A thermophilic α-galactosidase from Neosartorya fischeri P1 with high specific activity, broad substrate specificity and significant hydrolysis ability of soymilk

Bioresource Technology 153 (2014) 361–364

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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech

Short Communication

A thermophilic a-galactosidase from Neosartorya fischeri P1 with high specific activity, broad substrate specificity and significant hydrolysis ability of soymilk Huimin Wang, Pengjun Shi, Huiying Luo, Huoqing Huang, Peilong Yang, Bin Yao ⇑ Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, PR China

h i g h l i g h t s  One GH27

a-galactosidase was purified from N. fischeri with high specific activity.

 The enzyme showed optimal activities at 60–70 °C and pH 4.5.  The enzyme had strong abilities to degrade natural substrates and soymilk.  Its coding gene was cloned and expressed in P. pastoris with high yield.  rGal27A have cost-effective application potentials in feed and food industries.

a r t i c l e

i n f o

Article history: Received 5 October 2013 Received in revised form 14 November 2013 Accepted 25 November 2013 Available online 4 December 2013 Keywords: Neosartorya fischeri a-Galactosidase Thermophilic Broad substrate specificity

a b s t r a c t An extracellular a-galactosidase (Gal27A) with high specific activity of 423 U mg1 was identified in thermophilic Neosartorya fischeri P1. Its coding gene (1680 bp) was cloned and functionally expressed in Pichia pastoris. Sequence analysis indicated that deduced Gal27A contains a catalytic domain of glycoside hydrolase family 27. The native and recombinant enzymes shared some similar properties, such as pH optima at 4.5, temperature optima at 60–70 °C, resistance to most chemicals and saccharides, and great abilities to degrade raffinose and stachyose in soymilk. Considering the high yield (3.1 g L1) in P. pastoris, recombinant rGal27A is more favorable for industrial applications. This is the first report on purification and gene cloning of Neosartorya a-galactosidase. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction

a-Galactosidases (EC 3.2.1.22) are exo-glycosidases that catalyze the removal of a-1,6-linked terminal galactose residues from different substrates (Katrolia et al., 2012). Based on the sequence similarity, a-galactosidases have been classified as members of the glycoside hydrolase (GH) families 4, 27, 32, 36, 57, 97, and 110 (http://www.cazy.org/) (Cantarel et al., 2009). a-Galactosidases have been used in industrial processes of feed, food, and beet sugar production. Due to the specific requirements of each industry, a highly efficient and thermostable a-galactosidase with broad substrate specificity is in great demand (Berka et al., 2011). Thermophilic microorganisms are the main microbial sources of thermophilic and thermostable enzymes. However, only ⇑ Corresponding author. Address: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Beijing 100081, PR China. Tel.: +86 10 82106053; fax: +86 10 82106054. E-mail addresses: [email protected], [email protected] (B. Yao). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.11.078

a few a-galactosidases have been identified in thermophilic fungi, including Thermomyces lanuginosus (Puchart et al., 2000), Talaromyces emersonii (Janika et al., 2010), and Rhizomucor miehei (Katrolia et al., 2012). Soybeans are rich in proteins and have healthy benefits to both human and animals. However, the high concentrations of raffinose oligosaccharides (RFOs) in soybeans and other legumes cannot be digested by monogastric animals, and cause flatulence, gastrointestinal disturbance and low feed efficiency (Viana et al., 2009). Microbial a-galactosidases are widely added to soybean products to hydrolyze these soluble oligosaccharides to moderate the flatulence-causing property and improve the utilization of food and feed (Katrolia et al., 2012; Du et al., 2013). In the present study a thermophilic Neosartorya fischeri P1 was isolated from the acid wastewater of a tin mine in Yunnan, China. This fungus showed the ability to utilize soybean meal as the carbon source and secreted an extracellular a-galactosidase with high activity. The native a-galactosidase was purified, and the coding gene was cloned and successfully expressed in Pichia pastoris. Both

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H. Wang et al. / Bioresource Technology 153 (2014) 361–364

