Isolation and Identification of Two Phytase-producing Strains from Soybean Rhizosphere Soil and Cloning of Neutral Phytase Gene

doi:10.7506/spkx1002-6630-201311036

Abstract

Abstract:

Two strains capable of enhanced phytase production were isolated from soybean rhizosphere soil. According
to morphological observation and 16S rRNA sequence analysis, both were identified as Bacillus subtilis. From both
stains, phytase-encoding genes, phy(ycD) and phy(ycE), were cloned by polymerase chain reaction. Sequence analysis was
conducted with BLASTn and BLASTp programs, available on the National Center for Biotechnology Information database
(NCBI). The results revealed that Phy(ycD) and phy(ycE) genes shared 98% identity at nucleic acid level. Each of them
contained an open reading frame of 1149 bp encoding 382 amino acids. The deduced polypeptide phy(ycD) and phy(ycE)
belong to beta-propeller phytases (BPPs). A signal peptide consisting of 26 residues was found at the N- terminus. The two
sequences were distinguished from each other by five amino acid substitutions. Homology modeling of these phytases was
carired out using SWISS-MODEL. The software Spdbv was used to evaluate the constructed model. The results showed that
four amino acid substitutions were located in different secondary structures. The cloning of phy(ycD) and phy(ycE) can lay a
good foundation for basic research and industrial application of natural phytase.

Key words: phytase gene, cloning, enzyme characteristics, Bacillus subtilis

CLC Number:

Q812

YAO Ming-ze，LU Wen-liang，ZHANG Yao-hua，LIANG Ai-hua. Isolation and Identification of Two Phytase-producing Strains from Soybean Rhizosphere Soil and Cloning of Neutral Phytase Gene[J]. FOOD SCIENCE, 2013, 34(11): 163-167.

References

参考文献：
[1] Reddy N R, Sathe S K, Salunkhe D K. Phytates in legumes and cereals. Advances in food research, 1982, 28:1-92.
[2] Bohn L, Meyer A S, Rasmussen S K. Phytate: impact on environment and human nutrition. A challenge for molecular breeding. Journal of Zhejiang University Science B, 2008, 9(3):165-191.
[3] Mullaney E J, Ullah A H. The term phytase comprises several different classes of enzymes. Biochemical and biophysical research communications, 2003, 312(1):179-184.
[4] Oh B C, Choi W C, Park S, et al. Biochemical properties and substrate specificities of alkaline and histidine acid phytases. Applied microbiology and biotechnology, 2004, 63(4):362-372.
[5] Idriss E E, Makarewicz O, Farouk A, et al. Extracellular phytase activity of Bacillus amyloliquefaciens FZB45 contributes to its plant-growth-promoting effect. Microbiology, 2002, 148(Pt 7):2097-2109.
[6] Rao D E, Rao K V, Reddy V D. Cloning and expression of Bacillus phytase gene (phy) in Escherichia coli and recovery of active enzyme from the inclusion bodies. Journal of applied microbiology, 2008, 105(4):1128-1137.
[7] 丁强, 杨培龙, 黄火清, 等. 植酸酶发展现状和研究趋势[J]. 中国农业科技导报, 2010(03).
[8] Makarewicz O, Dubrac S, Msadek T, et al. Dual role of the PhoP approximately P response regulator: Bacillus amyloliquefaciens FZB45 phytase gene transcription is directed by positive and negative interactions with the phyC promoter. Journal of bacteriology, 2006, 188(19):6953-6965.
[9] 宋大新, 范长胜, 徐德强, 等. 微生物实验教程[ M ] 上海:复旦大学出版社, 1989:79-103.
[10] Oh B C, Chang B S, Park K H, et al. Calcium-dependent catalytic activity of a novel phytase from Bacillus amyloliquefaciens DS11. Biochemistry, 2001, 40(32):9669-9676.
[11] Kerovuo J, Lappalainen I, Reinikainen T. The metal dependence of Bacillus subtilis phytase. Biochemical and biophysical research communications. 2000, 268(2):365-369.
[12] Tran T T, Hashim S O, Gaber Y, et al. Thermostable alkaline phytase from Bacillus sp. MD2: effect of divalent metals on activity and stability. Journal of inorganic biochemistry, 2011, 105(7):1000-1007.
[13] Gulati H K, Chadha B S, Saini H S. Production and characterization of thermostable alkaline phytase from Bacillus laevolacticus isolated from rhizosphere soil. Journal of industrial microbiology & biotechnology, 2007, 34(1):91-98.
[14] Hu Y, Wang H N, Wu Q, et al. Enzymological properties of a phytase from Bacillus subtilis WHNB 02. J Southwest Agric Univ, 2005, 27(6):770-773.
[15] Zhang R, Yang P, Huang H, et al. Molecular and biochemical characterization of a new alkaline beta-propeller phytase from the insect symbiotic bacterium Janthinobacterium sp. TN115. Applied microbiology and biotechnology, 2011, 92(2):317-325.