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http://dx.doi.org/10.4014/jmb.0904.04003

Molecular Cloning and Expression of a Novel Protease-resistant GH-36 $\alpha$-Galactosidase from Rhizopus sp. F78 ACCC 30795  

Yanan, Cao (Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences)
Wang, Yaru (Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences)
Luo, Huiying (Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences)
Shi, Pengjun (Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences)
Meng, Kun (Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences)
Zhou, Zhigang (Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences)
Zhang, Zhifang (Biotechnology Research Institute, Chinese Academy of Agricultural Sciences)
Yao, Bin (Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences)
Publication Information
Journal of Microbiology and Biotechnology / v.19, no.11, 2009 , pp. 1295-1300 More about this Journal
Abstract
A 2,172-bp full-length gene (aga-F78), encoding a protease-resistant $\alpha$-galactosidase, was cloned from Rhizopus sp. F78 and expressed in Escherichia coli. The deduced amino acid sequence shared highest identity (45.0%) with an $\alpha$-galactosidase of glycoside hydrolase family 36 from Absidia corymbifera. After one-step purification with a Ni-NTA chelating column, the recombinant Aga-F78 migrated as a single band of ~82 and ~210 kDa on SDS-PAGE and nondenaturing gradient PAGE, respectively, indicating that the native structure of the recombinant Aga-F78 was a trimer. Exhibiting the similar properties as the authentic protein, purified recombinant Aga-F78 was optimally active at $50^{\circ}C$ and pH 4.8, highly pH stable over the pH range 5.0-10.0, more resistant to some cations and proteases, and had wide substrate specificity (pNPG, melidiose, raffinose, and stachyose). The recombinant enzyme also showed good hydrolytic ability to soybean meal, releasing galactose of $415.58\;{\mu}g/g$ soybean meal. When combined with trypsin, the enzyme retained over 90% degradability to soybean meal. These favorable properties make Aga-F78 a potential candidate for applications in the food and feed industries.
Keywords
$\alpha$-Galactosidase; molecular cloning; Rhizopus sp. F78; protease-resistant;
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1 Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254   DOI   PUBMED   ScienceOn
2 Fridjonsson, O., H. Watzlawick, A. Gehweiler, T. Rohrhirsch and R. Mattes. 1999. Cloning of the gene encoding a novel thermostable $\alpha$-galactosidase from Thermus brockianus ITI360. Appl. Environ. Microb. 65: 3955-3963   ScienceOn
3 Liu, Y. G. and R. F. Whittier. 1995. Thermal asymmetric interlaced PCR: Automatable amplification and sequencing of insert end fragment from PI and YAC clones for chromosome walking. Genomics 25: 674-681   DOI   ScienceOn
4 Luonteri, E., M. Tenkanen, and L. Viikari. 1998. Substrate specificities of Penicillium simplicissimum $\alpha$-galactosidases. Enzyme Microb. Technol. 22: 192-198   DOI   ScienceOn
5 Mi, S., K. Meng, Y. Wang, Y. Bai, T. Yuan, H. Luo, and B. Yao. 2007. Molecular cloning and characterization of a novel $\alpha$-galactosidase gene from Penicillium sp. F63 CGMCC 1669 and expression in Pichia pastoris. Enzyme Microb. Technol. 40: 1373-1380   DOI   ScienceOn
6 Somiari, R. I. and E. Balogh. 1993. Effect of soaking, cooking and crude $\alpha$-galactosidase treatment on the oligosaccharide content of cowpea flours. J. Sci. Food Agric. 61: 339-343   DOI   ScienceOn
7 Rai, M. and H. Padh. 2001. Expression systems for production of heterologous proteins. Curr. Sci. India 80: 1121-1128   ScienceOn
8 Linden, J. C. 1982. Immobilized $\alpha$-D-galactosidase in the sugar beet industry. Enzyme Microb. Technol. 4: 130-136   DOI   ScienceOn
9 Ademark, P., M. Larsson, F. Tjemeld, and H. Stalbrand. 2001. Multiple $\alpha$-galactosidases from Aspergillus niger: Purification, characterization and substrate specificities. Enzyme Microb. Technol. 29: 441-448   DOI   ScienceOn