enzymes were characterized and showed favorable properties, such as adaptability and stability over broader pH and temperature ranges, strong resistance to most chemicals, broad substrate specificity, and great capacity to eliminate RFOs from soymilk. 2. Methods 2.1. Fungal strain and culture conditions Strain P1 was identified as N. fischeri based on morphological characters and ITS rDNA sequence, and was deposited in the China General Microbiological Culture Collection under registration number CGMCC 3.15369. To induce a-galactosidase production, strain P1 was cultivated at 45 °C in the enzyme producing medium as described by Cao et al. (2009). After 6-day growth at 45 °C, the total a-galactosidase activity was 9.6 ± 0.1 U mL1 in culture supernatant. 2.2. Purification of the native a-galactosidase The culture supernatant (1.8 L) was collected by centrifugation (12,000g, 4 °C, 20 min), concentrated through a Vivaflow 200 membrane of 5-kDa molecular weight cutoff (Vivascience, Hannover, Germany), and loaded onto a HiTrap™ Sepharose XL FPLC column (GE Healthcare, Uppsala, Sweden) that was equilibrated with 20 mM McIlvaine buffer (40 mM Na2HPO4, 20 mM citric acid, pH 3.0). Proteins were eluted using a gradient of NaCl (0–1.0 M) in the same buffer. After one more chromatography on a FPLC Sephacryl S-200 HR column (GE Healthcare) equilibrated with 100 mM McIlvaine buffer (pH 4.5), the Gal27A was purified to electrophoretic homogeneity. SDS–PAGE (12.0%) and native PAGE (4–15%) were used to check the purity and molecular weights of Gal27A. Protein concentration was determined using a protein assay kit (Bio-Rad, Hercules, CA). The single protein band in the SDS–PAGE gel was trypsin-digested and identified using liquid chromatography–electrospray tandem mass spectrometry (LC–ESI-MS/MS) by Tianjin Biochip Co. Ltd. (Tianjin, China). 2.3. Cloning of the genomic DNA and cDNA of gal27A A primer set (GALF1: 50 -CATGACTGCCGCGAACGAGGTCGTC-30 , and GALR1: 50 -CTAGCATGCCCTCCCCACAACCAG-30 ) was designed based on the internal peptide sequences (IMTAANEVV and DHYSVELESHDVAALVVGR) obtained above, and used to amplify the partial fragment of gal27A with the total genomic DNA of strain P1 as template. The PCR products were ligated into pGEM-T Easy vector (Promega, Madison, WI) for sequencing and BLAST analysis. Thermal asymmetric interlaced-PCR (Liu and Whittier, 1995) was used to obtain the flanking regions. Total RNA was extracted and purified using the RNeasy plant mini kit (QIAGEN, Hilden, Germany) from the 6-day-old mycelia of strain P1 in the a-galactosidase producing medium. Full-length cDNA was obtained by reverse transcription (RT)-PCR and a primer set (GALF2: 50 -ATGACGACGTTTCTCTCTCTGACCAC-30 , and GALR2: 50 -CTAGCATGCCCTCCCCACAAC-30 ). The PCR products were subcloned into the pGEM-T Easy vector for sequencing. 2.4. Expression and purification of rGal27A The cDNA fragment of mature Gal27A was amplified by PCR with a specific primer set (GALF3: 50 -GGGACTAGTCTCGTTAGACCGGGCAATGTGGG-30 , and GALR3: 50 -GGGGCGGCCGCCTAGTG GTGGTGGTGGTGGTGGCATGCCCTCCCCACAACCAG-30 ). The PCR products were purified and digested with SpeI and NotI, and cloned into the corresponding sites of vector pPIC9 (Invitrogen). The recombinant expression plasmids were linearized with BglII, and

then expressed in P. pastoris GS115 competent cells according to the Pichia Expression Kit (Invitrogen). With 0.5% methanol induction, rGAL27A was secreted into the culture, which was further collected and purified by centrifugation, concentration and chromatograph as described above for native Gal27A. Recombinant rGAL27A was subjected to SDS–PAGE analysis and N-glycosylation removal with endo-b-N-acetylglucosaminidase H (Endo H) following the supplier’s instructions (New England Biolabs, Hitchin, UK). 2.5. Enzyme activity assay and biochemical characterization p-Nitrophenyl-a-D-galactopyranoside (pNPG; Sigma, St. Louis, MO) was used as the substrate. The enzymatic activities of Gal27A and rGal27A and their biochemical properties including pH/temperature adaptability and stability, resistance to chemicals and saccharides, kinetics, substrate specificity and hydrolysis products were determined according to Cao et al. (2009). One unit of a-galactosidase activity was defined as the amount of enzyme that released 1 lmol of pNP per min under pH 4.5, 70 °C (Gal27A) or 60 °C (rGal27A) for 5 min. All experiments were run in triplicate. 2.6. Soymilk treatment with enzymes One gram of defatted soybean flour was suspended in 10 mL of sodium citrate buffer (pH 4.5) and boiled for 5 min. After removal of the undissolved residues by centrifugation, 1 mL of soymilk was treated with 2 U of Gal27A or rGal27A at 50 °C for 3 h. The reaction was terminated by the addition of 200 lL of 300 mM Ba(OH)2 and 200 lL of 180 mM ZnSO4 (Anisha and Prema, 2007). The mixtures were centrifuged at 12,000g, 4 °C for 10 min to precipitate proteins. The amounts of galactose liberated in the supernatants were measured by high-performance liquid chromatography (HPLC). 2.7. Nucleotide sequence accession numbers The nucleotide sequences for the ITS gene and GH27 a-galactosidase gene (gal27A) of N. fischeri P1 were deposited into the GenBank database under the accession numbers KF640700 and KF640698, respectively. 3. Results and discussion 3.1. Purification and identification of native Gal27A from N. fischeri P1 The native a-galactosidase was purified to homogeneity with an 18.6-fold purification and a final yield of 3.7% through ion exchange chromatography and gel filtration (Table 1). The specific activity against pNPG was 423 U mg1, which is higher than most GH27 a-galactosidases but lower than that of Bispora sp. MEY-1 (581 U mg1; Wang et al., 2010). The enzyme showed a single band of approximately 55 kDa in SDS–PAGE (Fig. 1a) and two bands of about 230 and 440 kDa in native PAGE (Fig. 1b), indicating the enzyme to be a tetramer or octamer. LC–ESI-MS/MS analysis identified seven internal peptides, including DHYSVELESHDVAALVVGR, FHQDPVVGRPAHPYK, NPAPAGYDWR, MVPDPEKFPDGISGLADQIHDLGLK, AHFALWAAMKSPLIIGTALDSISQDHLAILSNK, IMTAANEVVNLGLK and TTGDITPSWPR. BLASTp analysis revealed that the native a-galactosidase from N. fischeri P1 was a member of GH27, thus designated Gal27A. 3.2. Gene cloning and sequence analysis The complete DNA and cDNA sequences of gal27A are 1680 and 1341 bp in length, respectively. Deduced Gal27A consists of a putative signal sequence (residues 1–22) and a catalytic domain

