Sato Vanessa Sayuri, Jorge João Atílio, Guimarães Luis Henrique Souza
Institute of Chemistry of Araraquara, UNESP, Rua Francisco Degni s/n, 14800-900, Araraquara, São Paulo, Brazil.
Faculty of Philosophy, Sciences and Letters of Ribeirão Preto, USP, Avenida Bandeirantes 3900, Monte Alegre, 14040-901, Ribeirão Preto, São Paulo, Brazil.
Appl Biochem Biotechnol. 2016 Jun;179(4):610-24. doi: 10.1007/s12010-016-2018-7. Epub 2016 Feb 23.
The Rhizopus microsporus var. microsporus biofilm was able to produce increased levels of an extracellular thermotolerant phytase using polyethylene and viscose as an inert support in both modified NBRIP medium and modified Khanna medium containing sugarcane bagasse as the carbon source. The enzyme production was strictly regulated by the phosphorus content with optimal production at 0.5 mM of sodium phytate and KH2PO4. The extracellular phytase, RMPhy1, was purified 4.18-fold with 4.78 % recovery using DEAE-cellulose and CM-cellulose. A single protein band with a molecular mass of 35.4 kDa was obtained when the samples were subjected to 10 % SDS-PAGE. The optimum temperature for activity was 55 °C and the optimum pH was 4.5. R. microsporus var. microsporus phytase exhibited high stability at 30 and 40 °C with a half-life of 115 min at 60 °C. The enzyme activity increased in the presence of Ca (2+) and was inhibited by Zn(2+), arsenate, and sodium phosphate. Phytase demonstrated high substrate specificity for sodium phytate with K m = 0.72 mM and V max = 94.55 U/mg of protein and for p-NPP with K m = 0.04 mM and V max = 106.38 U/mg of protein. The enzyme also hydrolyzed ATP, AMPc, glucose 6-phosphate, glucose 1-phosphate, and UDPG. This is the first report on phytase characterization delivered with biofilm technology. The properties of the enzyme account for its high potential for use in biotechnology and the possibility of application in different industrial sectors as feed in the future.
微小根毛霉微小根毛霉生物膜能够在含有甘蔗渣作为碳源的改良NBRIP培养基和改良Khanna培养基中，以聚乙烯和粘胶作为惰性载体，产生更高水平的胞外耐热植酸酶。酶的产生受到磷含量的严格调控，在0.5 mM植酸钠和KH₂PO₄时产量最佳。使用DEAE-纤维素和CM-纤维素对胞外植酸酶RMPhy1进行纯化，纯化倍数为4.18倍，回收率为4.78%。样品进行10% SDS-PAGE时，得到一条分子量为35.4 kDa的单一蛋白条带。酶活性的最适温度为55℃，最适pH为4.5。微小根毛霉微小根毛霉植酸酶在30℃和40℃时表现出高稳定性，在60℃时半衰期为115分钟。酶活性在Ca(2+)存在时增加，受到Zn(2+)、砷酸盐和磷酸钠的抑制。植酸酶对植酸钠表现出高底物特异性，K m = 0.72 mM，V max = 94.55 U/mg蛋白质；对p-NPP表现出高底物特异性，K m = 0.04 mM，V max = 106.38 U/mg蛋白质。该酶还能水解ATP、AMPc、葡萄糖6-磷酸、葡萄糖1-磷酸和UDPG。这是关于利用生物膜技术进行植酸酶表征的首次报道。该酶的特性表明其在生物技术领域具有很高的应用潜力，未来有可能应用于不同工业部门作为饲料。
