Objectives

Cold-active lipases which show high specific activity at low temperatures are attractive in industrial applications in terms of product stability and energy saving. We aimed to identify novel cold-active lipase suitable for oleates synthesis and bread making.

Results

A novel lipase gene (RmLipA) from Rhizopus microsporus was cloned and heterologously expressed in Pichia pastoris. The encoding sequence displayed 75% identity to the lipase from R. niveus. The highest extracellular lipase activity of 7931 U/mL was achieved in a 5-L fermentation. The recombinant enzyme (RmLipA) was optimally active at pH 8.0 and 20–25 °C, respectively, and stable over a wide pH range of 2.0–11.0. The enzyme was a cold-active lipase, exhibiting > 80% of its maximal activity at 0 °C. RmLipA was a sn-1,3 regioselective lipase, and preferred to hydrolyze pNP esters and triglycerides with relatively long chain fatty acids. RmLipA synthesized various oleates using oleic acid and different alcohols as substrates (> 95%). Moreover, it significantly improved the quality of bread by increasing its specific volume (21.7%) and decreasing its crumb firmness (28.6%).

Conclusions

A novel cold-active lipase gene from R. microsporus was identified, and its application potentials were evaluated. RmLipA should be a potential candidate in oleates synthesis and bread making industries.

中文翻译：

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适用于油酸合成和面包制作的小孢根霉冷活性脂肪酶的异源表达和生化表征

目标

在低温下表现出高比活性的冷活性脂肪酶在产品稳定性和节能方面在工业应用中具有吸引力。我们旨在确定适用于油酸合成和面包制作的新型冷活性脂肪酶。

结果

从小孢子根霉中克隆出一个新的脂肪酶基因( RmLipA )，并在毕赤酵母中异源表达。编码序列与来自R. niveus的脂肪酶显示出 75% 的同一性。7931 U/mL 的最高细胞外脂肪酶活性在 5-L 发酵中实现。重组酶 (RmLipA) 分别在 pH 8.0 和 20-25°C 时具有最佳活性，并且在 2.0-11.0 的宽 pH 范围内稳定。该酶是一种冷活性脂肪酶，在 0 °C 时表现出 > 80% 的最大活性。RmLipA 是一种sn -1,3 区域选择性脂肪酶，优选水解p具有相对长链脂肪酸的 NP 酯和甘油三酯。RmLipA 使用油酸和不同的醇作为底物 (> 95%) 合成了各种油酸盐。此外，它通过增加比容（21.7%）和降低面包屑硬度（28.6%）显着提高了面包的质量。

结论

从小孢子菌中鉴定出一种新的冷活性脂肪酶基因，并评估了其应用潜力。RmLipA 应该是油酸合成和面包制造行业的潜在候选者。