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H. Wang et al. / Bioresource Technology 153 (2014) 361–364 Table 1 Purification of native a-galactosidase Gal27A from N. fischeri P1. Purification step

Total volume (mL)

Total activity (U)

Total protein (mg)

Specific activity (U mg1)

Yield (%)

Purification folds

Crude extract HiTrap™ Sepharose XL Sephacryl S-200 HR

50 8.2 4.0

15521 1306 575

682.8 4.02 1.36

22.73 325 423

100 8.4 3.7

1.0 14.3 18.6

(a)

kDa 1 170 130 90 72 55 43 34

2

3

4

(b) kDa 660

10

3

440 232

26 17

2

1

140

• •

66

Fig. 1. Electrophoretic analysis of purified Gal27A and rGal27A. (a) SDS–PAGE analysis of native Gal27A and recombinant rGal27A. Lane 1, the low molecular weight markers; lane 2, purified Gal27A; lane 3, purified rGal27A; lane 4, deglycosylated rGal27A with Endo H treatment. (b) Native PAGE of native Gal27A. Lane 1, the high molecular weight markers; lane 2, native Gal27A stained with Coomassie Brilliant Blue; lane 3, native Gal27A hydrolyzed by 6-bromo-2-naphthyla-D-galactopyranoside and stained by Fast Blue B Salt.

of GH27 (residues 23–446), and shares 28–96% identities with functionally identified fungal a-galactosidases. The molecular weight and pI were estimated to be 49.2 kDa and 5.1, respectively. With Trichoderma reesei a-galactosidase (1SZN_A) as template, the homology-modeled structure of Gal27A was predicted to have a catalytic domain of (b/a)8-barrel fold, in which the putative catalytic residues Asp154 and Asp246 corresponding to the proton donor and acceptor, are located at the C-terminal side of the catalytic domain of Gal27A. 3.3. Expression and purification of recombinant Gal27A (rGal27A) Recombinant rGal27A was produced in the Pichia expression system with the total a-galactosidase activity of 70.6 ± 0.7 U mL1 in culture supernatant, which was about 7-folds higher than that of native Gal27A. The total secretary proteins of P. pastoris were high up to 3.2 g L1, and recombinant rGal27A constituted approximately 96% of them. Such high level of expression and unnecessary purification make rGal27A more cost-effective for commercialization. The specific activity of rGal27A was 234 U mg1 towards pNPG. As shown in Fig. 1a, rGal27A showed two bands of approximately 55.0 and 58.0 kDa, respectively. LC–ESI-MS/MS analysis indicated that both bands were rGal27A indeed. After treatment with Endo H, the two bands showed a reduction of 5.0 kDa in size, and the molecular weight of the larger band was still higher than the calculated value (53.0 kDa vs. 49.2 kDa). This discrepancy might be due to other post-translational modifications besides N-glycosylation (Daly and Hearn, 2005). 3.4. Biochemical characterization of Gal27A and rGal27A Most fungal a-galactosidases have an acidic pH optimum between pH 4.0 and 6.0. The pH optima of Gal27A and rGal27A fell

