草鱼肠道胰蛋白酶同工酶GT-B的纯化及性质

Purification and properties of trypsin isoform B from intestines of grass carp(Ctenopharyngodon idellus)

深入了解草鱼消化蛋白酶的性质有利于充分探讨草鱼内脏的可利用途径和研究草鱼消化生理学.将草鱼肠道用冰丙酮脱脂,用磷酸盐缓冲溶液提取,然后经过硫酸铵粗分级沉淀,再经过离子交换层析、凝胶层析等纯化,分离得到一种碱性蛋白酶.SDS-PAGE分析表明其相对分子质量为26400.SDS-底物-PAGE以及抑制实验等表明酶可以水解酪蛋白和N-苯甲酰-L-精氨酸乙酯盐酸盐(BAEE),其活性被大豆胰蛋白酶抑制剂(SBTI)和甲苯磺酰氟(PMSF)强烈抑制,表明这是一种胰蛋白酶,命名为GT-B.用BAEE作底物时,该酶的最适作用温度为45℃,在65℃加热20min,酶活性基本丧失;最适作用pH为8.5,在pH5.5～10.5范围内保持稳定,但在酸性环境里很容易失去活性.该酶的Vmax和Km值分别为3.33×103min-1和31.68μmol·mL-1,其生理转化效率高,为105.26mL·μmol-1·min-1.该酶催化水解合成底物时的活化能Ea比较低,为18.31kJ·mol-1.其活性不受Ca2+的影响,但是Ca2+能提高该酶的热稳定性.Zn2+,Cu2+,Hg2+和Al3+等的存在会使此酶活性降低.

Intestively understanding the properties of grass carp（Ctenopharyngodon idellus）digestive protease is useful to investigate the ways utilizing the gut from this species and learn its digestive physiology.An alkaline protease was separated from grass carp（Ctenopharyngodon idellus）intestines by following procedure：the grass carp intestines were defatted with cooled acetone（-20℃）,and extracted with 0.1 mol·L^-1 phosphate buffer（pH6.8）,the crude enzyme solution was fractionated with ammonium sulfate（10% to 40% saturation）;then the precipitation was purified with ion-exchange chromatography and gel-filtration chromatography.The SDS-PAGE electrophoresis showed that this enzyme had a relative molecular mass of 26400.The SDS-substrate-PAGE and the inhibition assays showed that it could hydrolyze casein and α-N-benzoyl-L-arginine ethyl ester·HCl（BAEE）,and it could be strongly inhibited by phenylmethylsulfonylfluoride（PMSF）and soybean trypsin inhibitor（SBTI）.These results indicated that it is a trypsin,named as GT-B.The GT-B had optimum temperature of 45℃ and optimum pH of 8.5 when hydrolyzing α-N-benzoyl-L-arginine ethyl ester·HCl（BAEE）.It lost 93.60% of its activity when heated for 20 min at 65℃,and it was stable at range of pH5.5 to 10.5 but became unstable under the acidic pH conditions.Vmax and Km of the GT-B were 3.33×10^3 min^-1 and 31.68 μmol·mL^-1,respectively.Its physiological efficiency was 105.26 mL·μmol^-1·min^-1,and its Arrhenius active energy was 18.31 kJ·mol^-1.The activity of GT-B was not influenced by Ca^2＋,but its thermo-stability could be improved in the presence of Ca^2＋.And its activity was reduced in the presence of Zn^2＋,Cu^2＋,Hg^2＋ and Al^3＋.