Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
FEBS Open Bio. 2014 Feb 3;4:168-74. doi: 10.1016/j.fob.2014.01.009. eCollection 2014.
Enzyme stability is an important parameter in biocatalytic applications, and there is a strong need for efficient methods to generate robust enzymes. We investigated whether stabilizing disulfide bonds can be computationally designed based on a model structure. In our approach, unlike in previous disulfide engineering studies, short bonds spanning only a few residues were included. We used cyclohexanone monooxygenase (CHMO), a Baeyer-Villiger monooxygenase (BVMO) from Acinetobacter sp. NCIMB9871 as the target enzyme. This enzyme has been the prototype BVMO for many biocatalytic studies even though it is notoriously labile. After creating a small library of mutant enzymes with introduced cysteine pairs and subsequent screening for improved thermostability, three stabilizing disulfide bonds were identified. The introduced disulfide bonds are all within 12 Å of each other, suggesting this particular region is critical for unfolding. This study shows that stabilizing disulfide bonds do not have to span many residues, as the most stabilizing disulfide bond, L323C-A325C, spans only one residue while it stabilizes the enzyme, as shown by a 6 °C increase in its apparent melting temperature.
酶稳定性是生物催化应用中的一个重要参数，因此需要高效的方法来产生稳定的酶。我们研究了是否可以基于模型结构来计算设计稳定二硫键。在我们的方法中，与以前的二硫键工程研究不同，我们包含了仅跨越几个残基的短键。我们使用环己酮单加氧酶（CHMO），即来自不动杆菌 sp. NCIMB9871 的 Baeyer-Villiger 单加氧酶（BVMO）作为目标酶。尽管这种酶众所周知不稳定，但它仍是许多生物催化研究的原型 BVMO。在创建了一个带有引入半胱氨酸对的突变酶的小文库，并随后筛选出提高热稳定性的文库后，确定了三个稳定的二硫键。引入的二硫键彼此之间的距离都在 12Å 以内，这表明该特定区域对于展开至关重要。这项研究表明，稳定二硫键不一定需要跨越许多残基，因为最稳定的二硫键 L323C-A325C 仅跨越一个残基，但可以稳定酶，其表观熔点升高了 6°C。
