Gene cloning, purification, and characterization of a heat-stable phytase from the fungus Aspergillus fumigatus

Phytase hydrolyzes phytate to release inorganic phosphate, which would decrease the addition of phosphorus to feedstuffs for monogastric animals and thus reduce environmental pollution. The gene encoding phytase from Bacillus sp. DS11 was cloned in Escherichia coli and its sequence determined. A 560-bp DNA fragment was used as a probe to screen the genomic library. It was obtained through PCR of Bacillus sp. DS11 chromosomal DNA and two oligonucleotide primers based on Nterminal amino acid sequences of the purified protein and the cyanogen bromide-cleaved 21-kDa fragment. The phy cloned was encoded by a 2.2-kb fragment. This gene comprises 1152 nucleotides and encodes a polypeptide of 383 amino acids with a deduced molecular mass of 41 808 Da. Phytase was produced to 20% content of total soluble proteins in E. coli BL21 (DE3) using the pET22b(+) vector with the inducible T7 promoter. This is the first nucleic sequence report on phytase from a bacterial strain. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V.

Section: Nucleotide Sequence Of Phymentioning

confidence: 99%

“…[8], Escherichia coli [9], Bacillus subtilis [10,11]. However, the cloning and sequencing of the phytase gene have been only reported for fungal phytases such as A. niger [12^14], A. fumigatus [15], A. terrus, and Myceliophthora thermophila [16] and there have been no reports about bacterial phytase genes until now.…”

Section: Introductionmentioning

confidence: 99%

Cloning of the thermostable phytase gene (phy) from Bacillus sp. DS11 and its overexpression in Escherichia coli

Kim¹

1998

“…It also reduces the amount of phosphorus in animal manure, thereby helping decrease phosphorus pollution in the environment. Not many phytases have been exploited in the feed industry because of several factors including high manufacturing costs, poor stability, and low specific activity of the enzyme in the environment of desired applications (Pasamontes et al, 1997;Rodriguez et al, 2000;Wang et al, 2007). Previously, phytases have been screened from various fungi in Thailand's BIOTEC culture collection.…”

Section: Introductionmentioning

confidence: 99%

Cell-surface phytase onPichia pastoriscell wall offers great potential as a feed supplement

Harnpicharnchai

Sornlake

Tang

et al. 2010

Cell-surface expression of phytase allows the enzyme to be expressed and anchored on the cell surface of Pichia pastoris. This avoids tedious downstream processes such as purification and separation involved with extracellular expression. In addition, yeast cells with anchored proteins can be used as a whole-cell biocatalyst with high value added. In this work, the phytase was expressed on the cell surface of P. pastoris with a glycosylphosphatidylinositol anchoring system. The recombinant phytase was shown to be located at the cell surface. The cell-surface phytase exhibited high activity with an optimal temperature at 50-55 degrees C and two optimal pH peaks of 3 and 5.5. The surface-displayed phytase also exhibited similar pH stability and pepsin resistance to the native and secreted phytase. In vitro digestibility test showed that P. pastoris containing cell-surface phytase released phosphorus from feedstuff at a level similar to secreted phytase. Yeast cells expressing phytase also provide additional nutrients, especially biotin and niacin. Thus, P. pastoris with phytase displayed on its surface has a great potential as a whole-cell supplement to animal feed.

“…A number of microorganisms, mainly of fungal and bacterial origin, are capable of phytase synthesis [7][8][9][10][11][12][13][14][15]. Recently, artificial phytases with superior thermostability were designed using semi-rational sequence comparison methods based upon several mesophilic homologues phytases from fungi (with sequences identity of about 50-70%) [16].…”

Section: Introductionmentioning

confidence: 99%

Gene cloning, expression and characterization of novel phytase from Obesumbacterium proteus

Zinin

Serkina

Gelfand

et al. 2004

The gene phyA encoding phytase was isolated from Obesumbacterium proteus genomic library and sequenced. The cleavage site of the PhyA signal peptide was predicted and experimentally proved. The PhyA protein shows maximum identity of 53% and 47% to phosphoanhydride phosphorylase from Yersinia pestis and phytase AppA from Escherichia coli, respectively. Based on protein sequence similarity of PhyA and its homologs, the phytases form a novel subclass of the histidine acid phosphatase family. To characterize properties of the PhyA protein, we expressed the phyA gene in E. coli. The specific activity of the purified recombinant PhyA was 310 U mg(-1) of protein. Recombinant PhyA showed activity at pH values from 1.5 through 6.5 with the optimum at 4.9. The temperature optimum was 40-45 degrees C at pH 4.9. The Km value for sodium phytate was 0.34 mM with a Vmax of 435 U mg(-1).