Neira-Vielma Alberto A, Aguilar Cristóbal N, Ilyina Anna, Contreras-Esquivel Juan C, Carneiro-da-Cunha María das Graça, Michelena-Álvarez Georgina, Martínez-Hernández José L
Group of Bioprocesses, Food Research Dept. School of Chemistry, Universidad Autónoma de Coahuila, Blvd. V. Carranza S/N. Col. República, CP 25280, Saltillo, Coahuila, México, México.
Departamento de Bioquímica, Universidade Federal de Pernambuco-UFPE, Av. Prof. Moraes Rego s/n, CEP 50.670-420, Recife, PE, Brazil.
Biotechnol Rep (Amst). 2017 Dec 15;17:49-54. doi: 10.1016/j.btre.2017.12.004. eCollection 2018 Mar.
In this study, an extracellular phytase produced by 7A-1, was biochemically characterized for possible industrial application. The enzyme was purified from a crude extract obtained by solid-state fermentation (SSF) of triticale waste. The extract was obtained by microfiltration, ultrafiltration (300, 100 and 30 kDa) and DEAE-Sepharose column chromatography. The molecular weight of the purified enzyme was estimated to be 89 kDa by SDS-PAGE. The purified enzyme was most active at pH 5.3 and 56 °C, and retained 50% activity over a wide pH range of 4 to 7. The enzymatic thermostability assay showed that the enzyme retained more than 70% activity at 80 °C for 60 s, 40% activity for 120 s and 9% after 300 s. The phytase showed broad substrate specificity, a value of 220 μM and of 25 μM/min. The purified phytase retained 50% of its activity with phosphorylated compounds such as phenyl phosphate, 1-Naphthyl phosphate, 2-Naphthyl phosphate, p-Nitrophenyl phosphate and Glycerol-2-phosphate. The inhibition of phytase activity by metal ions was observed to be drastically inhibited (50%) by Ca and was slightly inhibited (10%) by Ni, K, and Na, at 10 and 20 mM concentrations. A positive effect was obtained with Mg, Mn, Cu, Cd and Ba at 25 and 35% with stimulatory effect on the phytase activity.
在本研究中，对7A-1产生的一种胞外植酸酶进行了生物化学特性分析，以探讨其可能的工业应用。该酶从黑小麦废料固态发酵（SSF）获得的粗提物中纯化得到。粗提物通过微滤、超滤（300、100和30 kDa）以及DEAE-琼脂糖柱色谱法获得。通过SDS-PAGE估计纯化酶的分子量为89 kDa。纯化酶在pH 5.3和56°C时活性最高，在4至7的宽pH范围内保留50%的活性。酶的热稳定性测定表明，该酶在80°C下60秒保留超过70%的活性，120秒保留40%的活性，300秒后保留9%的活性。植酸酶表现出广泛的底物特异性，Km值为220μM，Vmax为25μM/分钟。纯化的植酸酶对磷酸化化合物如苯基磷酸、1-萘基磷酸、2-萘基磷酸、对硝基苯基磷酸和甘油-2-磷酸保留50%的活性。在10和20 mM浓度下，观察到金属离子对植酸酶活性的抑制作用，其中Ca显著抑制（50%），Ni、K和Na轻微抑制（10%）。Mg、Mn、Cu、Cd和Ba在25%和35%时对植酸酶活性有刺激作用，产生了积极影响。
