein concentration were achieved relative to previous reports (Table 4),
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surpassing the previously highest expression and activity of A. niger α-galactosidase by P.
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pastoris (4402.1 U/mL vs. 1299 U/mL) [18]. Expression level and productivity of P.
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pastoris-expressed TtGal27A are much higher than those of other α-galactosidases, making this α-galactosidase more favorable for industrial applications.
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The broad smeared bands obtained by SDS-PAGE of the fermentation broth supernatant
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were caused by glycosylation, which also occurs in other P. pastoris-expressed proteins [42,43]. In this study, TtGal27A molecular mass did not decrease obviously after de-O-glycosylation (Fig. 4). Similar phenomena have also been found in some other proteins
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heteroexpessed in P. pastoris [44–46]. Considering the obvious change of molecular mass of TtGal27A (from 57.5 kDa to 45.3 kDa), N-glycosylation should be the major glycosylation form of TtGal27A. It is usually difficult for glycosylated proteins to bind SDS [47], potentially causing relative higher stability in the presence of 1 mM SDS [13,16,48].
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Moreover, many reports have proved that glycosylation can enhance the resistance to proteolytic degradation of heterologous proteins expressed in P. pastoris [49–52] One mechanism which has been confirmed is the steric hindrance caused by glycan making the cleavage site inaccessible to the protease catalytic center [51,52]. In this study, TtGal27A was
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highly glycosylated and exhibited strong resistance (>90% activity) to various proteases. Therefore, the high glycosylation may contribute to protease-resistance of TtGal27A with the
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mechanism stated above.
The acid pH optimum (pH 4.5) of TtGal27A is similar to that of some other fungal
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α-galactosidases [4,14,19,41,53,54]. At pH 2.5, TtGal27A still displayed 36.0% of its
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maximal activity (Fig. 5A), whereas most other α-galactosidases show weak or no activity at
A
pH 3.0 [9,11–14,22,48]. In addition, TtGal27A was more stable at acidic pH than the
M
α-galactosidases from Streptomyces sp. S27 [16], Rhizopus sp. F78 ACCC 3079 [55],
ED
Gibberella sp. F75 [56], R. miehei CAU432 [14] and A. niger [7]. To use as feed additives, α-galactosidases that are more active and stable in the acidic gastric fluid are preferred [7,8].
PT
This acid adaptability might facilitate the application of TtGal27A in environments with
CC E
acidic pH. Furthermore, TtGal27A showed both a higher optimal temperature and better thermostability than many α-galactosidases [9,11,12,16,21,48,53,54]. Overall, due to its pH and temperature features, TtGal27A has good potential for application in industrial processes,
A
especially for the food and feed industry. The specific activity of TtGal27A towards pNPαGal was higher than that of many GH 27
α-galactosidases [4,13,19,23,24], but lower than specific activity of the enzymes from Termitomyces eurrhizus [57] and Tricholoma matsutake [58]. For raffinose, TtGal27A
17
exhibited comparable or higher specific activity than that of many other GH 27 α-galactosidases [13,23,56]. Substrate specificity (Table 2) suggests that TtGal27A prefers to hydrolyze the smaller oligosaccharide substrates. This property distinguishes it from most other GH 27 family α-galactosidases [4,12,13,19]. Regarding to the kinetic constants (Table
IP T
3), TtGal27A showed the highest affinity and catalytic efficiency towards pNPαGal, which is similar to most of α-galactosidase [9,11,12,59], followed by melibiose and raffinose.
SC R
Although the substrate affinity is relatively lower (higher Km value), the Vmax value is higher than many fungal α-galactosidases [12,60].
U
The competitive inhibitory effects of galactose have been found in many other fungal
N
GH 27 family α-galactosidases. The galactose Ki value of TtGal27A was lower than rAga27A
A
from Talaromyces leycettanus JCM12802 (19 mM) [19], ABGI from Agaricus bisporus (6.0
M
mM) [12], α-Gal1 from Talaromyces emersonii (2.77 mM) [24], but higher than the
ED
α-galactosidases such as PCGI from Pleurotus citrinopileatus (0.92 mM) [11] and α-Gal A from Penicillium canescens (1.0 ± 0.3 mM) [61]. The main inhibition type of melibiose is
PT
competitive inhibition and most of the α-galactosidases show lower Ki value than TtGal27A
CC E
[11,54]. Trisaccharide raffinose and tetrasaccharide stachyose did not inhibit the activity of TtGal27A. The different inhibition ability of these sugars might be explained by the steric hindrances which increase in parallel with the sugar molecular size in binding to the enzyme.
