P. Katrolia et al. / Journal of Molecular Catalysis B: Enzymatic 69 (2011) 112–119
119
100
90
80
70
60
50
40
30
20
10
0
over a broad pH range. Moreover, PaGalA was able to hydrolyze
lactose present in acid-whey and sweet-whey. Its lactose hydroly-
sis ability was found to be superior to two commercially available
-galactosidases suggesting its potential use in the hydrolysis of
lactose in whey and milk.
Acknowledgement
This work was supported by the New Century Excellent Talents
in University (NCET-08-0534).
References
[1] P.S. Panesar, R. Panesar, R.S. Singh, J.F. Kennedy, H. Kumar, J. Chem. Technol.
Biotechnol. 81 (2006) 530–543.
[2] G.T. Macfarlane, H. Steed, S. Macfarlane, J. Appl. Microbiol. 104 (2008) 305–344.
[3] C.S. Kim, E.S. Ji, D.K. Oh, Biotechnol. Lett. 25 (2003) 1769–1774.
[4] T.H. Nguyen, B. Splechtna, M. Steinbock, W. Kneifel, H.P. Lettner, K.D. Kulbe, D.
Haltrich, J. Agric. Food Chem. 54 (2006) 4989–4998.
0
1
2
3
4
5
6
7
8
Time (h)
[5] D. Todorova-Balvay, I. Stoilova, S. Gargova, M.A. Vijayalakshmi, J. Mol. Recognit.
19 (2006) 299–304.
[6] L. Fischer, C. Scheckermann, F. Wagner, Appl. Environ. Microbiol. 61 (1995)
1497–1501.
[7] J. Rogalski, J. Lobarzewski, Acta Biotechnol. 15 (1995) 211–222.
[8] M. Diaz, A.M. Pedregosa, J.R. de Lucas, S. Torralba, I.F. Monistrol, F. Laborda,
Microbiol. SEM 12 (1996) 585–592.
Fig. 6. Hydrolysis of lactose in whey by different -galactosidases. Comparison of
lactose hydrolysis with purified PaGalA (ꢂ) and -galactosidase from Aspergillus
oryzae (ꢂ) in acid whey (pH 4.8) as well as with PaGalA (ꢀ) and Lactozym (×) in
sweet whey (pH 6.6) was done at 40 ◦C. Samples at various time-intervals were
analyzed by HPLC for the amount of residual lactose.
[9] S.A. Shaikh, J.M. Khire, M.I. Khan, Biochem. Biophys. Acta 1472 (1999) 314–
322.
[10] V. Kumar, S. Ramakrishnan, T.T. Teeri, J.K. Knowles, B.S. Hartley, Nat. Biotechnol.
10 (1992) 82–85.
[11] W. Zhang, B. Yao, L. Wang, Microbiol. Bull. 18 (2002) 566–571.
[12] H. Wang, H. Luo, Y. Bai, Y. Wang, P. Yang, P. Shi, W. Zhang, Y. Fan, Y. Bin, J. Agric.
Food Chem. 57 (2009) 5535–5541.
[13] R. Daly, M. Hearn, J. Mol. Recognit. 18 (2005) 119–138.
[14] T.M. Rose, J.G. Henikoff, S. Henikoff, Nucleic Acids Res. 31 (2003) 3763–3766.
[15] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, J. Biol. Chem. (1975)
265–275.
6.6) and acid whey (pH 4.8) by PaGalA, Lactozym and A. oryzae -
galactosidase was evaluated. As shown in Fig. 6, 1.5 U ml−1 of the
-galactosidase from A. oryzae was able to hydrolyze 76.2% lac-
tose in acid whey (pH 4.8) at 40 ◦C. In comparison, PaGalA could
almost completely hydrolyze (97.4% hydrolysis) lactose in 8 h under
the same conditions. Thus, PaGalA was found to be better than
the commercial fungal enzyme in hydrolyzing lactose present in
acid whey. PaGalA also exhibited fairly good hydrolysis in sweet
whey resulting in 89.6% lactose hydrolysis after 8 h. The lactose
hydrolysis efficiency of PaGalA in sweet whey (89.6%) was found
which could hydrolyze 86.3% lactose after 8 h. Thus, PaGalA dis-
played better lactose hydrolytic capability over a wide pH range
than the commercially used -galactosidases.
