新型南极嗜冷真菌α-淀粉酶的烘焙工业功能表达及其磁性固定化。
Functional expression of a novel α-amylase from Antarctic psychrotolerant fungus for baking industry and its magnetic immobilization.
机构信息
State Key Lab of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, P.O.B.311, 130 Meilong Road, Shanghai, 200237, China.
Institute of Ocean and Earth Sciences, C308 Institute of Postgraduate Studies, University of Malaya, Kuala Lumpur, 50603, Malaysia.
出版信息
BMC Biotechnol. 2017 Feb 28;17(1):22. doi: 10.1186/s12896-017-0343-8.
BACKGROUND
α-Amylase plays a pivotal role in a broad range of industrial processes. To meet increasing demands of biocatalytic tasks, considerable efforts have been made to isolate enzymes produced by extremophiles. However, the relevant data of α-amylases from cold-adapted fungi are still insufficient. In addition, bread quality presents a particular interest due to its high consummation. Thus developing amylases to improve textural properties could combine health benefits with good sensory properties. Furthermore, iron oxide nanoparticles provide an economical and convenient method for separation of biomacromolecules. In order to maximize the catalytic efficiency of α-amylase and support further applications, a comprehensive characterization of magnetic immobilization of α-amylase is crucial and needed.
RESULTS
A novel α-amylase (AmyA1) containing an open reading frame of 1482 bp was cloned from Antarctic psychrotolerant fungus G. pannorum and then expressed in the newly constructed Aspergillus oryzae system. The purified recombinant AmyA1 was approximate 52 kDa. AmyA1 was optimally active at pH 5.0 and 40 °C, and retained over 20% of maximal activity at 0-20 °C. The K and V values toward soluble starch were 2.51 mg/mL and 8.24 × 10 mg/(mL min) respectively, with specific activity of 12.8 × 10 U/mg. AmyA1 presented broad substrate specificity, and the main hydrolysis products were glucose, maltose, and maltotetraose. The influence of AmyA1 on the quality of bread was further investigated. The application study shows a 26% increase in specific volume, 14.5% increase in cohesiveness and 14.1% decrease in gumminess in comparison with the control. AmyA1 was immobilized on magnetic nanoparticles and characterized. The immobilized enzyme showed improved thermostability and enhanced pH tolerance under neutral conditions. Also, magnetically immobilized AmyA1 can be easily recovered and reused for maximum utilization.
CONCLUSIONS
A novel α-amylase (AmyA1) from Antarctic psychrotolerant fungus was cloned, heterologous expression in Aspergillus oryzae, and characterized. The detailed report of the enzymatic properties of AmyA1 gives new insights into fungal cold-adapted amylase. Application study showed potential value of AmyA1 in the food and starch fields. In addition, AmyA1 was immobilized on magnetic nanoparticles and characterized. The improved stability and longer service life of AmyA1 could potentially benefit industrial applications.
背景
α-淀粉酶在广泛的工业过程中起着关键作用。为了满足生物催化任务不断增长的需求，人们付出了相当大的努力来分离极端微生物产生的酶。然而，关于来自耐冷真菌的α-淀粉酶的相关数据仍然不足。此外，由于其高消耗量，面包质量特别受到关注。因此，开发改善质地的淀粉酶可以将健康益处与良好的感官特性结合起来。此外，氧化铁纳米粒子为分离生物大分子提供了一种经济便捷的方法。为了最大限度地提高α-淀粉酶的催化效率并支持进一步的应用，对α-淀粉酶的磁性固定化进行全面表征是至关重要的。
结果
从南极耐冷真菌 G. pannorum 中克隆了一种新型的α-淀粉酶（AmyA1），其包含一个 1482 bp 的开放阅读框，然后在新构建的米曲霉系统中表达。纯化的重组 AmyA1 约为 52 kDa。AmyA1 在 pH 5.0 和 40°C 时活性最佳，在 0-20°C 时保留超过 20%的最大活性。对可溶性淀粉的 K 和 V 值分别为 2.51 mg/mL 和 8.24×10 mg/（mL min），比活力为 12.8×10 U/mg。AmyA1 表现出广泛的底物特异性，主要水解产物为葡萄糖、麦芽糖和麦芽四糖。进一步研究了 AmyA1 对面包质量的影响。应用研究表明，与对照相比，面包的比容增加了 26%，粘聚性增加了 14.5%，胶粘性降低了 14.1%。AmyA1 被固定在磁性纳米颗粒上并进行了表征。固定化酶在中性条件下表现出更好的热稳定性和增强的耐碱性。此外，磁性固定化 AmyA1 可以很容易地回收和重复使用，以实现最大利用。
结论
从南极耐冷真菌中克隆了一种新型的α-淀粉酶（AmyA1），在米曲霉中进行了异源表达，并对其进行了表征。AmyA1 酶学性质的详细报告为真菌冷适应淀粉酶提供了新的见解。应用研究表明，AmyA1 在食品和淀粉领域具有潜在的价值。此外，AmyA1 被固定在磁性纳米颗粒上并进行了表征。AmyA1 的稳定性提高和使用寿命延长可能有益于工业应用。
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