VKT多肽介导的固定化疏棉状嗜热丝孢菌脂肪酶催化制备生物柴油

doi:10.16085/j.issn.1000-6613.2023-0887

摘要/Abstract

摘要：

生物柴油是一种环境友好的生物液体燃料，对于酶法生产生物柴油，迫切需要找到廉价和高效的固定化脂肪酶。本研究利用固体结合肽（SBPs）VKT，将其与疏棉状嗜热丝孢菌脂肪酶（Thermomyces lanuginosus lipase，TLL）融合构建融合脂肪酶，并将其固定化在硅基材料上，获得了一种新型的生物催化剂。在所测试的硅基材料（ZSM-5、Na-Y、SAR-100、MCM-41和SiO₂微粉）中，固定在ZSM-5沸石上的TLL-VKT（TLL-VKT@ZSM-5）表现出最佳的固定化效率和最大负载，且具有优异的pH、温度、储存和洗脱稳定性。以TLL-VKT@ZSM-5为生物催化剂，对麻疯树籽油进行转酯化反应，48h生物柴油得率即达到93.9%。此外，TLL-VKT@ZSM-5还表现出较高的重复使用性能，在7次重复使用后，生物柴油得率依然保持71.9%。本研究的酶固定化方法具有简单高效、稳定性高和重复使用性能高等优点。本研究显示VKT肽在酶蛋白固定化的应用方面具有较好前景。

关键词: 疏棉状嗜热丝孢菌脂肪酶, 固定化, 硅基材料, 麻疯树籽油, 生物柴油, 固体结合肽

Abstract:

Biodiesel is an environmentally friendly bio-liquid fuel. For the enzymatic production of biodiesel, there is a pressing need for cost-effective and efficient immobilized lipase. In this study, solid-bound peptide VKT-fused Thermomyces lanuginosus lipase (TLL) was directly immobilized on unmodified silica-containing materials to create a novel biocatalyst. Among the silica-based materials (ZSM-5, Na-Y, SAR-100, MCM-41 and SiO₂ powder) tested, TLL-VKT immobilized on ZSM-5 zeolite (TLL-VKT@ZSM-5) showed the best immobilization efficiency and maximum loading, as well as exceptional stability in terms of pH, temperature, storage and elution. When used as a biocatalyst at 40℃ for 48 hours, TLL-VKT@ZSM-5 (1%, w/w, oil) resulted in a biodiesel yield of 93.9% from Jatropha curcas oil. Furthermore, TLL-VKT@ZSM-5 exhibited high reusability, with a biodiesel yield of 71.9% after 7 repeated uses. The immobilization method in this study offers the advantages of simplicity, high efficiency, high stability, and reusability. In the context of enzyme immobilization used in the production of various chemicals, VKT peptides present significant potential.

Key words: Thermomyces lanuginosus lipase, immobilization, silica-containing materials, Jatropha curcas oil, biodiesel, SBPs

中图分类号:

Q814.2

季骁彦, 许蕊, 王飞, 李迅. VKT多肽介导的固定化疏棉状嗜热丝孢菌脂肪酶催化制备生物柴油[J]. 化工进展, 2024, 43(6): 3285-3292.

JI Xiaoyan, XU Rui, WANG Fei, LI Xun. Direct immobilization of Thermomyces lanuginosus lipase mediated by VKT-peptide for efficient biodiesel production from Jatropha curcas oil[J]. Chemical Industry and Engineering Progress, 2024, 43(6): 3285-3292.

图/表 7

图1 TLL与TLL-VKT的SDS-PAGE图

图2 固定在不同硅基材料上的融合脂肪酶固定化效率

图3 游离脂肪酶和TLL-VKT@ZSM-5的酶活性质

表1 TLL-VKT@ZSM-5的洗脱稳定性实验

洗脱剂	相对蛋白洗脱率/%
1mol/L NaCl	2.0±0.1
2mol/L NaCl	2.5±0.4
3mol/L NaCl	5.6±0.2
4mol/L NaCl	6.3±0.2
5mol/L NaCl	12.7±0.9
1% Tween-20	1.6±0.2
2% Tween-20	1.3±0.2
3% Tween-20	4.4±1.5
1% SDS	75.8±2.5
2% SDS	100.0±1.1
3% SDS	100.0±0.9

