Bioresource Technology 153 (2014) 361–364

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Bioresource Technology

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Short Communication

A thermophilic a-galactosidase from Neosartorya fischeri P1 with highspecific activity, broad substrate specificity and significant hydrolysisability of soymilk

0960-8524/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.biortech.2013.11.078

⇑ Corresponding author. Address: Key Laboratory for Feed Biotechnology of theMinistry of Agriculture, Feed Research Institute, Chinese Academy of AgriculturalSciences, No. 12 Zhongguancun South Street, Beijing 100081, PR China. Tel.: +86 1082106053; fax: +86 10 82106054.

E-mail addresses: binyao@caas.cn, yaobin@caas.cn (B. Yao).

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 2013Received in revised form 14 November 2013Accepted 25 November 2013Available online 4 December 2013

Keywords:Neosartorya fischeria-GalactosidaseThermophilicBroad substrate specificity

a b s t r a c t

An extracellular a-galactosidase (Gal27A) with high specific activity of 423 U mg�1 was identified inthermophilic Neosartorya fischeri P1. Its coding gene (1680 bp) was cloned and functionally expressedin Pichia pastoris. Sequence analysis indicated that deduced Gal27A contains a catalytic domain ofglycoside hydrolase family 27. The native and recombinant enzymes shared some similar properties, suchas pH optima at 4.5, temperature optima at 60–70 �C, resistance to most chemicals and saccharides, andgreat abilities to degrade raffinose and stachyose in soymilk. Considering the high yield (3.1 g L�1) in P.pastoris, recombinant rGal27A is more favorable for industrial applications. This is the first report onpurification and gene cloning of Neosartorya a-galactosidase.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

a-Galactosidases (EC 3.2.1.22) are exo-glycosidases thatcatalyze the removal of a-1,6-linked terminal galactose residuesfrom different substrates (Katrolia et al., 2012). Based on thesequence similarity, a-galactosidases have been classified asmembers 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 requirementsof each industry, a highly efficient and thermostable a-galactosi-dase with broad substrate specificity is in great demand (Berkaet al., 2011). Thermophilic microorganisms are the main microbialsources of thermophilic and thermostable enzymes. However, only

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 bothhuman and animals. However, the high concentrations of raffinoseoligosaccharides (RFOs) in soybeans and other legumes cannot bedigested by monogastric animals, and cause flatulence, gastroin-testinal disturbance and low feed efficiency (Viana et al., 2009).Microbial a-galactosidases are widely added to soybean productsto hydrolyze these soluble oligosaccharides to moderate the flatu-lence-causing property and improve the utilization of food andfeed (Katrolia et al., 2012; Du et al., 2013).

In the present study a thermophilic Neosartorya fischeri P1 wasisolated from the acid wastewater of a tin mine in Yunnan, China.This fungus showed the ability to utilize soybean meal as the car-bon source and secreted an extracellular a-galactosidase with highactivity. The native a-galactosidase was purified, and the codinggene was cloned and successfully expressed in Pichia pastoris. Both

