Several putative lipase genes from the genome of the yeast Blastobotrys (Arxula) raffinosifermentans (adeninivorans) LS3 were overexpressed in the yeast itself and screened for the desymmetrization of the dicarboxylic acid diester diethyl adipate (DEA) into the monoester monoethyl adipate (MEA). MEA can serve as a monomeric spacer group for functional polymers used in medical chemistry and dental applications.

The selected lipase Alip2-c6hp was intracellularly located. After overexpression of the corresponding gene, it was purified and biochemically characterized using p-nitrophenyl butyrate as the substrate for standard activity tests. In fed-batch cultivation with constructed yeast strain B. raffinosifermentans G1212/YRC102-Alip2-c6h for large scale production of the Alip2-c6hp biocatalyst enzyme activities up to 674 U L⁻¹ were reached.

Several tested diesters were hydrolyzed selectively to monoesters. Under optimized conditions, the purified enzyme Alip2p-c6h converted 96 % of the substrate DEA to MEA within 30 min incubation, whereby only 1.6 % of the unwanted side-product adipic acid (AA) was formed. At room temperature the dicarboxylic acid esters diethyl malonate (DEM), diethyl succinate (DES), dimethyl adipate (DMA) and dimethyl suberate (DMSub) were completely hydrolyzed to their corresponding monoesters. A high yield of 87 % and 25 % could also be achieved with the dioldiesters 1,4-diacetoxybutane (DAB) and diacetoxyhexane (DAH).

In conclusion the potential of the lipase Alip2-c6hp expressed in B. raffinosifermentans is very promising for selective hydrolysis of DEA to MEA as well as for the production of other monoesters.

来自酵母Blastobotrys (Arxula) raffinosifermentans ( adeninivorans ) LS3 基因组的几个推定的脂肪酶基因在酵母本身中过表达，并筛选了二羧酸二酯己二酸二乙酯 (DEA) 去对称化为己二酸单酯单乙酯 (MEA)。MEA 可作为用于医学化学和牙科应用的功能性聚合物的单体间隔基团。

选定的脂肪酶 Alip2-c6hp 位于细胞内。在相应基因过表达后，使用对硝基苯丁酸作为标准活性测试的底物对其进行纯化和生化表征。在用构建的酵母菌株B. raffinosifermentans G1212/YRC102-Alip2-c6h 进行补料分批培养以大规模生产 Alip2-c6hp 生物催化剂酶的活性达到 674 UL ^-1。

几种测试的二酯选择性地水解成单酯。在优化条件下，纯化的酶 Alip2p-c6h 在 30 分钟温育内将 96% 的底物 DEA 转化为 MEA，从而仅形成 1.6% 的不需要的副产物己二酸 (AA)。在室温下，二羧酸酯丙二酸二乙酯 (DEM)、琥珀酸二乙酯 (DES)、己二酸二甲酯 (DMA) 和辛二酸二甲酯 (DMSub) 完全水解为其相应的单酯。使用二元酯 1,4-二乙酰氧基丁烷 (DAB) 和二乙酰氧基己烷 (DAH) 也可以获得 87% 和 25% 的高产率。

总之，在棉子疏草中表达的脂肪酶 Alip2-c6hp 的潜力对于 DEA 选择性水解为 MEA 以及其他单酯的生产非常有前景。