Patel Hardik, Gupte Shilpa, Gahlout Mayur, Gupte Akshaya
Department of Microbiology, N. V. Patel College of Pure and Applied Sciences, Vallabh Vidyanagar, 388 120, Gujarat, India.
3 Biotech. 2014 Feb;4(1):77-84. doi: 10.1007/s13205-013-0129-1. Epub 2013 Mar 16.
A native isolate of Pleurotus ostreatus HP-1 (Genbank Accession No. EU420068) was found to have an excellent laccase producing ability. The extracellular laccase was purified to electrophoretic homogeneity from copper sulphate induced solid-state fermentation medium by ammonium sulphate precipitation and ion-exchange chromatography. The enzyme was determined to be monomeric protein with an apparent molecular mass of 68,420 kDa, and an isoelectric point (pI) of 3.5. The inductively coupled plasma spectroscopy showed a presence of iron, zinc and copper in the purified enzyme. The absorption spectrum in the range of 200-700 nm showed the maximum absorption at 610 nm characteristic of fungal laccase and corresponding to the presence of type I copper atom. The laccase was stable at different temperatures up to 70 °C and retained 61 % activity at 50 °C. The enzyme reaction was inhibited by cysteine; sodium azide and EDTA. The enzyme oxidized various known laccase substrates, its lowest K value being for ortho-dianisidine and highest K and K/K for ABTS. The purified laccase exhibited different pH optima for different substrates. The N-terminal sequence did not show any similarity with N-terminal sequence of other species of genera Pleurotus.
一种平菇HP-1的本地分离株（Genbank登录号：EU420068）被发现具有出色的漆酶产生能力。通过硫酸铵沉淀和离子交换色谱法，从硫酸铜诱导的固态发酵培养基中纯化胞外漆酶至电泳纯。该酶被确定为单体蛋白，表观分子量为68,420 kDa，等电点（pI）为3.5。电感耦合等离子体光谱显示纯化后的酶中存在铁、锌和铜。200 - 700 nm范围内的吸收光谱显示在610 nm处有最大吸收，这是真菌漆酶的特征，对应于I型铜原子的存在。该漆酶在高达70°C的不同温度下稳定，在50°C时保留61%的活性。酶反应受到半胱氨酸、叠氮化钠和EDTA的抑制。该酶氧化各种已知的漆酶底物，其对邻联茴香胺的最低K值，对ABTS的K值和K / K值最高。纯化后的漆酶对不同底物表现出不同的最适pH值。N端序列与平菇属其他物种的N端序列没有任何相似性。