A
Most of GH 27 family α-galactosidases showed hydrolysis activity towards
galactomannans except for several cases [13,53,57]. For all of characterized GH 27 family α-galactosidases, there was no relevance between specific activity and galactomannan substrate M/G ratio. Some GH 27 family α-galactosidases from Irpex lacteus [60],
18
Talaromyces emersonii [24] and Agaricus bisporus [54] showed higher specific activity towards higher M/G substrate (i.e. locust bean gum or tara gum) than lower M/G substrate (i.e. guar gum). However, the GH 27 family α-galactosidases from Cyamopsis tetragonolobus (guar) [3], Tricholoma matsutake [58] and rice (α-Gal II) [62] showed reverse substrate
IP T
preference. Besides, several GH 27 family α-galactosidases such as α-Gal III from rice [62] and PCGI from Pleurotus citrinopileatus [11] showed similar specific activities towards
SC R
locust bean gum and guar gum. Most of GH 36 family α-galactosidases cannot hydrolyze
galactomannans [9,22,63] except several GH 36 family α-galactosidases [3,10,64], which
U
show higher specific activity towards locust bean gum than guar gum.
N
In the previous reports, only two or three kinds of galactomannan substrates (mostly
A
locust bean gum and guar gum) were used. However, it may be insufficient to reflect the
M
correlation between the substrate M/G ratio and the specific activity. Hence, in this study, five
ED
kinds of galactomannan substrate with M/G ratio ranging from 1.0 to 4.0 were used to investigate the TtGal27A’s hydrolysis ability towards different M/G ratio substrates (Table 2).
PT
TtGal27A exhibited higher specific activity towards galactomannan with higher M/G ratio.
CC E
The Trichoderma reesei α-galactosidase also prefer to hydrolyze higher M/G ratio substrate (locust bean gum) than or lower M/G ratio substrate (guar gum). Kim and Penner [65] put forward the opinion that the main factor influences the galactomannan substrate preference is
A
the extent of steric hindrance brought out by the side chains. The specific activity of TtGal27A towards various galactomannans with different M/G ratios could be explained by this assumption. Moreover, the hydrolysis activity towards various kind of galactomannans endow TtGal27A with potentials in gel rheological properties modification or antinutrient
19
factor removal since galactomannans can forming gels in food industry [66] and have antinutritive effect in feed industry [67,68]. Protease-resistant enzymes are more suitable for the treatment of agri-products in combination with proteases [6]. Moreover, protease-resistant enzymes perform better in the
IP T
gut environment, and are therefore more applicable for the food and feed industries [7,8]. TtGal27A was more stable in the presence of proteinase K than the α-galactosidases from
SC R
Agaricus bisporus [54], Bacillus megaterium [9], P. djamor [12], P. citrinopileatus [11], Rhizopus sp. F78 ACCC 30795 [55], and Gibberella sp. F75 [56]. For α-chymotrypsin,
U
subtilisin A and trypsin, the residual activities of TtGal27A were comparable to or higher than
N
those of most reported protease-resistant α-galactosidases [9,12,14,55,56]. Pepsin-resistant
A
enzymes retain their hydrolysis ability in the intestine [8]. Among the previously reported
M
α-galactosidases, only that from Bispora sp. MEY-1 showed pepsin resistance [23]. In this
ED
study, TtGal27A exhibited considerable resistance to pepsin (Fig. 6), conferring a further advantage of TtGal27A for food and feed applications.
PT
In conclusion, a novel α-galactosidase gene from T. terrestris was cloned and expressed
CC E
at high level in P. pastoris. TtGal27A showed good thermostability and was active and stable at acidic pH. It could hydrolyze RFOs and various galactomannans. In addition, the enzyme showed good resistance to many proteases. These features make TtGal27A's great potential in
A
the food and feed industries. Acknowledgements This work was supported in part by the Key Program of the National Natural Science Foundation of China (No. 31630096) and Program for Changjiang Scholars (No. T2014055).
20
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[69] S.M. Fitzsimons, D.M. Mulvihill, E.R. Morris, Large enhancements in thermogelation of whey protein isolate by incorporation of very low concentrations of guar gum, Food Hydrocoll. 22 (2008) 576–586. . [70] S. Gurkok, D. Cekmecelioglu, Z.B. Ogel, Optimization of culture conditions for
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Aspergillus sojae expressing an Aspergillus fumigatus α-galactosidase, Bioresour. Technol. 102 (2011) 4925–4929.