[16] U.K. Laemmli, Nature 227 (1970) 680–685.
[17] T. Haider, Q. Husain, Chem. Eng. Process. 48 (2009) 576–580.
[18] I. Roy, M.N. Gupta, Process Biochem. 39 (2003) 325–332.
[19] Y.M. Li, L.L. Lu, H.M. Wang, X.D. Xu, M. Xiao, Appl. Environ. Microbiol. 75 (2009)
5938–5942.
[20] B. Henrissat, G. Davies, Curr. Opin. Struct. Biol. 7 (1997) 637–644.
[21] Y. Ito, T. Sasaki, K. Kitamoto, C. Kumagai, K. Takahashi, K. Gomi, G. Tamura, J.
Gen. Appl. Microbiol. 48 (2002) 135–142.
PaGalA also possessed glycosyl transferase activity at higher
concentrations of lactose (Fig. 5B). -Galactosidases isolated from
various fungi as well as a few recombinant fungal -galactosidases
have been employed either as free enzymes or as immobilized onto
a support for production of galacto-oligosaccharides. We moni-
tored the rate of GOS synthesis up to 6 h at 50 ◦C in the presence of
30% lactose and our data suggests that a maximum yield of 19.7%
GOS was synthesized in 4 h.
[22] A.L. Rojas, R.A.P. Nagem, K.N. Neustroev, J. Mol. Biol. 343 (2004) 1281–1292.
[23] C. Gamauf, M. Marchetti, J. Kallio, T. Puranen, J. Vehmaanpera, G. Allmaier, C.
Kubicek, B. Seiboth, FEBS J. 274 (2007) 1691–1700.
[24] T. Yuan, P. Yang, Y. Wang, K. Meng, H. Luo, W. Zhang, N. Wu, Y. Fan, B. Yao,
Biotechnol. Lett. 30 (2008) 343–348.
[25] M. Wanarska, J. Kur, R. Pladzyk, M. Turkiewicz, Acta Biochim. Pol. 52 (2005)
781–787.
[26] E. Halbmayr, G. Mathiesen, T. Nguyen, T. Maischberger, C. Peterbauer, V. Eijsink,
D. Haltrich, J. Agric. Food Chem. 56 (2008) 4710–4719.
[27] S. Gargova, I. Pishtijski, I. Stoilova, Biotechnol. Biotechnol. Equip. 9 (1995)
47–51.
[28] S. O’Connell, G. Walsh, Appl. Microbiol. Biotechnol. 86 (2010) 517–524.
[29] Z. Nagy, T. Kiss, A. Szentirmai, S. Biro, Protein Express. Purif. 21 (2001) 24–29.
[30] Y. Tanaka, A. Kagamiishi, A. Kiuchi, T. Horiuchi, J. Biochem. 77 (1975)
241–247.
[31] R.L. Brandão, J.R. Nicoli, A.F. Figueiredo, J. Dairy Sci. 70 (1987) 1331–1337.
[32] R. Gonzalez, M. Monsan, Enzyme Microb. Technol. 13 (1991) 349–352.
[33] F. Widmer, J.L. Leuba, Eur. J. Biochem. 100 (1979) 559–567.
[34] S.A. Ansari, Q. Husain, J. Mol. Catal. B: Enzym. 63 (2010) 68–74.
[35] J. Szczodrak, J. Mol. Catal. B: Enzym. 10 (2000) 631–637.
4. Conclusions
A -galactosidase gene (PaGalA) from the acidophilic fungus, P.
aerugineus was cloned and expressed at high levels in P. pastoris. The
-galactosidase belongs to GH family 35 and shares high degree of
sequence similarity with other fungal -galactosidases. The puri-
fied PaGalA displayed optimum activity at acidic pH and stability