Cloning, expression and characterization of a linoleic acid isomerase from Propionibacterium acnes

LI Xun, GU Huaxiang, WANG Fei

JOURNAL OF NANJING FORESTRY UNIVERSITY ›› 2014, Vol. 38 ›› Issue (06) : 105-109.

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南京林业大学学报（自然科学版）
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JOURNAL OF NANJING FORESTRY UNIVERSITY ›› 2014, Vol. 38 ›› Issue (06) : 105-109. DOI: 10.3969/j.issn.1000-2006.2014.06.020

Cloning, expression and characterization of a linoleic acid isomerase from Propionibacterium acnes

  • LI Xun, GU Huaxiang, WANG Fei*
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Abstract

In order to study on the feasibility for the enzymatic synthesis of conjugated linoleic acid(CLA), the linoleic acid(LA)isomerase from Propionibacterium acnes was cloned and overexpressed in Escherichia coli, and characterized. The LA isomerase gene(pai)from P. acnes was improved by using codon optimization as E. coli codon usage. The DNA sequence encoding modified LA isomerase was cloned into pET-20b, yielding pET-20b-pai, and transformed into E. coli BL21(DE3). The recombinant LA isomerase had a molecular mass of 48 ku showing mainly in inclusion body on SDS-PAGE. The gene product was purified by Ni-NTA and the activity was 752.3 μmol/(min·mg)after induction and purification. At pH 7.0 and 35 ℃, the activity of enzyme reached the maximum. The purified enzyme was stable from pH 6.5 to pH7.5, and retained approx. 90% of its activity after 2 h at 35 ℃. A low-level inhibition of LA isomerase(22.4%)and(15.8%)was observed with Mn2+ and Zn2+(1 mmol/L), respectively. The recombinant LA isomerase had a Km of 1.13 mmol/L and kcat of 4.67 s-1 for linoleic acid. The result showed that the P. acnes linoleate isomerase can effectively catalyze LA to trans-10, cis-12 conjugated linoleic acid(t10, c12 CLA).

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LI Xun, GU Huaxiang, WANG Fei. Cloning, expression and characterization of a linoleic acid isomerase from Propionibacterium acnes[J]. JOURNAL OF NANJING FORESTRY UNIVERSITY. 2014, 38(06): 105-109 https://doi.org/10.3969/j.issn.1000-2006.2014.06.020

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