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[71] H. Shibuya, H. Nagasaki, S. Kaneko, S. Yoshida, G.G. Park, I. Kusakabe, H. Kobayashi, Cloning and high-level expression of α-galactosidase cDNA from Penicillium
A
CC E
PT
ED
M
A
N
U
purpurogenum, Appl. Environ. Microbiol. 64 (1998) 4489–4494.
31
Figure captions Fig. 1. Nucleotide and amino acid sequences of TtGal27A. For the nucleotide sequence, the exons and introns are shown in uppercase and lowercase letters, respectively. The start and stop codons are boxed (bold box). For the amino acid sequence, the predicted signal peptide is
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underlined. Putative N- and O-linked glycosylation sites are displayed on gray background and boxed, respectively. The translation termination site is indicated by an asterisk.
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Fig. 2. Multiple sequence alignments of TtGal27A with other GH 27 family α-galactosidases. Abbreviation of the α-galactosidases and NCBI GenBank accession numbers are as follows:
U
Thielavia terrestris CAU709 (T.t.ARI48112.1), Penicillium purpurogenum ATCC MYA 38
A
N
(P.p.AKH40275.1), Penicillium simplicissimum VTT-D-78090 (P.s.CAA08915.1), Aspergillus
M
niger N400 (A.n.CAB46229.1), Neosartorya fischeri P1 (N.f.AGV79321.1) and Umbelopsis vinacea (U.v.BAA33931.1). The conserved regions are boxed in frame. The putative
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nucleophile and proton donor residues are denoted by stars.
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Fig. 3. (A) Time course of α-galactosidase TtGal27A expression in Pichia pastoris by high
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cell-density fermentation in 5-L fermentor. Symbols: extracellular enzyme activity (filled triangle), extracellular protein concentration (filled square), cell wet weight (filled circle). Error bars indicate means ± SD (n = 3). (B) SDS–PAGE analysis of the culture broth supernatant.
A
Lane M is low-molecular-weight markers. Lanes 1–11 are the fermentation broth supernatant samples from 0 to 240-h induction at time intervals of 24 h. Fig. 4. Purification and deglycosylation of TtGal27A. Lane M, low-molecular-weight markers; lane 1, fermentation broth supernatant; lane 2, purified protein; lane 3, de-O-glycosylation of 32
purified TtGal27A; lane 4, purified protein deglycosylated by Endo H. The additional protein bands in the deglycosylated samples derived from O-glycosidase (147 kDa) and neuraminidase (43 kDa) (lane 3) or Endo H (29 kDa) (lane 4). Fig. 5. pH and temperature profiles of the purified TtGal27A. Optimal pH (A) and pH stability
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(B) was determined at 50°C in the 50 mM following buffers: glycine-HCl (open diamond, pH
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2.5–3.5), citrate (filled square, pH 3.0–6.0), acetate (filled circle, pH 4.0–5.5), 2-(N-morpholino)ethanesulfonic acid (MES, filled triangle, pH 5.5–6.5), phosphate (inverted open triangle, pH 6.0–8.0), Tris-HCl (filled diamond, pH 7.0–9.0), glycine-NaOH (inverted
U
filled triangle, pH 8.5–10.5). The optimal temperature (C) and thermal stability (D) were
A
N
carried out at different temperatures in 50 mM citrate buffer pH 4.5. Thermal inactivation of the
M
purified TtGal27A (E) was assayed after incubation at 50°C (filled triangle), 55°C (filled square), 60°C (filled circle) and 65°C (inverted filled triangle). 50°C, Y = -0.0006x + 4.5683, R2
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= 0.09988; 55°C, Y = -0.0018x + 4.3760, R2 = 0.9980; 60°C, Y = -0.0033x + 4.1896, R2 = 0.9847;
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65°C, Y = -0.0109x + 3.7161, R2 = 0.9986. All experiments were performed in triplicate and
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error bars indicate SD.
Fig. 6. Residual activity of TtGal27A after protease treatment for 30 min (A) and 60 min (B) at 37°C. Concentration of each protease and TtGal27A was 1 mg/mL and 0.1 mg/mL,
A
respectively. Residual activity was determined after the different incubation times at 37°C. Initial activity was set as 100% and relative activity was calculated. For the controls, TtGal27A was incubated in buffers without proteases. Error bars show means ± SD (n = 3).
