Discovery and Evaluation of High-activity Lipase and Its Application in Diacylglycerol Synthesis

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

Abstract

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

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.

Figures/Tables 8

References 38

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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

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

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

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

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］