| Phytase as a kind of green feed additive, could effectively improve the availability ofphytate-P and cut down the anti-nutrition function of phytate, which leads to the lowerproduction cost and more friendly to the environment. At present, the phytase major used in feedindustry are mesophilic and thermophilic phytase, which have a low catalytic activity at lowtemperatures compared with low-temperature phytase. While the physiological temperature ofmonogastric animals is39℃, and it is needed to has higher catalytic activity under the conditionof less than28℃in aquaculture. Therefore, the low-temperature phytase has great potential forfeed applications.Using two step methods, ten phytase producing microorganisms were isolated fromAntarctic deep-sea sediments. One isolate named JMUY14showed high phytase activity. It wasidentified as Rhodotorula sp. based on morphological, physiological and biochemicalcharacteristics,26S rRNA ITS sequence phylogenetic analysis. To our knowledge, this is thefirst report of a phytase from Rhodotorula sp.Singel-factor experiments and response surface methodology were applied for theoptimization of phytase production by Rhodotorula sp. JMUY14. The results were as follows:glucose4%, peptone2%, NaNO31.5%, KCl0.025%, MgSO4·7H2O0.025%, MnSO4·H2O0.002%, FeSO4·7H2O0.002%, fermentation time48h, fermentation temperature25℃, mediuminitial pH5.0, and inoculum size10%. By the analysis of singel-factor experiment results,fermentation temperature (℃), inoculum size(%) and peptone additive amount(%) were selectedas significant factors and optimized using a Box-Behnken Design (BBD) subsequently. Theresults were as follows: fermentation temperature was26℃, inoculnm size was10.4%, andpeptone additive amount was2.3%, respectively. Finally, the phytase activity reached205.447U/ml, which was in accordance with that of software predicted (201.948U/ml) and wasapproximately3.4times higher than the initial enzyme activity.The extracellular phytase of Rhodotorula sp. JMUY14was purified by ammonium sulfateprecipitation, DEAE Sepharose Fast Flow anion exchange, SP Sepharose Fast Flow cationexchange and Sephadex G-100gel filtration with the specific activity of31635U/mg. Theactivity recovery was5%with purification folds of15.16. The following main results of phytasecharacterization were obtained:(1) The isoelectric point (pI) and molecular weight were4.33and 63kDa, respectively;(2) The optimum temperature was50℃and retained phytase activity of17.5%and85%at10℃and37℃respectively, which showed that it was a low-temperaturephytase. The residual activities were64.8%and41%after being incubated at45℃and50℃for50min, respectively. When it was incubated at55℃for10min,20min,30min,40min,50min, theresidual phytase activities were45.7%,36.2%,22.9%,21.0%, and12.4%, respectively. Whileincubated at60℃or65℃for10min let to phytase inactivation;(3) The optimum pH was5.0and it was stable at pH2.0～8.0, which reflected the pH stability of this phyase. It could play arole in the gastrointestinal tract of monogastric animals;(4) Phytase activity was stimulated byMg2+, Fe2+, Fe3+, K+, Na+, Ca2+, EDTA, EGTA and inhibited by Cu2+, Zn2+, Mn2+, Ag+, PMSF,Phenylgloxal hydrate, and SDS. Phenylgloxal hydrate was the specificity inhibitor of histidineacid phosphatase (HAP). Therefore, the phytase of Rhodotorula sp. JMUY14should belong toHAP;(5) After being incubated in media containing0.1mg/ml pepsin and trypsin at37℃for1h,the residual activity were92.2%and75.6%, respectively, indicating its strong protease resistance;(6) Kenetic analysis indicated a high-affinity apparent Km value of0.247mM/0.817mM and anapparent Vmax value of0.0265mM/min and0.0218mM/min for phytate and sodium phytate,respectively. |
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