33
Table 1 Purification of TtGal27A from Pichia pastoris fermentation culture. Total activity Total protein Specific
Purification
Recovery
step
(U)a
(mg)
activity (U/mg)
factor
(%)b
Culture
12784.8
24.3
526.1
1.0
100
3233.6
4.3
752.0
Q-sepharose
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supernatant
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Purification
1.4
fast flow
25.3
Enzymatic activity assays were carried out at 60°C in 50 mM citrate pH 4.5 for 10 min.
b
Recovery is the ratio of residual enzyme activity to initial total enzyme activity as
A
N
U
a
A
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ED
M
percentage.
34
Table 2 Specific activity of TtGal27A towards different substratesa. Specific activityb
Relative
(U/mg)
activityc (%)
pNPαGal
752.0 ± 14.7
100.0 ± 0.0
Oligosaccharide
Melibiose
4.0 ± 0.1
0.5 ± 0.0
substratese
Raffinose
2.3 ± 0.0
Stachyose
1.3 ± 0.0
alactomannan
Locust bean gum
8.7 ± 0.0
substratesf
Cassia gum
5.1 ± 0.1
M/G ratiod
Substrate
0.2 ± 0.0
-
U
N
A
M
-
1.2 ± 0.0
3.9-4.0 [66]
0.7 ± 0.0
ca.
3.0
[66,69] 0.3 ± 0.0
ca. 2.8 [69]
Guar gum
1.0 ± 0.0
0.1 ± 0.0
1.6–1.8 [66]
Fenugreek gum
0.3 ± 0.0
0.0 ± 0.0
1.0–1.1 [66]
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Activity was determined at 60°C in 50 mM citrate pH 4.5 for 10 min.
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a
0.3 ± 0.0
2.1 ± 0.0
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Tara gum
-
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substrate
-
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Chromogenic
Data represent means ± SD (n = 3).
c
Data represent means ± SD (n = 3) relative to samples using pNPαGal as substrate.
d
Molar ratio of mannose (M) to galactose (G).
e
Final concentration was 4 mM.
f
Final concentration was 0.4%.
A
b
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Table 3 Kinetic parameters for the purified TtGal27Aa Vmax (μmol min-1 mg-1)
Km (mM)
kcat (s-1)
kcat/Km (mM-1 s-1)
pNPαGal
1050 ± 21.7
1.4 ± 0.1
1.01
0.719
melibiose
54.2 ± 0.5
39.5 ± 1.3
0.05
0.0013
raffinose
144.7 ± 5.5
268.0 ± 7.5
0.14
0.0005
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Substrate
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a All assays were conducted for 5 min at 50°C in 50 mM citrate buffer (pH 4.5). Mean values
A
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ED
M
A
N
U
and SD for three independent experiments are shown.
36
Table 4 Heterologous expression of α-galactosidases in different hosts. Microorganism source
Expression host
Extracellular
Extracellular
Reference
enzyme activity protein
385708
ATCC11906
Aspergillus niger
Pichia
-a
sojae 10.4
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Aspergillus fumigatus IMI Aspergillus
concentration (g/L)
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(U/mL)
pastoris 1299
U
KM71H
[70]
0.533
[18]
0.54
[56]
Escherichia coli BL21
1.42
Neosartorya fischeri P1
P. pastoris GS115
11.2
-a
[4]
550.2
-a
[20]
63
0.21
[71]
111
-a
[21]
1953.9
5.1
[22]
4402.13
8.3
This
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zalesk Saccharomyces
purpurogenum no. 618
cerevisiae WS3-2A
F63 P. pastoris GS115
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sp.
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Penicillium
Penicillium
A
janczewskii P. pastoris X-33
M
Penicillium
N
Gibberella sp. F75
CGMCC 1669 Rhizomucor
miehei P. pastoris GS115
A
CAU432
Thielavia
terrestris P. pastoris GS115
CAU709 a
study No data available. 37
A ED
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CC E
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SC R
U
N
A
M
.
Fig. 1.
38
Fig. 2.
39
A ED
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CC E
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SC R
U
N
A
M
Fig. 3.
40
A ED
PT
CC E
IP T
SC R
U
N
A
M
A ED
PT
CC E Fig. 4.
41
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SC R
U
N
A
M
Fig. 5.
42
A ED
PT
CC E
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SC R
U
N
A
M
A
Fig. 6.
43
ED
PT
CC E
IP T
SC R
U
N
A
M