4824 J. Agric. Food Chem., Vol. 58, No. 8, 2010
Chen et al.
band on SDS-PAGE, one peak by gel filtration HPLC, and
two peaks by reverse-phase HPLC. Attempting to N-terminal
sequence the recombinant enzyme failed probably due to
glycosylation issues. Considering that the biochemical pro-
perties of recombinant enzyme in the present study are similar
to those of the wild-type, it is assumed that the recombinant
enzyme was also proteolyzed after synthesis and formed a
heterosubunit protein. If this is correct, it may be presumed that
the protease which hydrolyzes the pro-R-glucosidase has no strict
substrate specificity. It not only exists in A. niger but also exists
in P. pastoris. Further information is needed to support this
hypothesis.
In summary, cDNA of A. niger R-glucosidase was cloned,
expressed and characterized in detail. The expression level of 2.07
U/mL and 2.21 U/mg protein obtained in the culture media in the
present study represents the highest yield of A. niger R-glucosi-
dase reported so far. Detailed biochemical characterization
demonstrated that the recombinant enzyme from P. pastoris
was similar to that of native R-glucosidase from A. niger,
suggesting that the recombinant enzyme has been successfully
post-translationally modified in the P. pastoris expression system.
Further enhancement of the yield of the recombinant R-glucosi-
dase by fermentation technology is currently underway in our
laboratory, and these studies will provide the basis for the
application ofrecombinant R-glucosidaseinthe industryofIMOs
production.
(10) Prodanovic, R.; Milosavic, N.; Sladic, D.; Zlatovic, M.; Bozic, B.;
Velickovic, T. C.; Vujcic, Z. Transglucosylation of hydroquinone
catalysed by alpha-glucosidase from baker’s yeast. J. Mol. Catal. B:
Enzym. 2005, 35 (4-6), 142–146.
(11) Nakamura, A.; Nishimura, I.; Yokoyama, A.; Lee, D.-G.; Hidaka,
M.; Masaki, H.; Kimura, A.; Chiba, S.; Uozumi, T. Cloning and
sequencing of an alpha-glucosidase gene from Aspergillus niger and
its expression in A. nidulans. J. Biotechnol. 1997, 53, 75–84.
(12) Ogawa, M.; Nishio, T.; Minoura, K.; Uozumi, T.; Wada, M.;
Hashimoto, N.; Kawachi, R.; Oku, T. Recombinant alpha-glucosi-
dase from Aspergillus niger. Overexpression by Emericella nidulans,
purification and Characterization. J. Appl. Glycosci. 2006, 53, 13–16.
(13) Lee, D. G.; Nishimura-Masuda, I.; Nakamura, A.; Hidaka, M.;
Masaki, H.; Uozumi, T. Overproduction of aphla-glucosidase in
Aspergillus niger transformed with the cloned gene aglA. J. Gen.
Appl. Microbiol. 1998, 44, 177–181.
(14) Ruanglek, V.; Sriprang, R.; Ratanaphan, N.; Tirawongsaroj, P.;
Chantasigh, D.; Tanapongpipat, S.; Pootanakit, K.; Eurwilaichitr,
L. Cloning, expression, characterization and high cell-density pro-
duction of recombinant endo-1,4-β-xylanase from Aspergillus niger
in Pichia pastoris. Enzyme Microb. Technol. 2007, 41 (1-2), 19–25.
(15) Macauley-Patrick, S.; Fazenda, M. L.; McNeil, B.; Harvey, L. M.
Heterologous protein production using the Pichia pastoris expres-
sion system. Yeast 2005, 22 (4), 249–270.
(16) Tong, X.; Tang, Q.; Wu, Y.; Wu, J.; Chen, J. Cloning of the gene
encoding aphla-glucosidase from Aspergillus niger and its expression
in Pichia pastoris. Acta Microbiol. Sin. 2009, 49 (2), 262–268.
(17) Chantasingh, D.; Pootanakit, K.; Champreda, V.; Kanokratana, P.;
Eurwilaichitr, L. Cloning, expression and characterization of a
xylanase 10 from Aspergillus terreus (BCC129) in Pichia pastoris.
Protein Expression Purif. 2006, 46 (1), 143–149.
LITERATURE CITED
(18) Nashiru, O.; Koh, S.; Lee, S.-Y.; Lee, D.-S. Novel R-glucosidase
from extreme thermophile Thermus caldophilus GK24. J. Biochem.
Mol. Biol. 2001, 34 (4), 347–354.
(19) Chen, X.; Cao, Y.; Ding, Y.; Lu, W.; Li, D. Cloning, functional
expression and characterization of Aspergillus sulphureus beta-
mannanase in Pichia pastoris. J. Biotechnol. 2007, 128 (3), 452–461.
(20) Tseng, S. J.; Hsu, J. P. A Comparison of the Parameter Estimating
Procedures for the Michaelis-Menten Model. J. Theor. Biol. 1990,
145 (4), 457–464.