362 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 temperatureranges, strong resistance to most chemicals, broad substratespecificity, 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 morphologicalcharacters and ITS rDNA sequence, and was deposited in the ChinaGeneral Microbiological Culture Collection under registration num-ber CGMCC 3.15369. To induce a-galactosidase production, strainP1 was cultivated at 45 �C in the enzyme producing medium as de-scribed by Cao et al. (2009). After 6-day growth at 45 �C, the totala-galactosidase activity was 9.6 ± 0.1 U mL�1 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 200membrane of 5-kDa molecular weight cutoff (Vivascience,Hannover, Germany), and loaded onto a HiTrap™ Sepharose XLFPLC column (GE Healthcare, Uppsala, Sweden) that wasequilibrated with 20 mM McIlvaine buffer (40 mM Na2HPO4,20 mM citric acid, pH 3.0). Proteins were eluted using a gradientof NaCl (0–1.0 M) in the same buffer. After one more chromatogra-phy on a FPLC Sephacryl S-200 HR column (GE Healthcare) equili-brated with 100 mM McIlvaine buffer (pH 4.5), the Gal27A waspurified to electrophoretic homogeneity. SDS–PAGE (12.0%) andnative PAGE (4–15%) were used to check the purity and molecularweights of Gal27A. Protein concentration was determined using aprotein assay kit (Bio-Rad, Hercules, CA). The single protein bandin the SDS–PAGE gel was trypsin-digested and identified usingliquid 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 designedbased on the internal peptide sequences (IMTAANEVV and DHYS-VELESHDVAALVVGR) obtained above, and used to amplify the par-tial fragment of gal27A with the total genomic DNA of strain P1 astemplate. The PCR products were ligated into pGEM-T Easy vector(Promega, Madison, WI) for sequencing and BLAST analysis. Ther-mal asymmetric interlaced-PCR (Liu and Whittier, 1995) was usedto obtain the flanking regions.

Total RNA was extracted and purified using the RNeasy plantmini kit (QIAGEN, Hilden, Germany) from the 6-day-old myceliaof strain P1 in the a-galactosidase producing medium. Full-lengthcDNA was obtained by reverse transcription (RT)-PCR and a primerset (GALF2: 50-ATGACGACGTTTCTCTCTCTGACCAC-30, and GALR2:50-CTAGCATGCCCTCCCCACAAC-30). The PCR products were sub-cloned into the pGEM-T Easy vector for sequencing.

2.4. Expression and purification of rGal27A

The cDNA fragment of mature Gal27A was amplified by PCRwith a specific primer set (GALF3: 50-GGGACTAGTCTCGTTA-GACCGGGCAATGTGGG-30, and GALR3: 50-GGGGCGGCCGCCTAGTGGTGGTGGTGGTGGTGGCATGCCCTCCCCACAACCAG-30). The PCRproducts were purified and digested with SpeI and NotI, and clonedinto the corresponding sites of vector pPIC9 (Invitrogen). Therecombinant expression plasmids were linearized with BglII, and

then expressed in P. pastoris GS115 competent cells according tothe Pichia Expression Kit (Invitrogen). With 0.5% methanol induc-tion, rGAL27A was secreted into the culture, which was further col-lected and purified by centrifugation, concentration andchromatograph as described above for native Gal27A. RecombinantrGAL27A was subjected to SDS–PAGE analysis and N-glycosylationremoval with endo-b-N-acetylglucosaminidase H (Endo H) follow-ing 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 Gal27Aand rGal27A and their biochemical properties including pH/tem-perature adaptability and stability, resistance to chemicals and sac-charides, kinetics, substrate specificity and hydrolysis productswere determined according to Cao et al. (2009). One unit ofa-galactosidase activity was defined as the amount of enzyme thatreleased 1 lmol of pNP per min under pH 4.5, 70 �C (Gal27A) or60 �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 ofsodium citrate buffer (pH 4.5) and boiled for 5 min. After removalof the undissolved residues by centrifugation, 1 mL of soymilk wastreated with 2 U of Gal27A or rGal27A at 50 �C for 3 h. The reactionwas terminated by the addition of 200 lL of 300 mM Ba(OH)2 and200 lL of 180 mM ZnSO4 (Anisha and Prema, 2007). The mixtureswere centrifuged at 12,000g, 4 �C for 10 min to precipitate proteins.The amounts of galactose liberated in the supernatants weremeasured by high-performance liquid chromatography (HPLC).

