高活性脂肪酶的发掘、评价及在甘油二脂合成中应用

doi:10.13560/j.cnki.biotech.bull.1985.2025-0392

摘要/Abstract

摘要：

目的针对国产脂肪酶催化效率低、热稳定性差等问题，筛选高活性脂肪酶菌株，解析催化特性用于酶法高效制备甘油二酯。方法采用中性红橄榄油平板初筛及对硝基苯酚比色法复筛的方法，从富含油脂的土壤中筛选高产脂肪酶菌株，结合形态学与16S rDNA序列分析进行菌种鉴定，通过PCR扩增获得高活性脂肪酶基因序列，探究其酶学性质，建立无溶剂体系评价其甘油二酯合成效率。结果从18份土壤样品中成功发掘到一株高产脂肪酶菌株E12C，胞外酶活为（80 826.4±1 838.9）U/L，经形态学与16S rDNA序列鉴定为洋葱伯克霍尔德菌（Burkholderia cepacia），命名为B. cepacian OCRI-Lip100，已保藏于中国典型培养物保藏中心。该脂肪酶命名为Lip-12c，最适反应温度为60 ℃、最适反应pH为9.0，在此条件下酶活为（190 761.2±5 181.5）U/L，比活力为（39 254.5±271.3）U/g蛋白，显著高于进口脂肪酶，在40-70 ℃和pH 5.0-10.0范围可维持较高活性，金属离子Na⁺和Mg²⁺对酶活性提升效果显著。在无溶剂体系中40 ℃酶法水解橄榄油4 h，甘油二酯含量达33.5%。结论土壤中发掘的高产脂肪酶菌株B. cepacian OCRI-Lip100，在无溶剂体系中展现高效的甘油二酯合成能力，不仅丰富了现有脂肪酶菌种资源库，还为功能脂质的高效生物制造提供了技术支撑。

关键词: 脂肪酶, 洋葱伯克霍尔德菌, 分离鉴定, 酶学性质, 甘油二酯

Abstract:

Objective Aiming to address the challenges of low catalytic efficiency and insufficient thermal stability in domestic lipases, this study focuses on screening high-activity lipase-producing strains, analyzing its catalytic properties, and applying it to efficient enzymatic production of diacylglycerol. Method High-yield lipase-producing strains were screened from oil-rich soil using neutral red olive oil plates for primary screening and the p-nitrophenol colorimetric method for secondary screening. Strain identification was performed by combining morphological analysis with 16S rDNA sequence analysis. The high-activity lipase gene sequence was obtained through polymerase chain reaction (PCR) amplification, followed by investigating enzymatic properties. A solvent-free system was established to evaluate diglyceride synthesis efficiency. Result A high-yield lipase-producing strain E12C was successfully isolated from 18 soil samples, exhibiting extracellular enzyme activity of 80 826.4 ± 1 838.9 U/L. Morphological and 16S rDNA sequence analysis were combined to identify the strain as Burkholderia cepacia, designated as B. cepacian OCRI-Lip 100, which has been deposited in the China Center for Type Culture Collection (CCTCC). The lipase, named Lip-12c, showed optimal activity at 60 ℃ and pH 9.0, with enzyme activity reaching (190 761.2 ± 5 181.5) U/L and specific activity of (39 254.5 ± 271.3) U/g protein under these conditions, significantly higher than imported lipases. It maintained high activity within 40-70 ℃ and pH 5.0-10.0, with Na⁺ and Mg²⁺ markedly enhancing its activity. In a solvent-free system, enzymatic hydrolysis of olive oil at 40 ℃ for 4 h yielded 33.5% diacylglycerol. Conclusion The high-yield lipase-producing strain B. cepacian OCRI-Lip 100, isolated from the soil, demonstrates efficient diacylglycerol synthesis in a solvent-free system. This achievement not only enriches the existing resource pool of lipase-producing strains but also provides technical support for the efficient biomanufacturing of functional lipids.

Key words: lipase, Burkholderia cepacia, isolation and identification, enzymatic property, diacylglycerol

徐远志, 胡珊, 代思泽, 游帅, 郑明明, 单凯. 高活性脂肪酶的发掘、评价及在甘油二脂合成中应用[J]. 生物技术通报, 2025, 41(11): 177-189.

XU Yuan-zhi, HU Shan, DAI Si-ze, YOU Shuai, ZHENG Ming-ming, SHAN Kai. Discovery and Evaluation of High-activity Lipase and Its Application in Diacylglycerol Synthesis[J]. Biotechnology Bulletin, 2025, 41(11): 177-189.

