新型嗜热耐碱脂肪酶的纯化表征及应用

张昕怡; 许蕊; 王钰棋; 张瑜; 王飞; 李迅

doi:10.11949/0438-1157.20200285

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新型嗜热耐碱脂肪酶的纯化表征及应用

生物化学工程与技术 | 更新时间：2026-07-24

- 新型嗜热耐碱脂肪酶的纯化表征及应用
- Purification and characterization of novel thermo-alkaline lipase and its application
- 化工学报 2020年71卷第11期页码：5246-5255
- 作者机构：
  
  1.南京林业大学化学工程学院，江苏南京 210037
  2.江苏省生物质绿色燃料与化学品重点实验室，江苏南京 210037
- 作者简介：
  
  张昕怡（1996—），女，硕士研究生，gypsophilaz@163.com
  李迅（1975—），女，博士，教授，xunlee@njfu.edu.cn
- 基金信息：
  
  国家重点研发计划(2019YFB1504002)
- DOI：10.11949/0438-1157.20200285
  中图分类号： Q 81
- 收稿：2020-03-18，
  
  修回：2020-07-07，
  
  网络首发：2020-11-03，
  
  纸质出版：2020-11-05
- 稿件说明：
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张昕怡, 许蕊, 王钰棋, 张瑜, 王飞, 李迅. 新型嗜热耐碱脂肪酶的纯化表征及应用[J]. 化工学报, 2020, 71(11): 5246-5255

ZHANG Xinyi, XU Rui, WANG Yuqi, ZHANG Yu, WANG Fei, LI Xun. Purification and characterization of novel thermo-alkaline lipase and its application[J]. CIESC Journal, 2020, 71(11): 5246-5255
张昕怡, 许蕊, 王钰棋, 张瑜, 王飞, 李迅. 新型嗜热耐碱脂肪酶的纯化表征及应用[J]. 化工学报, 2020, 71(11): 5246-5255 DOI： 10.11949/0438-1157.20200285.

ZHANG Xinyi, XU Rui, WANG Yuqi, ZHANG Yu, WANG Fei, LI Xun. Purification and characterization of novel thermo-alkaline lipase and its application[J]. CIESC Journal, 2020, 71(11): 5246-5255 DOI： 10.11949/0438-1157.20200285.

摘要

将来源于解脂嗜热互营杆菌（

Thermosyntropha lipolytica

）的脂肪酶（

LipA）基因

tll1

导入大肠杆菌BL21（DE3）中表达，通过热处理和镍柱亲和层析获得纯酶，并对其酶学性质进行研究。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）显示

LipA分子量为53×10

，其最适反应温度为65℃，最适反应pH为8.0。在55~65℃范围内酶活较高且比较稳定；在pH

7.0~11.0于室温保存1 h后，残留相对酶活仍达80%以上。1 mmol/L 金属离子Zn

、Fe

和试剂SDS，0.05%（质量分数）Tween 80，对酶活力具有强烈的抑制作用，残留相对酶活皆低于15%；1 mmol/L Mg

、Mn

对酶活力表现出轻微的激活作用。由底物专一性实验可得，该酶对辛酸对硝基苯酯（C8）和癸酸对硝基苯酯（C10）偏好明显。以棕榈酸对硝基苯酯（

NPP）为底物，该酶动力学参数

值为0.23 mmol/L，

max

为33.50 mmol/(L

min)，

cat

为22.83

-1

。以重组脂肪酶为催化剂在无溶剂体系中制备生物柴油，含水率20%，酶加量200 U/g油，醇油比为4∶1的条件下，在55℃催化大豆油反应48 h，收率可达91.75%。

Abstract

In order to excavate thermo-alkaline lipases from bacterial living in extreme conditions

we try to express new gene from

Thermosyntropha lipolytica

DSM 11003

an anaerobic

thermophilic

alkali-tolerant bacterium which grows in alkaline hot springs Lake Bogoria in Kenya and explore its application in biodiesel production. The lipase gene (

tll

1) of 1434 bp were ligated at the

Nco

I /

EcoR

I sites of the expression vectors pET28a to yield the construct of pET28a-TLL1. The strain harboring pET28a-TLL1 was cultivated for expression at 25℃

the specific activity of 1.99 U/mg protein were detected in disrupted cells. The recombinant lipase

LipA was purified by a simple two-step procedure involving heat treatment and Ni-chelating affinity chromatography. The subunit of purified

LipA showed a molecular mass of 53×10

on 12% sodium dodecyl sulfate polyacrylamide gel electrophoresi

s (SDS-PAGE). The purified

LipA exhibited optimal activity at 65℃ and pH 8.0 and it was stable from 55℃ to 65℃. The enzyme remained above 80% of its original activity at pH ranging from 7.0 to 11.0 and at room temperature for 1 h. The activity of

LipA was little unaffected by Co

and Ni

and a little activated by Mg

and Mn

but were significantly inhibited by Zn

and SDS

and Tween 80 under the assay conditions. The purified recombinant

LipA had a specific activity of 22.11 U/mg protein using

-nitrophenyl palmitate (

NPP) as substrate. Determined by Sigma-Plot of reaction rate on

NPP

the

was 0.23 mmol/L

the

max

was 33.50 mmol/(L

min)

and the

cat

was 22.83 s

-1

. The enzyme was also active towards

NPP

-nitrophenyl laurate (

NPL)

-nitrophenyl myristate (

-NPM) and

-nitrophenyl caproate (

NPC)

moreover

LipA exhibited a strong preference for

-nitrophenol decanoate (

-NPD) and

-nitrophenyl octoate (

-NPO). Using recombinant lipase as a catalyst to prepare biodiesel in a solvent-free system

with a water content of 20%

an enzyme dosage of 200 U/g oil

and an alcohol-to-oil ratio of 4∶1

catalyzed soybean oil reaction at 55℃ for 48 h

the yield can reach 91.75%.

关键词

Keywords

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