Navone Laura, Vogl Thomas, Luangthongkam Pawarisa, Blinco Jo-Anne, Luna-Flores Carlos H, Chen Xiaojing, von Hellens Juhani, Mahler Stephen, Speight Robert
Faculty of Science, Queensland University of Technology, Brisbane, QLD, Australia.
ARC Centre of Excellence in Synthetic Biology, Queensland University of Technology, Brisbane, QLD, Australia.
Biotechnol Biofuels. 2021 Mar 31;14(1):80. doi: 10.1186/s13068-021-01936-8.
Phytases are widely used commercially as dietary supplements for swine and poultry to increase the digestibility of phytic acid. Enzyme development has focused on increasing thermostability to withstand the high temperatures during industrial steam pelleting. Increasing thermostability often reduces activity at gut temperatures and there remains a demand for improved phyases for a growing market.
In this work, we present a thermostable variant of the E. coli AppA phytase, ApV1, that contains an extra non-consecutive disulfide bond. Detailed biochemical characterisation of ApV1 showed similar activity to the wild type, with no statistical differences in k and K for phytic acid or in the pH and temperature activity optima. Yet, it retained approximately 50% activity after incubations for 20 min at 65, 75 and 85 °C compared to almost full inactivation of the wild-type enzyme. Production of ApV1 in Pichia pastoris (Komagataella phaffi) was much lower than the wild-type enzyme due to the presence of the extra non-consecutive disulfide bond. Production bottlenecks were explored using bidirectional promoters for co-expression of folding chaperones. Co-expression of protein disulfide bond isomerase (Pdi) increased production of ApV1 by ~ 12-fold compared to expression without this folding catalyst and restored yields to similar levels seen with the wild-type enzyme.
Overall, the results show that protein engineering for enhanced enzymatic properties like thermostability may result in folding complexity and decreased production in microbial systems. Hence parallel development of improved production strains is imperative to achieve the desirable levels of recombinant protein for industrial processes.
植酸酶在商业上广泛用作猪和家禽的膳食补充剂，以提高植酸的消化率。酶的开发主要集中在提高热稳定性，以承受工业蒸汽制粒过程中的高温。提高热稳定性通常会降低酶在肠道温度下的活性，对于不断增长的市场而言，对改良植酸酶仍有需求。
在本研究中，我们展示了大肠杆菌AppA植酸酶的一种热稳定变体ApV1，它含有一个额外的非连续二硫键。对ApV1的详细生化特性分析表明，其活性与野生型相似，对植酸的k和K 、pH活性最佳值和温度活性最佳值均无统计学差异。然而，在65、75和85℃孵育20分钟后，它仍保留了约50%的活性，而野生型酶几乎完全失活。由于存在额外的非连续二硫键，毕赤酵母（Komagataella phaffi）中ApV1的产量远低于野生型酶。使用双向启动子共表达折叠伴侣来探究生产瓶颈。与没有这种折叠催化剂的表达相比，共表达蛋白二硫键异构酶（Pdi）使ApV1的产量提高了约12倍，并将产量恢复到与野生型酶相似的水平。
总体而言，结果表明，通过蛋白质工程增强酶的热稳定性等特性可能会导致折叠复杂性增加，并降低微生物系统中的产量。因此，必须同时开发改良的生产菌株，以实现工业生产所需水平的重组蛋白。
