Cloning and characterization of a novel phytase from wastewater treatment yeast Hansenula fabianii J640 and expression in Pichia pastoris.
Phosphohydrolysis of organic phosphorus compounds by acid phosphatases (EC 3.1.3.1 and EC 3.1.3.2) is an important method for efficient removal of phosphorus from high concentration organic wastewater. Another important method is supplementation of animal feed with phytase (EC 3.1.3.8 and EC 3.1.3.26), which improves the availability of phytate-phosphates (phosphate that are hydrolyzed by phytases), making it possible to add less phosphate to animal feed and resulting in the excretion of less phosphorus by the animals. In the present study, we purified a novel phytase from the wastewater treatment yeast Hansenula fabianii J640 (Hfphytase), cloned the 1456 bp open reading frame (ORF) encoding Hfphytase, and characterized Hfphytase. The molecular weight of Hfphytase after deglycosylation by PNGaseF was 49 kDa. The optimal pH and temperature for enzyme activity were 4.5 and 50 degrees C, respectively. Hfphytase exhibits 40% identity with Debaryomyces castellii phytase, 37% identity with Aspergillus niger PhyB, and 34% identity with Saccharomyces cerevisiae Pho5p. Recombinant Hfphytase was transformed and expressed in Pichia pastoris. The yield was 23 g/l by jar fermenter cultivation. The marked phosphohydrolysis activity exhibited by Hfphytase on six substrates (pNP-P, sodium phytate, glucose-1 phosphate, glucose-6 phosphate, alpha-glycerophosphate and beta-glycerophosphate) indicated that it is a non-specific acid phosphatase.
Watanabe
Takashi
T
Graduate School of Biosphere Science, Hiroshima University, Kagamiyama 1-4-4 Higashihiroshima, Hiroshima 739-8527, Japan.
Ikeda
Hiroko
H
Masaki
Kazuo
K
Fujii
Tsutomu
T
Iefuji
Haruyuki
H
D010832:6-Phytase / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D001483:Base Sequence; D001709:Biotechnology / Q000379:methods; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D005285:Fermentation; D005821:Genetic Techniques; D019298:Glucose-6-Phosphate / Q000737:chemistry; D005994:Glycerophosphates / Q000737:chemistry; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics* / Q000378:metabolism*; D014871:Water Microbiology; D018508:Water Purification
D017931:DNA Primers; D005994:Glycerophosphates; D019298:Glucose-6-Phosphate; C029620:alpha-glycerophosphoric acid; D010832:6-Phytase; C031463:beta-glycerophosphoric acid