University of Ioannina, Department of Chemistry, Ioannina 45110, Greece.
Bioresour Technol. 2012 Nov;123:214-20. doi: 10.1016/j.biortech.2012.06.090. Epub 2012 Jul 4.
A novel extracellular hydrolase of ∼45kDa molecular mass was purified from Kluyveromyces marxianus IFO 0288 cultures and characterized as serine protease. The K(m)-value of protease (designated protease-KM-IFO-0288-A), which was found active in media containing elevated [NaCl] but lacking EDTAK(2), decreased with increasing [Ca(2+)]. The protease maintained considerable activity at the range of 10-60°C and pH 6.00-10.25, with optimum k(cat)/K(m)-value at 35.5°C and pH 7.75. It was strongly affected by specific irreversible inhibitors of serine proteases while was unaffected by inhibitors of cysteine proteases. Significant rate constants, activation energies, and proton inventories were estimated from the profiles of Michaelis-Menten parameters, versus pH, temperature and deuterium atom fraction, in the hydrolysis of Suc-AAPF-pNA showing that protease-KM-IFO-0288-A performs catalysis via a charge-relay system. The properties of protease-KM-IFO-0288-A suggest that K. marxianus represents a valuable source of extracellular protease of biotechnological interest which, given its GRAS status, could find several important applications.
从马克斯克鲁维酵母IFO 0288 培养物中纯化出一种新型的约 45kDa 分子量的细胞外水解酶，并将其鉴定为丝氨酸蛋白酶。蛋白酶（命名为蛋白酶-KM-IFO-0288-A）的 K(m)值在含有高[NaCl]但缺乏 EDTAK(2)的介质中是活跃的，随着[Ca(2+)]的增加而降低。该蛋白酶在 10-60°C 和 pH 6.00-10.25 的范围内保持相当高的活性，在 35.5°C 和 pH 7.75 时具有最佳的 k(cat)/K(m)-值。它受到丝氨酸蛋白酶特异性不可逆抑制剂的强烈影响，而不受半胱氨酸蛋白酶抑制剂的影响。通过 Suc-AAPF-pNA 水解的米氏参数与 pH、温度和氘原子分数的关系的轮廓，估计了显著的速率常数、活化能和质子库存，表明蛋白酶-KM-IFO-0288-A 通过电荷中继系统进行催化。蛋白酶-KM-IFO-0288-A 的性质表明，马克斯克鲁维酵母是具有生物技术意义的细胞外蛋白酶的有价值的来源，鉴于其 GRAS 地位，它可以找到几个重要的应用。
