Bipolaris australiensis HD-1胆红素氧化酶纯化、鉴定及其酶学性质

目的]为了获得B.bipolaris HD-1胆红素氧化酶纯品,确定酶的同源性及其常规酶学性质.方法]将B.bipolaris HD-1发酵液依次通过硫酸铵盐析、阴离子交换层析和凝胶过滤层析确定蛋白的纯度、浓度和酶活力;通过基质辅助激光解析电离飞行时间质谱（MALDI-TOF）法获得氨基酸序列,并利用Mascot软件在NCBI蛋白库中的比对,以确定同源蛋白来源;用SDS-PAGE方法测其表观分子量;用等电聚焦方法测定等电点;用Linwerver-Burk双倒数作图法测定米氏常数;并对该酶的酶学性质进行了常规的测定.结果]获得具有胆红素氧化酶酶活性的电泳纯单体蛋白,酶活为46 000 U/L,比活为1.15 U/mg;MALDI-TOF共获得135个氨基酸,该蛋白与来源于Hel-minthosporium tritici-vulgaris的漆酶前体蛋白序列同源性达100％;蛋白的表观分子量为68 kDa;pI为4.1;它对ABTS和胆红素的米氏常数分别为Km（ABTS） =1.8×10-5 mol/L（pH 3.0）和Km（bilirnbin）=2.7×10-5 mol/L（pH 7.5）;对胆红素的最适催化活性为36℃,酶在-40℃下可保持2年,酶活保持在90％以上,在pH 8 ～9.5下可保持相当高的催化胆红素活性,能够耐受高浓度的硝酸钠和尿素,低浓度的叠氮化钠对酶活有促进作用而高浓度则抑制,对氯化钠、溶解氧敏感.结论]该蛋白具有典型的胆红素氧化酶特性,又存在明显的不同.

Purification,Identification and Properties of Bilirubin Oxidase from Bipolaris australiensis HD-1

Objective] The research aimed to obtain a high purity protein of BOD from B.bipolaris HD-1,and determined its homology and the common properties.Method] The supernatant of B.bipolaris HD-1 broth was performed by ammonium sulfate salting out,DEAE-sepharose fast flow anion exchange chromatography and SephadexG-75 gel filtration chromatography respectively.The purity of this protein was determined by SDS-PAGE.The specific activity was assayed in Tris-HCl buffer at 37 ℃ （pH 7.4）.The amino acid residues was determined by Matrix-assisted laser desorption ionization time of flight mass spectrometry（MALDI-TOF） and the homology was obtained with software Mascot on the databank of NCBI.The molecular weight was determined by SDS-PAGE.The pI was determined by means of isoelectric focusing.The kinetic parameters for this enzyme were determined by Linwerver-Burk double-reciprocal plots.The common properties of BOD were assayed by conventional methods.Result] A single band was obtained by SDS-PAGE.The enzyme activity was 46 000 U/L.The specific activity was 1.15 U/mg.A 135 amino acid residue was obtained.There was a 100％ homologous with a laccase precousors from Helminthosporium tritici-vulgaris.The molecular weight was 68 kDa.The pI was 4.1.The kinetic paranmeters for this enzyme were Km（ABTs） =1.8 × 10-5mol/L at pH 3.0 and Km（bitintbin） =2.7 × 10-5 mnol/L at pH 7.5 respectively.The common properties of this enzyme shown as：the enzyme had a optimum temperature to catalyze bilirubin to biliverdin at 36 ℃,it was stable at-40 ℃ or lower and keep activity up to 90％ for two years,it could keep a high activity in range of pH from 8 to 9.5,the enzyme had activity in condition that higher concentration of sodium nitrate and urea,however,the enzyme was inhibited by high concentration of sodium aside,there was a contrary results at lower concentration.It was sensitive for sodium chloride and dissolved oxygen（DO）.Conclusion] BOD from B.australiensis HD-1 exhibited a typical characteristic of BODs,and at that time a significandy different existed.