图/表 8

表1 PCR中用到的主要引物

Table 1 Main primers used in PCR

引物名称 Primer name	引物序列 Sequence of primer（5′-3′）	功能 Function
27-F	AGAGTTTGATCCTGGCTCAG	扩增16S rDNA序列用于菌种鉴定
1492-R	TACGGCTACCTTGTTACGACTT	扩增16S rDNA序列用于菌种鉴定
F	CGTGCTTCACTCCGCATT	扩增脂肪酶前段序列bclipF
R	GACCGGGTCTTCCGCA	扩增脂肪酶前段序列bclipF
12C-F	TTCGTCAATGTATTCGGCA	扩增脂肪酶后段序列bclipR
11-R	GAGCGCATCGAGATACGC	扩增脂肪酶后段序列bclipR
KZ-F	CAAATGGGTCGCGGATCCGAATTCATGGCGAGTGCGCCCGCCGAC	扩增完整脂肪酶序列bclip12c
KZ-R	TCGAGTGCGGCCGCAAGCTTTCACACGCCCGCGAGCTTCA	扩增完整脂肪酶序列bclip12c

图1 产脂肪酶菌株的发掘和生长曲线A：产脂肪酶菌株在平板上的变色圈；B：各菌株的脂肪酶活力；C：菌株生长曲线。不同字母表示有显著差异（P≤0.05），下同

Fig. 1 Excavation and growth curve of lipase-producing strainsA: Discoloration circle of lipase-producing strains on the plate. B: Lipase activity of each strain. C: Strain growth curve. Different letters indicate significant differences (P≤0.05), the same below

图2 菌株E12C的鉴定A：E12C菌株在LB平板上的菌落形态；B：在400 nm尺寸下的扫描电镜；C：E12C菌株的16S rDNA系统发育树

Fig. 2 Identification of strain E12CA: Colony morphology of E12C strain on LB plate. B: Scanning electron microscope at 400 nm. C: Phylogenetic tree of16S rDNA for strain E12C

图3 脂肪酶Lip-12c的基因鉴定A：脂肪酶基因序列bclipF和bclipR；B：成熟脂肪酶基因序列bclip12c；C：脂肪酶Lip-12c的SDS-PAGE结果分析；D：预测的脂肪酶Lip-12c三维结构；E：脂肪酶Lip-12c的系统发育树图；F：信号肽预测结果

Fig. 3 Identification of lipase Lip-12c geneA: Lipase gene sequence bclipF and bclipR. B: Mature lipase gene sequence bclip12c. C: Analysis of SDS-PAGE results of lipase Lip-12c. D: Predicted three-dimensional structure of lipase Lip-12c. E: Phylogenetic tree of lipase Lip-12c. F: Signal peptide prediction results

图4 酶学性质分析A：pH对酶活的影响；B：温度对酶活的影响；C：金属离子对酶活的影响；D：底物链长特异性

Fig. 4 Analysis of enzymatic propertiesA: Effect of pH on enzyme activity. B: Effect of temperature on enzyme activity. C: Effect of metal ions on enzyme activity. D: Substrate chain length specificity

表2 有机溶剂对脂肪酶Lip-12c活力的影响

Table 2 Effect of organic solvents on the activity of lipase lip-12c

有机溶剂 Organic solvent	油水分配系数 Log P	相对酶活 Relative activity （%）	有机溶剂 Organic solvent	油水分配系数 Log P	相对酶活 Relative activity （%）
无有机溶剂	-	100 ± 1.44^ab	乙酸	-0.17	70.62 ± 1.08^g
正己烷	3.5	85.37 ± 1.77^de	丙酮	-0.24	77.44 ± 2.71^f
环己烷	3.44	88.50 ± 1.03^d	乙醇	-0.31	91.69 ± 1.48^c
二氯甲烷	1.25	77.01 ± 0.71^f	乙腈	-0.33	90.66 ± 2.97^cd
乙醚	0.85	103.68 ± 1.29^a	甲醇	-0.77	97.23 ± 0.76^bc
乙酸乙酯	0.68	81.44 ± 0.64^e	丙三醇	-1.76	87.42 ± 0.74^d
异丙醇	0.05	79.94 ± 1.16^ef

表3 不同脂肪酶的比活力对比

Table 3 Comparison of specific activity of different lipases

脂肪酶 Lipase	蛋白浓度 Protein concentration （mg/mL）	比活力 Specific activity （U/g protein）	酶来源 Enzyme source
Lip-12c	1.57 ± 0.04^b	39 254.5 ± 271.3^a	本研究
Candida rugosa lipase AY 400SD	2.49 ± 0.12^a	6 171.1 ± 507.3^b	商品酶
C. rugosa lipase CRL	1.21 ± 0.18^b	3 376.6 ± 223.3^c
C. rugosa lipase AY SD	0.27 ± 0.05^c	298.0 ± 31.3^d
C. rugosa lipase AYS	0.26 ± 0.05^c	220.1 ± 15.5^d
Geobacillus thermocatenulatus KCTC	-	22 700	［25］
Aeromonas caviae LipT51	-	34 200	［24］

图5 脂肪酶Lip-12c水解产物气相色谱图

Fig. 5 GC of hydrolysis products of lipase Lip-12c hydrolysates

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