蔗糖异构酶PalI在解脂耶氏酵母中的表面展示

Effective surface display of sucrose isomerase PalI on the cells of Yarrowia lipolytica

摘要:

【背景】蔗糖异构酶(PalI)生物转化蔗糖是目前生产异麦芽酮糖的主要方法，但在生产过程中存在的蔗糖异构酶转化蔗糖副产物比例较高、游离酶需要分离纯化等问题限制了异麦芽酮糖工业生产的应用。【目的】构建蔗糖异构酶PalI在解脂耶氏酵母(Yarrowia lipolytica) Po1g中的表面展示菌株，以降低蔗糖异构酶转化蔗糖的副产物比例及其纯化成本。【方法】为获得具有生产PalI能力的Y. lipolytica Po1g表面展示菌株，通过重叠延伸PCR将克雷伯氏菌(Klebsiella singaporensis) LX3的PalI的编码基因PalI与全基因合成的来自Y. lipolytica细胞壁的锚定蛋白Pir1融合，转入Y. lipolytica Po1g中构建表面展示菌株。利用3,5-二硝基水杨酸(3,5-dinitrosalicylic acid，DNS)比色定糖法测定表面展示的PalI酶活力并对其酶学性质进行探究，通过高效液相色谱法分析其转化蔗糖的产物。【结果】构建了蔗糖异构酶表面展示菌株Pir1-PalI/Po1g，经DNS法测得展示在Y. lipolytica Po1g表面的PalI酶活力为(4 694.6±56.6) U/g-菌体干重(dry cell weight，DCW)，该酶的最适反应温度及pH值分别为45 ℃和pH 6.0，在20-55℃，pH 3.5-9.0区间内较稳定。对该酶转化蔗糖产物进行分析，结果表明以异麦芽酮糖为主的二糖产物与单糖副产物的比例可达到91:9。【结论】本研究构建了表面展示PalI的Y. lipolytica Po1g菌株，为异麦芽酮糖的工业化生产奠定了基础。

关键词:蔗糖异构酶;解脂耶氏酵母;表面展示系统;酵母细胞壁锚定蛋白Pir1;酶学性质

Abstract:

[Background] Bioconversion via sucrose isomerase (PalI) is currently a preferred method to produce isomaltulose. However, this method still has some shortcomings, such as the formation of multiple products and the tedious purification process of PalI from cell extracts. [Objective] We sought to achieve efficient isomaltulose production via constructing a surface display strain for PalI on Y. lipolytica Po1g and reduce the proportion of by-products and purification cost. [Methods] The gene PalI encoding sucrose isomerase PalI from Klebsiella singaporensis LX3 was fused with the gene Pir1 encoding the anchoring protein Pir1 from the yeast cell wall by overlap extension PCR. The fused gene was then transferred into Y. lipolytica Po1g for the surface display of PalI. The enzymatic properties of the surface-displayed PalI were investigated via colorimetry with 3,5-dinitrosalicylic acid (DNS), and the products of sucrose conversion were detected by high performance liquid chromatography. [Results] The PalI surface display strain Pir1-PalI/Po1g was successfully constructed, and the highest activity of the displayed PalI reached (4 694.6±56.6) U/g of dry cell weight. The displayed PalI was stable at a broad range of 20-55 ℃ and pH 3.5-9.0, with the best performance at pH 6.0 and 45 ℃. Moreover, the displayed PalI led to significantly lower proportion of monosaccharide by-products during the bioconversion process than the free PalI. [Conclusion] Using Y. lipolytica Po1g as the host, we constructed a recombinant strain for the surface display of PalI, which provided a basis for the industrial production of isomaltulose.

Key words:sucrose isomerase;Yarrowia lipolytica;surface display system;yeast cell wall-anchoring protein Pir1;enzymatic properties