A comparative approach to recombinantly produce the plant enzyme horseradish peroxidase in Escherichia coli.
A comparative approach to recombinantly produce the plant enzyme horseradish peroxidase in Escherichia coli.
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DOI：
10.1016/j.jbiotec.2017.03.003
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发表时间：
2017-04-20
影响因子：
4.1
通讯作者：
Spadiut O
中科院分区：
文献类型：
--
作者：
Gundinger T;Spadiut O
辣根过氧化物酶(HRP)被广泛应用于各种生物技术和医学领域。由于其从植物中分离出来的缺点，以往的研究主要是以大肠杆菌作为重组表达宿主。然而，复性包涵体的产量和周质中活性酶的表达都没有超过每升培养液10毫克的最终滴度。因此，传统的植物生产HRP的方法仍然盛行。在这项研究中，我们回顾了HRP在大肠杆菌中的重组生产，并对两种策略进行了研究和比较：(A)HRP作为包涵体(IBS)的生产和随后的复性以及(B)在周质中生产活性HRP。事实上，无论采用哪种方法，我们都能在大肠杆菌中生产HRP。复性得率为10%，最终滴度为10 0 mg L−-1发酵液，周质中每升发酵液可产生48 mg活性辣根过氧化物酶。在生化性质方面，可溶性HRP的催化活性和稳定性显著降低，这可能是由于本研究中使用的融合伙伴DsbA所致。复性后的HRP表现出与植物HRP相似的底物亲和力，催化效率降低了11倍，热稳定性降低了2倍。综上所述，我们开发了一个HRP工程和生产工具箱。我们建议通过定向进化或半理性蛋白质设计来设计HRP，在大肠杆菌的周质中表达HRP，允许直接筛选改进的变体，最后以IB的形式大量生产这些变体，然后进行折叠。
Horseradish peroxidase (HRP) is used in various biotechnological and medical applications. Since its isolation from plant provides several disadvantages, the bacterium Escherichia coli was tested as recombinant expression host in former studies. However, neither production from refolded inclusion bodies nor active enzyme expression in the periplasm exceeded final titres of 10 mg per litre cultivation broth. Thus, the traditional way of production of HRP from plant still prevails. In this study, we revisited the recombinant production of HRP in E. coli and investigated and compared both strategies, (a) the production of HRP as inclusion bodies (IBs) and subsequent refolding and (b) the production of active HRP in the periplasm. In fact, we were able to produce HRP in E. coli either way. We obtained a refolding yield of 10% from IBs giving a final titre of 100 mg L−1 cultivation broth, and were able to produce 48 mg active HRP per litre cultivation broth in the periplasm. In terms of biochemical properties, soluble HRP showed a highly reduced catalytic activity and stability which probably results from the fusion partner DsbA used in this study. Refolded HRP showed similar substrate affinity, an 11-fold reduced catalytic efficiency and 2-fold reduced thermal stability compared to plant HRP. In conclusion, we developed a toolbox for HRP engineering and production. We propose to engineer HRP by directed evolution or semi-rational protein design, express HRP in the periplasm of E. coli allowing straight forward screening for improved variants, and finally produce these variants as IB in high amounts, which are then refolded.