Enhanced thermal stability of the β-galactosidase BgaB from Bacillus circulans by cyclization mediated via SpyTag/SpyCatcher interaction and its use in galacto-oligosaccharides synthesis

• Published in: International journal of biological macromolecules Vol. 222; pp. 2341 - 2352
• Main Authors: Han, Mingming, Xu, Chenxi, Guan, Bo, Hu, Youzhen, Kazlauskas, Romas J., Li, Xu, Ni, Yongqing
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 01.12.2022
• Subjects: Aggregation; Galacto-oligosaccharides; SpyTag/SpyCatcher; Thermal stability; β-Galactosidase; Aggregation; Galacto-oligosaccharides; β-Galactosidase; Thermal stability; SpyTag/SpyCatcher
• ISSN: 0141-8130; 1879-0003
• DOI: 10.1016/j.ijbiomac.2022.10.021

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. •β-Galactosidase BgaB with an N- and C-termini distance of 20.2 Å was cyclized.•The cyclized β-galactosidase CBB showed an increased melting temperature.•Cyclization conferred β-galactosidase BgaB enhanced resilience to aggregation.•Synthesis of GOS with CBB at low initial lactose concentration gave higher yields.