10 Clarke, J. H., K. Davidson, J. E. Rixon, J. R. Halstead, M. P. Fransen, H. J. Gilbert, and G. P. Hazlewood. 2000. A comparison of enzyme-aided bleaching of softwood paper pulp using combinations of xylanase, marmanase and $\alpha$-galactosidase. Appl. Microbiol. Biotechnol. 53: 661-667   DOI   ScienceOn
11 Baik, S. H., K. Saito, A. Yokota, K. Asano, and F. Tomita. 2000. Molecular cloning and high-level expression in Escherichia coli of fungal $\alpha$-galactosidase from Abisidia corymbifera IFO8084. J. Biosci, Bioeng. 90: 168-173   ScienceOn
12 Varbanets, L. D., V. M. Malanchuk, T. T. Buglova, and R. A. Kuhlmann. 2001. Penicillium sp. 23 alpha-galactosidase: Purification and substrate specificity. Carbohydr. Polym. 44: 357-363   DOI   ScienceOn
13 Ishiguro, M., K. Kaneko, A. Kuno, Y. Koyama, S. Yoshida, G G Park, Y. Sakakibara, I. Kusakabe, and H. Kobayashi. 2001. Purification and characterization of the recombinant Thermus sp. strain T2 $\alpha$-galactosidase expressed in Escherichia coli. Appl. Environ. Microbial. 67: 1601-1606   DOI   ScienceOn
14 Li, S., W. D. Kim, S. Kaneko, P. A. Prema, M. Nakajima, and H. Kobayashi. 2007. Expression of rice (Oryza sativa L. var. Nipponbare) $\alpha$-galactosidase genes in Escherichia coli and characterization. Biosci. Biotechnol. Biochem. 71: 520-526   DOI   ScienceOn
15 Mulimani, V. H. and Rarnalingam. 1995. Enzymic hydrolysis of raffinose and stachyose in soymilk by alpha-galactosidase from Gibberella fujikuroi. Biochem. Mol. Biol. Int. 36: 897-905   PUBMED   ScienceOn
16 Graham, G. C., P. Mayers, and R. J. Henry. 1994. A simplified method for the preparation of fungal genomic DNA for PCR and RAPD analysis. Biotechniques 16: 48-50   PUBMED   ScienceOn
17 Soh, C., Z. M. Ali, and H. Lazan. 2006. Characterisation of an $\alpha$-galactosidase with potential relevance to ripening related texture changes. Phytochemistry 67: 242-254   DOI   ScienceOn
18 Suryani, T. Kimura, K. Sakka, and K. Ohmiya, 2003. Cloning, sequencing and expression of the gene encoding the Clostridium stercorarium $\alpha$-galactosidase Aga36A in Escherichia coli. Biosci. Biotech Biochem. 67: 2160-2166   DOI   ScienceOn
19 Tsuboi, K. 2007. Enzyme replacement therapy in patients with Fabry's disease. J. Int. Med. Res. 35: 574-581   DOI   PUBMED   ScienceOn
20 Bishop, D. F., D. H. Calhoun, H. S. Bemtein, P. Hantzopoulos, M. Quinn, and R. J. Desnick. 1986. Human $\alpha$-galactosidase A: Nucleotide sequence of a cDNA clone encoding the mature enzyme. Proc. Natl. Acad. Sci. U.S.A. 83: 4859-4863   DOI   ScienceOn
21 Civas, A., R. Eberhard, P. Le Dizet, and F. Petek. 1984. Glycosidases induced in Aspergillus tamarii. Mycelial alpha-D-galactosidases. J. Biochem. 219: 849-855   ScienceOn
22 Puchal, F. 1999. Role of feed enzyme in poultry nutrition examined. Feedstuffs: 11: 12-14
23 Kondoh, K., K. Morisaki, W. Kim, G. Park, S. Kaneko, and H. Kobayashi. 2005. Cloning and expression of the gene encoding Streptomyces coelicolor A3(2) alpha-galactosidase belonging to family 36. Biotechnol. Lett. 27: 641-647   DOI   ScienceOn
24 Zhang, Y., F. Gong, G. Bao, H. Gao, S. Ji, Y. Tan, et al. 2007. B to O erythrocyte conversion by the recombinant $\alpha$-galactosidase. Chin. Med. J. 120: 1145-1150   PUBMED   ScienceOn
25 Veldman, A. V., W. A. G. Veen, D. Barug, and P. A. Van Paridon. 1993. Effects of $\alpha$-galactosidase in feed on ileal piglet digestive physiology. J. Anim. Physiol. Anim. Nutr. 69: 57-65   DOI   ScienceOn
26 Igbasan, F. A., W. Guenter, and B. A. Slominski. 1997. The effects of pectinase and $\alpha$-galactosidase supplementation on the nutritive value of peas for broiler chickens. Can. J. Anim. Sci. 77: 537-539   DOI   ScienceOn
27 Cao, Y., P. Yang, P. Shi, Y. Wang, H. Luo, K. Meng, et al. 2007. Purification and characterization of a novel protease-resistant $\alpha$-galactosidase from Rhizopus sp. F78 ACCC 30795. Enzyme Microb. Technol. 41: 835-841   DOI   ScienceOn
28 Ghazi, S., J. A. Rooke, and H. Galbraith. 2003. Improvement of the nutritive value of soybean meal by protease and $\alpha$-galactosidase treatment in broiler cockerels and broiler chicks. Brit. Poult. Sci. 44: 410-418   DOI   ScienceOn