新型嗜热耐碱脂肪酶的纯化表征及应用

doi:10.11949/0438-1157.20200285

摘要/Abstract

摘要：

将来源于解脂嗜热互营杆菌（Thermosyntropha lipolytica）的脂肪酶（TlLipA）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TlLipA分子量为53×10³，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Zn²⁺、Fe³⁺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 Mg²⁺、Mn²⁺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（p-NPP）为底物，该酶动力学参数K_m值为0.23 mmol/L，V_max为33.50 mmol/(L·min)，k_cat为22.83 _S^-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%。}

关键词: 生物催化, 生物柴油, 生物燃料, 脂肪酶, 嗜热耐碱酶, 酶学性质

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 (tll1) 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 TlLipA was purified by a simple two-step procedure involving heat treatment and Ni-chelating affinity chromatography. The subunit of purified TlLipA showed a molecular mass of 53×10³ on 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The purified TlLipA 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 TlLipA was little unaffected by Co²⁺, K⁺, Na⁺, and Ni²⁺, and a little activated by Mg²⁺ and Mn²⁺, ^{but were significantly inhibited by Zn²⁺, Fe³⁺, and SDS, and Tween 80 under the assay conditions. The purified recombinant TlLipA had a specific activity of 22.11 U/mg protein using p-nitrophenyl palmitate (p-NPP) as substrate. Determined by Sigma-Plot of reaction rate on p-NPP, the K_m was 0.23 mmol/L, the V_max was 33.50 mmol/(L·min), and the k_cat was 22.83 s^-1. The enzyme was also active towards p-NPP, p-nitrophenyl laurate (p-NPL), p-nitrophenyl myristate (p-NPM) and p-nitrophenyl caproate (p-NPC), moreover TlLipA exhibited a strong preference for p-nitrophenol decanoate (p-NPD) and p-nitrophenyl octoate (p-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%.}

Key words: biocatalysis, biodiesel, biofuel, lipase, thermo-alkaline enzyme, enzymatic properties

中图分类号:

Q 81

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.

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

图/表 13

图1 重组脂肪酶 TlLipA 的SDS-PAGE 分析M—蛋白质标准分子量；1—质粒pET28a转化BL21(DE3) 重组菌的全细胞液；2—质粒pET28a转化BL21(DE3) 重组菌的上清液；3—质粒pET28a-TLL1转化BL21(DE3) 重组菌的全细胞液；4—质粒pET28a-TLL1转化BL21(DE3) 重组菌的上清液；5—质粒pET28a-TLL1转化BL21(DE3) 重组菌的沉淀；6—质粒pET28a-TLL1转化BL21(DE3)重组菌热处理后的上清液；7—质粒pET28a-TLL1转化BL21(DE3)重组菌的Ni2+亲和柱纯化后的酶液

Fig.1 SDS-PAGE analysis of the recombinant lipase TlLipA

表1 重组脂肪酶 TlLipA 的纯化

Table 1 The purification of recombinant lipase TlLipA

纯化步骤

总体积/ml

总酶活/U

比酶活/

(U/mg)

图2 温度对重组脂肪酶TlLipA酶活力的影响

Fig.2 Effect of temperature on lipase activity (a) and stability (b)

图3 pH对重组脂肪酶TlLipA酶活力的影响

Fig.3 Effect of pH on lipase activity (a) and stability (b)

表2 金属离子和化学试剂对酶活性的影响

Table 2 Effects of ions and reagents on the activity of the lipase

金属离子和化学试剂

相对酶活/%

control

Fe³⁺

Co²⁺

Zn²⁺

Mn²⁺

Ca²⁺

K⁺

Mg²⁺

Na⁺

Ni²⁺

Cu²⁺

PMSF

LAS

Tween 80

AEO-7

EDTA

SDS

Triton X-100

100±3.15

11.71±2.49

90.84±1.72

9.73±1.34

113.24±3.04

81.24±7.04

90.27±4.60

108.55±7.11

88.76±5.46

87.63±3.68

69.31±6.50

86.39±3.46

72.62±4.37

8.19±2.16

16.36±2.73

15.90±2.61

2.28±1.44

61.15±1.07

图4 重组脂肪酶TlLipA的底物特异性

Fig.4 The activities of TlLipA toward substrates with different acyl chain lengths

图5 重组脂肪酶TlLipA反应速率和底物浓度的关系

Fig.5 Relationship of reaction velocity and substrate concentration of lipase

表3 各嗜热耐碱脂肪酶动力学参数比较

Table 3 Comparison of kinetic parameters of various thermo-alkaline lipases

脂肪酶来源	最适温度/℃	最适pH	K_m/(mmol/L)	V_max/(mmol/(L·min))	文献
T. lipolytica(TlLipA)	65	8	0.23	33.50	本文
G. thermodenitrificans AV-5	65	9	0.44	0.556	[12]
T. Anoxybacillus flavithermus HBB 134	50	9	0.084	1.29	[17]
Staphylococcus aureus	40	8	1.57	3.25	[27]
Thermotoga maritima	70	7.5	8	2.50	[28]
Geobacillus sp.	65	8.5	14	17.86	[29]
Streptomyces thermocarboxydus ME168	50	8	0.28	16.54	[30]

图6 气相色谱图1—棕榈酸甲酯；2—十七烷酸甲酯；3—硬脂酸甲酯；4—油酸甲酯；5—亚油酸甲酯；6—亚麻酸甲酯

Fig.6 Gas chromatography spectrometry of standard sample mixture and reaction production

图7 反应溶剂体系的比较

Fig.7 Comparison of reaction systems

图8 醇油比对收率的影响

Fig.8 Effect of methanol to oil ratio on the yield

图9 含水率对收率的影响

Fig.9 Effect of water content on the yield

表4 各嗜热耐碱脂肪酶制备生物柴油比较

Table 4 Comparison of preparation of biodiesel using various thermo-alkaline lipases

脂肪酶来源	反应温度/℃	反应时间/h	醇油比	原料油	生物柴油收率/%	文献
T. lipolytica(TlLipA)	55	48	4∶1	大豆油	91.75	本文
G. thermodenitrificans AV-5	65	48	3∶1	废餐饮油	76	[12]
G.thermodenitrificans AV-5	65	48	3∶1	椰子油	45.5	[12]
Bacillus sp.	55	40	3∶1	Oedogonium sp. oil.	76	[13]
Idiomarina sp. W33	60	60	4∶1	麻风树油	84	[36]

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