图4 TLL-VKT@ZSM-5的储存稳定性

图5 TLL-VKT@ZSM-5催化麻疯树籽油制备生物柴油

图6 TLL-VKT@ZSM-5在生物柴油制备中的可重复使用性

参考文献 53

1	ZDARTA J, MEYER A, JESIONOWSKI T, et al. A general overview of support materials for enzyme immobilization: Characteristics, properties, practical utility[J]. Catalysts, 2018, 8(2): 92.
2	TAHSIRI Z, NIAKOUSARI M, HOSSEINI S M H, et al. Magnetic layered double hydroxide nanosheet as a biomolecular vessel for enzyme immobilization[J]. International Journal of Biological Macromolecules, 2022, 209: 1422-1429.
3	ZENG Qi, LI Qi, SUN Di, et al. Alcalase microarray base on metal ion modified hollow mesoporous silica spheres as a sustainable and efficient catalysis platform for proteolysis[J]. Frontiers in Bioengineering and Biotechnology, 2020, 8: 565.
4	CHEN Jing, LENG Juan, YANG Xiai, et al. Enhanced performance of magnetic graphene oxide-immobilized laccase and its application for the decolorization of dyes[J]. Molecules, 2017, 22(2): 221.
5	SMITH S, GOODGE K, DELANEY M, et al. A comprehensive review of the covalent immobilization of biomolecules onto electrospun nanofibers[J]. Nanomaterials, 2020, 10(11): 2142.
6	ZENG Shuo, SHI Jinwei, FENG Anchao, et al. Modification of electrospun regenerate cellulose nanofiber membrane via atom transfer radical polymerization (ATRP) approach as advanced carrier for laccase immobilization[J]. Polymers, 2021, 13(2): 182.
7	DICOSIMO R, MCAULIFFE J, POULOSE A J, et al. Industrial use of immobilized enzymes[J]. Chemical Society Reviews, 2013, 42(15): 6437.
8	KATSIMPOURAS C, STEPHANOPOULOS G. Enzymes in biotechnology: Critical platform technologies for bioprocess development[J]. Current Opinion in Biotechnology, 2021, 69: 91-102.
9	TANG Zhenghua, PALAFOX-HERNANDEZ J P, Wing-Cheung LAW, et al. Biomolecular recognition principles for bionanocombinatorics: An integrated approach to elucidate enthalpic and entropic factors[J]. ACS Nano, 2013, 7(11): 9632-9646.
10	ZERNIA S, OTT F, BELLMANN-SICKERT K, et al. Peptide-mediated specific immobilization of catalytically active cytochrome P450 BM3 variant[J]. Bioconjugate Chemistry, 2016, 27(4): 1090-1097.
11	CARE A, BERGQUIST P L, SUNNA A. Solid-binding peptides: Smart tools for nanobiotechnology[J]. Trends in Biotechnology, 2015, 33(5): 259-268.
12	JANCIK PROCHAZKOVA A, SALINAS Y, YUMUSAK C, et al. Cyclic peptide stabilized lead halide perovskite nanoparticles[J]. Scientific Reports, 2019, 9: 12966.
13	CARE A, NEVALAINEN H, BERGQUIST P L, et al. Effect of Trichoderma reesei proteinases on the affinity of an inorganic-binding peptide[J]. Applied Biochemistry and Biotechnology, 2014, 173(8): 2225-2240.
14	SEKER U O S, SHARMA V K, AKHAVAN S, et al. Engineered peptides for nanohybrid assemblies[J]. Langmuir, 2014, 30(8): 2137-2143.
15	KUANG Zhifeng, KIM S N, CROOKES-GOODSON W J, et al. Biomimetic chemosensor: Designing peptide recognition elements for surface functionalization of carbon nanotube field effect transistors[J]. ACS Nano, 2010, 4(1): 452-458.
16	BANSAL R, ELGUNDI Z, GOODCHILD S C, et al. The effect of oligomerization on a solid-binding peptide binding to silica-based materials[J]. Nanomaterials, 2020, 10(6): 1070.
17	RAMACHANDRAN B, CHAKRABORTY S, KANNAN R, et al. Immobilization of hyaluronic acid from Lactococcus lactis on polyethylene terephthalate for improved biocompatibility and drug release[J]. Carbohydrate Polymers, 2019, 206: 132-140.
18	RÜBSAM K, STOMPS B, BÖKER A, et al. Anchor peptides: A green and versatile method for polypropylene functionalization[J]. Polymer, 2017, 116: 124-132.
19	RÜBSAM K, DAVARI M, JAKOB F, et al. KnowVolution of the polymer-binding peptide LCI for improved polypropylene binding[J]. Polymers, 2018, 10(4): 423.
20	LIU Chang, STEER D L, SONG Haipeng, et al. Superior binding of proteins on a silica surface: Physical insight into the synergetic contribution of polyhistidine and a silica-binding peptide[J]. The Journal of Physical Chemistry Letters, 2022, 13(6): 1609-1616.
