Trabelsi Sahar, Sahnoun Mouna, Elgharbi Fatma, Ameri Rihab, Ben Mabrouk Sameh, Mezghani Monia, Hmida-Sayari Aïda, Bejar Samir
Laboratory of Microbial Biotechnology and Engineering Enzymes (LMBEE), Centre of Biotechnology of Sfax (CBS), University of Sfax, Sidi Mansour Road Km 6, P.O. Box 1177, 3018, Sfax, Tunisia.
Mol Biol Rep. 2019 Feb;46(1):921-932. doi: 10.1007/s11033-018-4548-2. Epub 2018 Dec 7.
A synthetic cDNA-AmyA gene was cloned and successfully expressed in Pichia pastoris as a His-tagged enzyme under the methanol inducible AOX1 promoter. High level of extracellular amylase production of 72 U/mL was obtained after a 72 h induction by methanol. As expected, the recombinant strain produced only the AmyA isoform since the host is a protease deficient strain. Besides, the purified r-AmyA showed a molecular mass of 54 kDa, the same pH optimum equal to 5.6 but a higher thermoactivity of 60 °C against 50 °C for the native enzyme. Unlike AmyA which maintained 50% of its activity after a 10-min incubation at 60 °C, r-AmyA reached 45 min. The higher thermoactivity and thermostability could be related to the N-glycosylation. The r-AmyA activity was enhanced by 46% and 45% respectively in the presence of 4 mM Fe and Mg ions. This enzyme was more efficient in bread-making since such ions were reported to have a positive impact on the nutriment quality and the rheological characteristics of the wheat flour dough. The thermoactivity/thermostability as well as the iron and magnesium activations could also be ascribed to the presence of an additional C-terminal loop containing the His tag.
一个合成的cDNA-AmyA基因被克隆，并在甲醇诱导的AOX1启动子下作为带有His标签的酶在毕赤酵母中成功表达。甲醇诱导72小时后，胞外淀粉酶产量达到72 U/mL的高水平。正如预期的那样，由于宿主是蛋白酶缺陷型菌株，重组菌株只产生AmyA同工型。此外，纯化后的r-AmyA分子量为54 kDa，最适pH值相同，均为5.6，但与天然酶相比，热活性更高，在60℃时为60℃，而天然酶在50℃时。与AmyA在60℃孵育10分钟后仍保持50%的活性不同，r-AmyA达到了45分钟。更高的热活性和热稳定性可能与N-糖基化有关。在4 mM铁离子和镁离子存在下，r-AmyA活性分别提高了46%和45%。这种酶在面包制作中更有效，因为据报道这些离子对小麦粉面团的营养质量和流变学特性有积极影响。热活性/热稳定性以及铁和镁的激活作用也可能归因于含有His标签的额外C末端环的存在。
