Han Mingming, Xu Chenxi, Guan Bo, Hu Youzhen, Kazlauskas Romas J, Li Xu, Ni Yongqing
School of Food Science and Technology, Shihezi University, Shihezi, China.
School of Food Science and Technology, Shihezi University, Shihezi, China.
Int J Biol Macromol. 2022 Dec 1;222(Pt B):2341-2352. doi: 10.1016/j.ijbiomac.2022.10.021. Epub 2022 Oct 7.
Cyclization of proteins using SpyTag/SpyCatcher is a novel approach to increase their thermal stability. In this paper, we test this approach on two β-galactosidases from Bacillus circulans, BgaB and BgaC, and find that BgaB was stabilized while BgaC was not. Wild-type BgaB precipitated completely upon heating above 70 °C, but after SpyRing cyclization, it remained soluble after heating to 90 °C. Similarly, wild-type BgaB retained only 50 % activity after heating at 60 °C for 10 min, but this increased to 80 % after SpyRing cyclization. In contrast, cyclization decreased the stability of BgaC. After SpyRing cyclization, BgaC only retained 2 % activity after 20-min incubation at 55 °C, whereas the wild-type BgaC retained 25 % activity. One reason for the different effect of cyclization may the shorter distance between the N- and C-termini in BgaB (20.2 Å) as compared to BgaC (43.7 Å). The intrinsic fluorescence and circular dichroism spectra suggested that SpyRing cyclization of BgaB did not significantly change its conformation or secondary structure. SpyRing cyclized BgaB yielded similar amounts and compositions of galacto-oligosaccharides using a high initial lactose concentration (40 %, w/v), but a slightly higher amount at low initial lactose concentration (5 %, w/v) suggesting increased transgalactosylation activity.
利用SpyTag/SpyCatcher对蛋白质进行环化是一种提高其热稳定性的新方法。在本文中，我们对来自环状芽孢杆菌的两种β-半乳糖苷酶BgaB和BgaC测试了这种方法，发现BgaB的稳定性增强而BgaC没有。野生型BgaB在加热至70℃以上时会完全沉淀，但经过SpyRing环化后，加热至90℃仍可保持溶解状态。同样，野生型BgaB在60℃加热10分钟后仅保留50%的活性，但SpyRing环化后这一比例增加到了80%。相比之下，环化降低了BgaC的稳定性。经过SpyRing环化后，BgaC在55℃孵育20分钟后仅保留2%的活性，而野生型BgaC保留了25%的活性。环化产生不同效果的一个原因可能是BgaB的N端和C端之间的距离（20.2 Å）比BgaC（43.7 Å）短。内在荧光和圆二色光谱表明，BgaB的SpyRing环化并没有显著改变其构象或二级结构。使用高初始乳糖浓度（40%，w/v）时，SpyRing环化的BgaB产生的低聚半乳糖的量和组成相似，但在低初始乳糖浓度（5%，w/v）时，其产生的量略高，表明转半乳糖基化活性增强。