(1) Kimura, A.; Takata, M.; Sakai, O.; Matsui, H.; Takai, N.;
Takayanagi, T.; Nishimura, I.; Uozumi, T.; Chiba, S. Complete
amino acid sequence of crystalline alpha-glucosidase from Aspergil-
lus niger. Biosci., Biotechnol., Biochem. 1992, 56 (8), 1368–1370.
(2) Kita, A.; Matsui, H.; Somoto, A.; Kimura, A.; Takata, M.; Chiba, S.
Substrate specificity and subsite affinities of crystalline alpha-glu-
cosidase from Aspergillus niger. Agric. Biol. Chem. 1991, 55 (9),
2327–2335.
(3) Kato, N.; Suyama, S.; Shirokane, M.; Kato, M.; Kobayashi, T.;
Tsukagoshi, N. Novel alpha-glucosidase from Aspergillus nidulans
with strong transglycosylation activty. Appl. Environ. Microbiol.
2002, 68 (3), 1250–1256.
(21) Olusanya, O.; Olutiola, P. O. Characterisation of maltase from
enteropathogenic Escherichia coli. FEMS Microbiol. Lett. 1986, 36
(2-3), 239–244.
(22) Yamasaki, Y.; Suzuki, Y.; Ozawa, J. Certain properties of alpha-
glucosidase from Mucor racemosus. Agric. Biol. Chem. 1997, 41,
1559–1565.
(23) Raasch, C.; Streit, W.; Schanzer, J.; Bibel, M.; Gosslar, U.; Liebl, W.
Thermotoga maritima Agla, an extremely thermostable NADþ,
Mn2þ, and thiol-dependent alpha-glucosidase. Extremophiles 2000,
4 (4), 189–200.
(24) Cregg, J. M.; Cereghino, J. L.; Shi, J.; Higgins, D. R. Recombinant
protein expression in Pichia pastoris. Mol. Biotechnol. 2000, 16 (1),
23–52.
(4) Mala, S.; Dvorakova, H.; Hrabal, R.; Kralova, B. Towards regio-
selective synthesis of oligosaccharides by use of alpha-glucosidases
with different substrate-specificity. Carbohydr. Res. 1999, 322, 209–
218.
(5) Fernandez-Arrojo, L.; Marin, D.; De Segura, A. G.; Linde, D.;
Alcalde, M.; Gutierrez-Alonso, P.; Ghazi, I.; Plou, F. J.; Fernandez-
Lobato, M.; Ballesteros, A. Transformation of maltose into pre-
biotic isomaltooligosaccharides by a novel alpha-glucosidase from
Xantophyllomyces dendrorhous. Process Biochem. 2007, 42 (11),
1530–1536.
(25) Lee, S. S.; He, S.; Withers, S. G. Identification of the catalytic
nucleophile of the Family 31 alpha-glucosidase from Aspergillus
niger via trapping of a 5-fluoroglycosyl-enzyme intermediate. Bio-
chem. J. 2001, 359, 381–386.
(6) McCleary, V. B.; Gibson, T. S.; Sheehan, H.; Casey, A.; Horgan, L.;
O’Flaherty, J. Purification, properties and industrial significance of
transglucosidase from Aspergillus niger. Carbohydr. Res. 1989, 185
(1), 147–162.
(7) Duan, K. J.; Sheu, D. C.; Lin, M. T.; Hsueh, H. C. Reaction
mechanism of isomaltooligosaccharides synthesis by alpha-glucosi-
dase from Aspergillus carbonarious. Biotechnol. Lett. 1994, 16 (11),
1151–1156.
Received for review January 6, 2010. Revised manuscript received
March 17, 2010. Accepted March 23, 2010. This work was supported
financially by the National Outstanding Youth Foundation of China
(20625619), Research Program of State Key Laboratory of Food
Science and Technology (SKLF-MB-200802, SKLF-TS-200910,
SKLF-KF-200909), Program of Innovation Team of Jiangnan
University (2008CXTD01), Public Topic of Key Laboratory of
Industrial Biotechnology, Ministry of Education, Jiangnan University
(KLIB-KF200904) and the Key Program of National Natural Science
Foundation of China (No. 20836003).
(8) Mori, T.; Nishimoto, T.; Okura, T.; Chaen, H.; Fukuda, S. Purifica-
tion and characterization of cyclic maltosyl-(1f 6)-maltose hydro-
lase and alpha-glucosidase from an Arthrobacter globiformis strain.
Biosci., Biotechnol., Biochem. 2008, 72 (7), 1673–1681.
(9) Li, y.-J.; Zhao, G.-Y.; Du, W.; Zhang, T.-J. Effect of dietary
isomaltooligosaccharides on nutrient digestibility and concentration
of glucose, insulin, cholesterol and triglycerides in serum of growing
pigs. Anim. Feed Sci. Technol. 2009, 151 (3-4), 312–315.