Purification and characterization of a novel cold-adapted phytase from Rhodotorula mucilaginosa strain JMUY14 isolated from Antarctic.
A yeast producing a cold-adapted phytase was isolated from Antarctic deep-sea sediment and identified as a Rhodotorula mucilaginosa strain JMUY14 of basidiomycetous yeasts. It was cultured in fermentation optimized by a response surface methodology based on the Box-Behnken design. The maximum activity of phytase reached 205.447 U ml(-1), which was close to the predicted value of 201.948 U ml(-1) and approximately 3.4 times higher than its initial activity. The extracellular phytase was purified by 15.2-fold to homogeneity with a specific activity of 31,635 U mg(-1) by (NH4 )2 SO4 precipitation, and a combination of DEAE Sepharose Fast Flow, SP Sepharose Fast Flow, and Sephadex G-100. The molecular weight of the purified enzyme was estimated to be 63 kDa and its pI was 4.33. Its optimal temperature and pH were 50 °C and 5.0, respectively. Its activity was 85% at 37 °C, and showed good stability at pH 3.0 ∼ 7.0. When compared with mesophilic counterparts, the phytase not only exhibited a higher activity during 20 ∼ 30 °C but also had a low Km (247 µM) and high kcat (1394 s(-1)). The phytase activity was slightly stimulated in the presence of Mg(2+), Fe(2+), Fe(3+), K(+), Na(+), Ca(2+), EDTA, and EGTA and moderately inhibited by Cu(2+), Zn(2+), Mn(2+), Ag(+), PMSF, SDS, and phenylgloxal hydrate. It was resistant to both pepsin and trypsin. Since the phytase produced by the R. mucilaginosa JMUY14 showed a high specific activity, good pH stability, strong protease resistance, and high activity at low temperature, it has great potential for feed applications, especially in aquaculture.
College of Food and Bioengineering, Jimei University, Xiamen, China.|Fujian Provincial Key Laboratory of Food Microbiology and Enzyme Engineering, Xiamen, China.
Peng
P
Yu
College of Food and Bioengineering, Jimei University, Xiamen, China.|Fujian Provincial Key Laboratory of Food Microbiology and Enzyme Engineering, Xiamen, China.
Xue-Ting
XT
Wang
College of Food and Bioengineering, Jimei University, Xiamen, China.|Fujian Provincial Key Laboratory of Food Microbiology and Enzyme Engineering, Xiamen, China.
Jing-Wen
JW
Liu
D010832:6-Phytase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D000222:Adaptation, Physiological; D000595:Amino Acid Sequence; D000864:Antarctic Regions; D017756:Aquaculture; D003080:Cold Temperature; D004795:Enzyme Stability; D005285:Fermentation; D019015:Geologic Sediments / Q000382:microbiology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D010434:Pepsin A / Q000378:metabolism; D010802:Phylogeny; D012248:Rhodotorula / Q000201:enzymology* / Q000302:isolation & purification / Q000502:physiology; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism
D010832:6-Phytase; D014357:Trypsin; D010434:Pepsin A
Antarctic yeast; Cold-adapted phytase; Identification; Phytase purification and characterization