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. 2020 Dec;10(12):523.
doi: 10.1007/s13205-020-02517-6. Epub 2020 Nov 10.

High-efficiency expression of the thermophilic lipase from Geobacillus thermocatenulatus in Escherichia coli and its application in the enzymatic hydrolysis of rapeseed oil

Jun Zhang  1   2 , Miao Tian  1   2 , Pengmei Lv  1 , Wen Luo  1 , Zhiyuan Wang  1 , Jingliang Xu  1   3 , Zhongming Wang  1
Affiliations

High-efficiency expression of the thermophilic lipase from Geobacillus thermocatenulatus in Escherichia coli and its application in the enzymatic hydrolysis of rapeseed oil

Jun Zhang et al. 3 Biotech. 2020 Dec.

Abstract

Long-chain fatty acids are widely used in food and chemical industries, and the enzymatic preparation of fatty acids is considered an environmentally friendly process. In the present study, long-chain fatty acids were prepared by the enzymatic hydrolysis of rapeseed oil with a genetically engineered lipase. Because thermophilic lipase has strong stability at higher temperatures, it was more suitable for the industrial production of long-chain fatty acids. Therefore, the thermophilic lipase BTL2 from Geobacillus thermocatenulatus was efficiently expressed in E. coli BL21(DE3) cells with an enzyme activity of 39.50 U/mg followed by gene codon optimisation. Experimental results showed that the recombinant lipase BTL2 exhibited excellent resistance to certain organic solvents (n-hexane, benzene, ethanol, and butanol). The metal cation Ca2+ and the non-ionic surfactant Triton-100X enhanced enzyme activity by 7.36% and 56.21% respectively. Moreover, the acid value of the liberated long-chain fatty acids by hydrolysing rapeseed oil was approximately 161.64 mg KOH/g at 50 °C in 24 h, the hydrolytic conversion rate was 91.45%, and the productivity was approximately 6.735 mg KOH/g h. These results suggested that the recombinant lipase BTL2 has excellent hydrolytic performance for rapeseed oil and showed great potential for the enzymatic preparation of long-chain fatty acids.

Keywords: Enzymatic hydrolysis; Escherichia coli; Rapeseed oil; Thermophilic lipase.

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Figures

Fig. 1
Fig. 1
BTL2-pET28b ( +) recombinant plasmid construction (a), and protein purification profile of recombinant lipase BTL2 by SDS-PAGE (b). MW molecular weight marker, IN Input samples, FT flow-through samples, W washes samples (W1–W3), E eluted fractions samples (E1–E9). 2 μg was the loading amount of purified recombinant BTL2
Fig. 2
Fig. 2
Effects of pH on recombinant lipase BTL2. The optimal pH was measured at 60 °C with varying buffer pH; lipase BTL2 was incubated in various pH buffers for 1 h before enzyme activity analysis to verify pH stability
Fig. 3
Fig. 3
Effects of temperature on recombinant lipase BTL2. The optimal temperature was measured at pH 8.0 with varying temperatures; lipase BTL2 was incubated at various temperatures for 1 h before lipase activity analysis to verify temperature stability
Fig. 4
Fig. 4
Reaction time curve of recombinant lipase BTL2 to release long-chain fatty acids. The hydrolysis reaction of rapeseed oil catalysed by recombinant lipase BTL2 was carried out under 50 °C in 32 h
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