A recombinant putative lipoxygenase from Burkholderia thailandensis with a specific activity of 26.4 U mg(-1) was purified using HisTrap affinity chromatography. The native enzyme was a 75-kDa dimer with a molecular mass of 150 kDa. The enzyme activity and catalytic efficiency (k cat/K m) were the highest for linoleic acid (k cat of 93.7 s(-1) and K m of 41.5 μM), followed by arachidonic acid, α-linolenic acid, and γ-linolenic acid. The enzyme was identified as an omega-6 linoleate lipoxygenase (or a linoleate 13S-lipoxygenase) based on genetic and HPLC analyses as well as substrate specificity. The reaction conditions for the enzymatic production of 13-hydroxy-9,11(Z,E)-octadecadienoic acid (13-HODE) were optimal at pH 7.5, 25 °C, 20 g l(-1) linoleic acid, 2.5 g l(-1) enzyme, 0.1 mM Cu(2+), and 6% (v/v) methanol. Under these conditions, linoleate 13-lipoxygenase from B. thailandensis produced 20.8 g l(-1) 13-HODE (70.2 mM) from 20 g l(-1) linoleic acid (71.3 mM) for 120 min, with a molar conversion yield of 98.5% and productivity of 10.4 g l(-1) h(-1). The molar conversion yield and productivity of 13-HODE obtained using B. thailandensis lipoxygenase were 151 and 158% higher, respectively, than those obtained using commercial soybean lipoxygenase under the optimum conditions for each enzyme at the same concentrations of substrate and enzyme.
A new biotransformation process for the production of the flavor lactone was developed by using permeabilized Waltomyces lipofer, which was selected as an efficient ␥-dodecalactone-producing yeast among 10 oleaginous yeast strains. The optimal reaction conditions for ␥-dodecalactone production by permeabilized W. lipofer cells were pH 6.5, 35°C, 200 rpm, 0.7 M Tris, 60 g/liter of 10-hydroxystearic acid, and 30 g/liter of cells. Under these conditions, nonpermeabilized cells produced 12 g/liter of ␥-dodecalactone after 30 h, with a conversion yield of 21% (wt/wt) and a productivity of 0.4 g/liter/h, whereas permeabilized cells obtained after sequential treatments with 50% ethanol and 0.5% Triton X-100 produced 46 g/liter of ␥-dodecalactone after 30 h, with a conversion yield of 76% (wt/wt) and a productivity of 1.5 g/liter/h. These values were 3.7-and 3.8-fold higher than those obtained using nonpermeabilized cells. These are the highest reported concentration, conversion yield, and productivity for the production of the bioflavor lactone.
Specialized proresolving mediators (SPMs), such as resolvins and maresins, resolve inflammation and protect against infection at trace amounts generated by neutrophils and macrophages in humans. Thus, SPMs have been leading compounds in treatment of inflammation and infection. These mediators have been synthesized using chemical methods, which have disadvantages such as use of toxic chemical reagents, many (20−30) step reactions, and expensive processes. Here, we first discovered a human SPM-producing microorganism and found a 15-lipoxygenase (15-LOX) from the microorganism that produces SPMs via double oxygenation from polyunsaturated fatty acids. The 15-LOX was altered to 12-LOX using structure-based engineering. We performed the quantitative biocatalytic synthesis of SPMs by wild-type 15-LOX and engineered 12-LOX. Owing to the high double-oxygenating activities of LOXs, we succeeded in the one-enzyme biotransformation of docosahexaenoic acid and docosapentaenoic acid into the SPM resolvin D5 (RvD5, 7S,17S-dihydroxydocosahexaenoic acid) and RvD5 n−3 DPA (7S,17Sdihydroxydocosapentaenoic acid) (>40% conversion), respectively, by Escherichia coli expressing wild-type 15-LOX and into the 7Sepimer of maresin (MaR) 1 (7S,14S-dihydroxydocosahexaenoic acid) and 7S-epimer of MaR1 n−3 DPA (7S,14S-dihydroxydocosapentaenoic acid) (>20% conversion), respectively, by E. coli expressing engineered 12-LOX. Our study contributes to the environmentally friendly synthesis of medicinally important SPMs.
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