21	CARE A, CHI Fei, BERGQUIST P L, et al. Biofunctionalization of silica-coated magnetic particles mediated by a peptide[J]. Journal of Nanoparticle Research, 2014, 16(8): 1-9.
22	LI Lulu, LONG Liangkun, DING Shaojun. Direct affinity-immobilized phenolic acid decarboxylase by a linker peptide on zeolite for efficient bioconversion of ferulic acid into 4-vinylguaiacol[J]. ACS Sustainable Chemistry & Engineering, 2020, 8(39): 14732-14742.
23	YU Seung-Hye, KUMAR M, KIM Il Won, et al. A comparative analysis of in vitro toxicity of synthetic zeolites on IMR-90 human lung fibroblast cells[J]. Molecules, 2021, 26(11): 3194.
24	GUTIÉRREZ-LÓPEZ A N, MENA-CERVANTES V Y, GONZÁLEZ-ESPINOSA M A, et al. Green and fast biodiesel production at room temperature using soybean and Jatropha curcas L. oils catalyzed by potassium ferrate[J]. Journal of Cleaner Production, 2022, 372: 133739.
25	KALITA P, BASUMATARY B, SAIKIA P, et al. Biodiesel as renewable biofuel produced via enzyme-based catalyzed transesterification[J]. Energy Nexus, 2022, 6: 100087.
26	WANG Qian, GUO Xuan, GE Meiling, et al. Engineering balanced anions coupling with tailored functional groups of poly(ionic liquid)s immobilized lipase enables effective biodiesel production[J]. Molecular Catalysis, 2022, 531: 112673.
27	Roberto FERNANDEZ-LAFUENTE. Lipase from Thermomyces lanuginosus: Uses and prospects as an industrial biocatalyst[J]. Journal of Molecular Catalysis B: Enzymatic, 2010, 62(3/4): 197-212.
28	MONTEIRO R R C, ARANA-PEÑA S, ROCHA T N DA, et al. Liquid lipase preparations designed for industrial production of biodiesel. Is it really an optimal solution?[J]. Renewable Energy, 2021, 164: 1566-1587.
29	XU Rui, CHEN Zirong, CHEN Yanyang, et al. Multiple strategies for high-efficiency expression of Thermomyces lanuginosus lipase in Pichia pastoris and production of biodiesel in solvent-free system[J]. Fuel, 2023, 333: 126246.
30	AMESHO K T T, LIN Yuan-Chung, CHEN Chinen, et al. Kinetics studies of sustainable biodiesel synthesis from Jatropha curcas oil by exploiting bio-waste derived CaO-based heterogeneous catalyst via microwave heating system as a green chemistry technique[J]. Fuel, 2022, 323: 123876.
31	GUTIÉRREZ-LÓPEZ A N, MENA-CERVANTES V Y, GARCÍA-SOLARES S M, et al. NaFeTiO₄/Fe₂O₃-FeTiO₃ as heterogeneous catalyst towards a cleaner and sustainable biodiesel production from Jatropha curcas L. oil[J]. Journal of Cleaner Production, 2021, 304: 127106.
32	GIWA A, ADEYEMI I, DINDI A, et al. Techno-economic assessment of the sustainability of an integrated biorefinery from microalgae and Jatropha: A review and case study[J]. Renewable and Sustainable Energy Reviews, 2018, 88: 239-257.
33	GO A W, SUTANTO S, ZULLAIKAH S, et al. A new approach in maximizing and direct utilization of whole Jatropha curcas L. kernels in biodiesel production — Technological improvement[J]. Renewable Energy, 2016, 85: 759-765.
34	YAN Jinyong, ZHENG Xianliang, DU Lei, et al. Integrated lipase production and in situ biodiesel synthesis in a recombinant Pichia pastoris yeast: An efficient dual biocatalytic system composed of cell free enzymes and whole cell catalysts[J]. Biotechnology for Biofuels, 2014, 7(1): 1-8.
35	NICOLÁS P, LASSALLE V L, FERREIRA M L. Quantification of immobilized Candida antarctica lipase B (CALB) using ICP-AES combined with Bradford method[J]. Enzyme and Microbial Technology, 2017, 97: 97-103.
36	TIAN Kaiyuan, TAI Kee, B Jian Wei CHUA, et al. Directed evolution of Thermomyces lanuginosus lipase to enhance methanol tolerance for efficient production of biodiesel from waste grease[J]. Bioresource Technology, 2017, 245: 1491-1497.
37	ZHANG Meiling, Seung-Hyun JUN, Youngho WEE, et al. Activation of crosslinked lipases in mesoporous silica via lid opening for recyclable biodiesel production[J]. International Journal of Biological Macromolecules, 2022, 222: 2368-2374.
