罗伦隐球酵母脂肪酶纯化与ANS快速测定脂肪酶热稳定性方法的建立
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摘要
脂肪酶作为一种重要的工业用酶,在造纸、医药、食品、日用化工、能源开发等领域有着广泛的研究与应用。作者通过过离子交换柱Sepharose Fast Flow纯化了罗伦隐球酵母B40 (Cryptococcus laurentii B40)脂肪酶,获得了纯度超过95%的脂肪酶液,SDS-PAGE电泳显示其相对分子质量约为44.3KDa,该脂肪酶测序结果显示N端的12个氨基酸序列为：D-F-G-P-I-T-I-Y-T-P-P-A。酶学性质分析表明该脂肪酶是一种中低温碱性脂肪酶,较适作用温度为25℃,最适作用pH为10.2,Tm为40.90℃,能够催化水解中短碳链(12碳以下)的甘油三酯,对混合碳类甘油三酯也有一定的催化作用。分析了不同金属离子和表面活性剂对该酶的影响,发现重金属离子对该酶活性的发挥有较强的抑制作用,而能促进脂质溶解的试剂则能较好地提高酶的催化效用。
脂肪酶的热稳定性是脂肪酶的重要性质之一,热稳定性好意味着储存方便,便于运输,能够耐受工业生产应用中的高温环境等。寻找一种简单快速的脂肪酶热稳定性测定法对筛选热稳定脂肪酶的工作有着重要的作用,它能够大大节省实验成本,提高实验效率,缩短研究到生产实践的周期。本文报道了一种简捷、高通量的脂肪酶热稳定性测定法——ANS脂肪酶稳定性快速测定方法的建立,该方法可将测定时间由传统的NaOH滴定法的几天缩短为几小时,而且非常适合高通量脂肪酶热稳定性的测定。
As one of the most widely use industry enzyme, lipase (EC 3.1.1.3) was used in medicine, food, daily chemical industry and other industrial fields. The lipase from Cryptococcus laurentii B40, which was reported as a lipase, was purified by DEAE Sepharose fast flow ion exchange column. The purified protein shows a single band on SDS-PAGE with the molecular about 44.3KDa estimated by SDS-PAGE electrophoresis. The N-terminal sequence of it is D-F-G-P-I-T-I-Y-T-P-P-A. The optimum pH and temperature are 10.2 and 25℃respectively. The enzyme could catalyse triglyceride below 12 carbon and could activated by some reagents like Sodium Dehydrocholate, but inhibited by many heavy metal such as Hg2+
Lipase is a widely used industry enzyme but usually with poor thermo-stability. One of the methods to improve the thermo-stability is generating mutations by mutagenesis or DNA Shuffling and selecting the useful mutants from a mutant library. It is necessary to develop a rapid method to measure the thermo-stability of lipases since the traditional reaction activity analysis. Here we develop a rapid and high throughput ANS Fluorescence signal measurement to evaluate the thermo-stability of the lipases:incubate lipase at 25-65℃for 30min, then combine 0.20mg/mL lipase,0.05mM 1,8-ANS (8-Anilino-l-naphthalenesulfonic acid, ANS) in the buffer of 20mM Tris-HCl, 100mM-500mM NaCl, pH7.2. Read fluorescence signal at EX 378nm, EM 465nm with fluorescence photometer or plate reader. Then calculate the Tm with GraphPad Prism5.0. We tested PEL (Penicillium expansum lipase, mutant PEL-ep8-K115R and PEL-ep8-K202A with above method and got the similar Tm value that measured by traditional method.
脂肪酶的热稳定性是脂肪酶的重要性质之一,热稳定性好意味着储存方便,便于运输,能够耐受工业生产应用中的高温环境等。寻找一种简单快速的脂肪酶热稳定性测定法对筛选热稳定脂肪酶的工作有着重要的作用,它能够大大节省实验成本,提高实验效率,缩短研究到生产实践的周期。本文报道了一种简捷、高通量的脂肪酶热稳定性测定法——ANS脂肪酶稳定性快速测定方法的建立,该方法可将测定时间由传统的NaOH滴定法的几天缩短为几小时,而且非常适合高通量脂肪酶热稳定性的测定。
As one of the most widely use industry enzyme, lipase (EC 3.1.1.3) was used in medicine, food, daily chemical industry and other industrial fields. The lipase from Cryptococcus laurentii B40, which was reported as a lipase, was purified by DEAE Sepharose fast flow ion exchange column. The purified protein shows a single band on SDS-PAGE with the molecular about 44.3KDa estimated by SDS-PAGE electrophoresis. The N-terminal sequence of it is D-F-G-P-I-T-I-Y-T-P-P-A. The optimum pH and temperature are 10.2 and 25℃respectively. The enzyme could catalyse triglyceride below 12 carbon and could activated by some reagents like Sodium Dehydrocholate, but inhibited by many heavy metal such as Hg2+
Lipase is a widely used industry enzyme but usually with poor thermo-stability. One of the methods to improve the thermo-stability is generating mutations by mutagenesis or DNA Shuffling and selecting the useful mutants from a mutant library. It is necessary to develop a rapid method to measure the thermo-stability of lipases since the traditional reaction activity analysis. Here we develop a rapid and high throughput ANS Fluorescence signal measurement to evaluate the thermo-stability of the lipases:incubate lipase at 25-65℃for 30min, then combine 0.20mg/mL lipase,0.05mM 1,8-ANS (8-Anilino-l-naphthalenesulfonic acid, ANS) in the buffer of 20mM Tris-HCl, 100mM-500mM NaCl, pH7.2. Read fluorescence signal at EX 378nm, EM 465nm with fluorescence photometer or plate reader. Then calculate the Tm with GraphPad Prism5.0. We tested PEL (Penicillium expansum lipase, mutant PEL-ep8-K115R and PEL-ep8-K202A with above method and got the similar Tm value that measured by traditional method.
引文
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