within this range, which were both 4.5. They were stable in broader pH ranges (more than 80% activity at pH 3.0–10.0) than other fungal a-galactosidases (generally pH 4.0–8.0). The enzymes were active over a broad temperature range (30–90 °C), and Gal27A showed better adaptability and stability than rGal27A at 70 °C. The decrease in the temperature optima after Pichia expression has been reported for the a-galactosidases Agl1 from Penicillium sp. F63 and a-Gal1 from T. emersonii (Mi et al., 2007; Janika et al., 2010). In view of enzyme properties, Gal27A is favorable for application in a wider range of industrial processes. Gal27A and rGal27A were resistant to most tested chemicals except for Ag+ and SDS as occurred to most other a-galactosidases (Viana et al., 2009). Ag+ may attack the free cysteine residues in the active site and interfere with substrate interaction by binding in the catalytic pocket, thereby interfering with the substrateenzyme interaction (Fujimoto et al., 2003). Fe3+ enhanced the activities of two enzymes by >30%. Of five tested saccharides, D-galactose is a competitive inhibitor, and sucrose, fructose, glucose, and xylose had little effects on enzyme activity.

3.5. Kinetics, substrate specificities and analysis of hydrolysis products The Km, Vmax and kcat values were 1.52 mM, 1000.3 lmol min1 mg1 and 820.2 s1 for Gal27A, and 0.8 mM, 449.5 lmol min1 mg1 and 368.6 s1 for rGal27A, respectively. rGal27A had a lower Km value than Gal27A, which suggested that rGal27A has a higher affinity to substrate pNPG. However, Gal27A exhibited higher catalytic efficiency (kcat/Km) than rGal27A and could hydrolyze pNPG at a faster rate. Gal27A and rGal27A had similar activities towards synthetic substrates, exhibiting highest activities on pNPG (100%), but no activities on other nitrophenyl derivatives. Gal27A and rGal27 had different abilities to hydrolyze natural galacto-oligosaccharides. After treatment with 1 U mL1 of Gal27A, 667.6, 428.9, and 447.8 lg of galactose were released from 1 mg of melibiose, raffinose, and stachyose, respectively. Under the same conditions, rGal27A released 255.4, 883.3, and 476.3 lg of galactose, respectively. Interestingly, both Gal27A and rGal27A acted on the side chains of some polymeric substrates, such as locust bean gum, guar gum and konjac flour (data not shown). The substrate specificity result indicated that Gal27A is a member of the a-galactosidase group with high activity against polymers (Katrolia et al., 2012).

3.6. Soymilk hydrolysis with Gal27A and rGal27A The amounts of raffinose and stachyose in soymilk were determined to be 0.93 and 12.68 mg mL1, respectively, by HPLC. After treatment with Gal27A at 50 °C for 3 h, the raffinose and stachyose contents were decreased by 69.9% and 94.5%, respectively. Under the same conditions, rGal27A removed 72.5% of raffinose and 54.8% of stachyose in soymilk. Their performances in RFOs removal are a little wrose than that of Aspergillus and Rhizomucor a-galactosidases (Ferreira et al., 2011; Katrolia et al., 2012), which might be ascribed to different soybean products or less incubation time.

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4. Conclusions One extracellular GH27 a-galactosidase was purified from N. fischeri P1 with high specific activity. To increase its yield, the coding gene was cloned and efficiently expressed in P. pastoris. The enzymes had similar pH adaptability and stability, chemical and saccharides resistance, and hydrolysis abilities to degrade RFOs in soymilk. Each enzyme had its own advantages. Gal27A had higher specific activity and temperature optimum, and better thermostability. But rGal27A had greater affinity to substrates and high yield in Pichia expression system. From the industrial point of view, rGal27A is more favorable for cost-effective application in the feed and food industries. Acknowledgements This research was supported by the Agricultural Science and Technology Conversion Funds (SQ2012EC3260006), the National High Technology Research and Development Program of China (863 program, 2012AA022208), and the National Science and Technology Support Program (2011BADB02). References Anisha, G.S., Prema, P., 2007. Production of a-galactosidase by a novel actinomycete Streptomyces griseoloalbus and its application in soymilk hydrolysis. World J. Microbiol. Biotechnol. 23, 859–864. Berka, R.M., Grigoriev, I.V., Otillar, R., Salamov, A., Grimwood, J., Reid, I., Ishmael, N., John, T., Darmond, C., Moisan, M.C., 2011. Comparative genomic analysis of the thermophilic biomass-degrading fungi Myceliophthora thermophila and Thielavia terrestris. Nat. Biotechnol. 29, 922–927. Cantarel, B.L., Coutinho, P.M., Rancurel, C., Bernard, T., Lombard, V., Henrissat, B., 2009. The carbohydrate-active enzymes database (CAZy): an expert resource for Glycogenomics. Nucleic Acids Res. 37, D233–D238. Cao, Y., Wang, Y., Luo, H., Shi, P., Meng, K., Zhou, Z., Zhang, Z., Yao, B., 2009. Molecular cloning and expression of a novel protease-resistant GH-36

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