38	OKUMURA K, SATO K, KAMIOKA K, et al. Direct immobilization of triphenylphosphine palladium complexes on the external surface of zeolite Β[J]. Microporous and Mesoporous Materials, 2019, 288: 109571.
39	COSTA-SILVA T A, CARVALHO A K F, SOUZA C R F, et al. Highly effective Candida rugosa lipase immobilization on renewable carriers: Integrated drying and immobilization process to improve enzyme performance[J]. Chemical Engineering Research and Design, 2022, 183: 41-55.
40	GIRELLI A M, CHIAPPINI V. Renewable, sustainable, and natural lignocellulosic carriers for lipase immobilization: A review[J]. Journal of Biotechnology, 2023, 365: 29-47.
41	PARANDI E, SAFARIPOUR M, ABDELLATTIF M H, et al. Biodiesel production from waste cooking oil using a novel biocatalyst of lipase enzyme immobilized magnetic nanocomposite[J]. Fuel, 2022, 313: 123057.
42	NÁJERA-MARTÍNEZ E F, MELCHOR-MARTÍNEZ E M, SOSA-HERNÁNDEZ J E, et al. Lignocellulosic residues as supports for enzyme immobilization, and biocatalysts with potential applications[J]. International Journal of Biological Macromolecules, 2022, 208: 748-759.
43	ZHAO Junxin, MA Maomao, YAN Xianghui, et al. Immobilization of lipase on β-cyclodextrin grafted and aminopropyl-functionalized chitosan/Fe₃O₄ magnetic nanocomposites: An innovative approach to fruity flavor esters esterification[J]. Food Chemistry, 2022, 366: 130616.
44	KHOOBI M, KHALILVAND-SEDAGHEH M, RAMAZANI A, et al. Synthesis of polyethyleneimine (PEI) and β-cyclodextrin grafted PEI nanocomposites with magnetic cores for lipase immobilization and esterification[J]. Journal of Chemical Technology & Biotechnology, 2016, 91(2): 375-384.
45	RIOS N S, NETO D M A, DOS SANTOS J C S, et al. Comparison of the immobilization of lipase from Pseudomonas fluorescens on divinylsulfone or p-benzoquinone activated support[J]. International Journal of Biological Macromolecules, 2019, 134: 936-945.
46	RABBANI G, AHMAD E, AHMAD A, et al. Structural features, temperature adaptation and industrial applications of microbial lipases from psychrophilic, mesophilic and thermophilic origins[J]. International Journal of Biological Macromolecules, 2023, 225: 822-839.
47	MEHDI W A, MEHDE A A, ÖZACAR M, et al. Characterization and immobilization of protease and lipase on chitin-starch material as a novel matrix[J]. International Journal of Biological Macromolecules, 2018, 117: 947-958.
48	KHAN M F, KUNDU D, HAZRA C, et al. A strategic approach of enzyme engineering by attribute ranking and enzyme immobilization on zinc oxide nanoparticles to attain thermostability in mesophilic Bacillus subtilis lipase for detergent formulation[J]. International Journal of Biological Macromolecules, 2019, 136: 66-82.
49	ZHANG Huaxia, LIU Tianshu, ZHU Yawei, et al. Lipases immobilized on the modified polyporous magnetic cellulose support as an efficient and recyclable catalyst for biodiesel production from Yellow horn seed oil[J]. Renewable Energy, 2020, 145: 1246-1254.
50	OTARI S V, PATEL S K S, KALIA V C, et al. One-step hydrothermal synthesis of magnetic rice straw for effective lipase immobilization and its application in esterification reaction[J]. Bioresource Technology, 2020, 302: 122887.
51	ZHANG Jun, CHEN Xiaoyan, Pengmei LYU, et al. Bionic-immobilized recombinant lipase obtained via bio-silicification and its catalytic performance in biodiesel production[J]. Fuel, 2021, 304: 121594.
52	MIAO Changlin, YANG Lingmei, WANG Zhongming, et al. Lipase immobilization on amino-silane modified superparamagnetic Fe₃O₄ nanoparticles as biocatalyst for biodiesel production[J]. Fuel, 2018, 224: 774-782.
53	ABDULMALEK S A, LI Kai, WANG Jianhua, et al. Enhanced performance of Rhizopus oryzae lipase immobilized onto a hybrid-nanocomposite matrix and its application for biodiesel production under the assistance of ultrasonic technique[J]. Fuel Processing Technology, 2022, 232: 107274.

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