2.7. Nucleotide sequence accession numbers

The nucleotide sequences for the ITS gene and GH27 a-galacto-sidase gene (gal27A) of N. fischeri P1 were deposited into theGenBank database under the accession numbers KF640700 andKF640698, 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 withan 18.6-fold purification and a final yield of 3.7% through ion ex-change chromatography and gel filtration (Table 1). The specificactivity against pNPG was 423 U mg�1, which is higher than mostGH27 a-galactosidases but lower than that of Bispora sp. MEY-1(581 U mg�1; Wang et al., 2010). The enzyme showed a single bandof approximately 55 kDa in SDS–PAGE (Fig. 1a) and two bands ofabout 230 and 440 kDa in native PAGE (Fig. 1b), indicating the en-zyme to be a tetramer or octamer. LC–ESI-MS/MS analysis identi-fied seven internal peptides, including DHYSVELESHDVAALVVGR,FHQDPVVGRPAHPYK, NPAPAGYDWR, MVPDPEKFPDGISGLAD-QIHDLGLK, AHFALWAAMKSPLIIGTALDSISQDHLAILSNK, IMTAA-NEVVNLGLK and TTGDITPSWPR. BLASTp analysis revealed thatthe native a-galactosidase from N. fischeri P1 was a member ofGH27, thus designated Gal27A.

3.2. Gene cloning and sequence analysis

The complete DNA and cDNA sequences of gal27A are 1680 and1341 bp in length, respectively. Deduced Gal27A consists of aputative signal sequence (residues 1–22) and a catalytic domain

Table 1Purification of native a-galactosidase Gal27A from N. fischeri P1.

Purification step Total volume (mL) Total activity (U) Total protein (mg) Specific activity (U mg�1) Yield (%) Purification folds

Crude extract 50 15521 682.8 22.73 100 1.0HiTrap™ Sepharose XL 8.2 1306 4.02 325 8.4 14.3Sephacryl S-200 HR 4.0 575 1.36 423 3.7 18.6

•

•

•

660

440

232

140

66

kDa1 2 31 2 3kDa

17013090725543

34

26

17

4(a) (b)

10

Fig. 1. Electrophoretic analysis of purified Gal27A and rGal27A. (a) SDS–PAGEanalysis of native Gal27A and recombinant rGal27A. Lane 1, the low molecularweight 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 withCoomassie Brilliant Blue; lane 3, native Gal27A hydrolyzed by 6-bromo-2-naphthyl-a-D-galactopyranoside and stained by Fast Blue B Salt.

H. Wang et al. / Bioresource Technology 153 (2014) 361–364 363

of GH27 (residues 23–446), and shares 28–96% identities withfunctionally identified fungal a-galactosidases. The molecularweight and pI were estimated to be 49.2 kDa and 5.1, respectively.With Trichoderma reesei a-galactosidase (1SZN_A) as template, thehomology-modeled structure of Gal27A was predicted to have acatalytic domain of (b/a)8-barrel fold, in which the putative cata-lytic residues Asp154 and Asp246 corresponding to the proton do-nor and acceptor, are located at the C-terminal side of the catalyticdomain of Gal27A.

3.3. Expression and purification of recombinant Gal27A (rGal27A)

Recombinant rGal27A was produced in the Pichia expressionsystem with the total a-galactosidase activity of 70.6 ± 0.7 U mL�1

in culture supernatant, which was about 7-folds higher than that ofnative Gal27A. The total secretary proteins of P. pastoris were highup to 3.2 g L�1, and recombinant rGal27A constituted approxi-mately 96% of them. Such high level of expression and unnecessarypurification make rGal27A more cost-effective for commercializa-tion. The specific activity of rGal27A was 234 U mg�1 towardspNPG. As shown in Fig. 1a, rGal27A showed two bands of approx-imately 55.0 and 58.0 kDa, respectively. LC–ESI-MS/MS analysisindicated that both bands were rGal27A indeed. After treatmentwith 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 thanthe calculated value (53.0 kDa vs. 49.2 kDa). This discrepancymight be due to other post-translational modifications besidesN-glycosylation (Daly and Hearn, 2005).

3.4. Biochemical characterization of Gal27A and rGal27A

Most fungal a-galactosidases have an acidic pH optimum be-tween 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 broad-er pH ranges (more than 80% activity at pH 3.0–10.0) than otherfungal a-galactosidases (generally pH 4.0–8.0). The enzymes wereactive over a broad temperature range (30–90 �C), and Gal27Ashowed better adaptability and stability than rGal27A at 70 �C.The decrease in the temperature optima after Pichia expressionhas been reported for the a-galactosidases Agl1 from Penicilliumsp. F63 and a-Gal1 from T. emersonii (Mi et al., 2007; Janikaet al., 2010). In view of enzyme properties, Gal27A is favorablefor application in a wider range of industrial processes.

Gal27A and rGal27A were resistant to most tested chemicalsexcept for Ag+ and SDS as occurred to most other a-galactosidases(Viana et al., 2009). Ag+ may attack the free cysteine residues in theactive site and interfere with substrate interaction by binding inthe catalytic pocket, thereby interfering with the substrate-enzyme interaction (Fujimoto et al., 2003). Fe3+ enhanced theactivities 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 min�1 mg�1 and 820.2 s�1 for Gal27A, and 0.8 mM,449.5 lmol min�1 mg�1 and 368.6 s�1 for rGal27A, respectively.rGal27A had a lower Km value than Gal27A, which suggested thatrGal27A has a higher affinity to substrate pNPG. However, Gal27Aexhibited higher catalytic efficiency (kcat/Km) than rGal27A andcould hydrolyze pNPG at a faster rate.

Gal27A and rGal27A had similar activities towards syntheticsubstrates, exhibiting highest activities on pNPG (100%), but noactivities on other nitrophenyl derivatives. Gal27A and rGal27had different abilities to hydrolyze natural galacto-oligosaccha-rides. After treatment with 1 U mL�1 of Gal27A, 667.6, 428.9, and447.8 lg of galactose were released from 1 mg of melibiose, raffi-nose, and stachyose, respectively. Under the same conditions, rGa-l27A released 255.4, 883.3, and 476.3 lg of galactose, respectively.Interestingly, both Gal27A and rGal27A acted on the side chains ofsome polymeric substrates, such as locust bean gum, guar gum andkonjac flour (data not shown). The substrate specificity result indi-cated that Gal27A is a member of the a-galactosidase group withhigh activity against polymers (Katrolia et al., 2012).

3.6. Soymilk hydrolysis with Gal27A and rGal27A

The amounts of raffinose and stachyose in soymilk were deter-mined to be 0.93 and 12.68 mg mL�1, respectively, by HPLC. Aftertreatment with Gal27A at 50 �C for 3 h, the raffinose and stachyosecontents were decreased by 69.9% and 94.5%, respectively. Underthe same conditions, rGal27A removed 72.5% of raffinose and54.8% of stachyose in soymilk. Their performances in RFOs removalare a little wrose than that of Aspergillus and Rhizomucor a-galacto-sidases (Ferreira et al., 2011; Katrolia et al., 2012), which might beascribed to different soybean products or less incubation time.

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

4. Conclusions

One extracellular GH27 a-galactosidase was purified from N.fischeri P1 with high specific activity. To increase its yield, the cod-ing gene was cloned and efficiently expressed in P. pastoris. The en-zymes had similar pH adaptability and stability, chemical andsaccharides resistance, and hydrolysis abilities to degrade RFOsin soymilk. Each enzyme had its own advantages. Gal27A had high-er specific activity and temperature optimum, and better thermo-stability. But rGal27A had greater affinity to substrates and highyield in Pichia expression system. From the industrial point of view,rGal27A is more favorable for cost-effective application in the feedand food industries.

Acknowledgements

This research was supported by the Agricultural Science andTechnology Conversion Funds (SQ2012EC3260006), the NationalHigh Technology Research and Development Program of China(863 program, 2012AA022208), and the National Science and Tech-nology Support Program (2011BADB02).

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