Wenqi Zhao,Meihuan Yao,Huidan Liu,Tingru Du,Zhijun Guo,Binbin Zhang,Yujie Wang,Guangyue Bai
Tailoring of the arylboronic acid structures is essential for developing specific monosaccharide (MS) recognition receptors, in which the designed boronic acid derivative should have an appropriate pKa, a stereostructure matched with the target MS, and an easily detectable method. Herein, we report a novel bis(m-benzylammonium) boronic acid with an ethyl spacer (BTEAB) as the receptor molecule to MS. Owing to its unique lyotropic acidity and intrinsic fluorescence characteristics in aqueous solution, BTEAB provides a new strategy for the design of MS sensors. The cation−π interaction promotes the self-assembly behavior of BTEAB and the lyotropic acidity and fluorescence quenching induced by aggregation. The aggregation behavior of BTEAB was confirmed by Rayleigh light scattering, fluorescence, and 1H NMR, and its lyotropic acidity is quantified by the decrease in its acid dissociation constant pKa. It was found that the esterification of BTEAB with three model MSs (fructose, glucose, and galactose) does not destroy the aggregates, indicating that the lyotropic acidity is an applicable characteristic for regulating the pH value of MS recognition. The affinity of BTEAB with MS and the structures of BTEAB-MS ester were studied mainly by isothermal titration calorimetry (ITC), fluorescence and NMR. The interaction mechanism of BTEAB with different MS was proposed. This work provides new insights into MS recognition under about physiological pH adjusted by diarylboronic acid aggregation.
β-1,3-Galactanases selectively degrade β-1,3-galactan, thus it is an attractive enzyme technique to map high-galactan structure and prepare galactooligosaccharides. In this work, a gene encoding exo -β-1,3-galactanase (PxGal43) was screened form Paenibacillus xylanexedens , consisting of a GH43 domain, a CBM32 domain and α-L-arabinofuranosidase B (AbfB) domain. Using β-1,3-galactan (AG-II-P) as substrate, the recombined enzyme expressed in Escherichia coli BL21 (DE3) exhibited an optimal activity at pH 7.0 and 30 °C. The enzyme was thermostable, retaining >70 % activity after incubating at 50 °C for 2 h. In addition, it showed high tolerance to various metal ions, denaturants and detergents. Substrate specificity indicated that PxGal43 hydrolysis only β-1,3-linked galactosyl oligosaccharides and polysaccharides, releasing galactose as an exo -acting manner. The function of the CBM32 and AbfB domain was revealed by their sequential deletion and suggested that their connection to the catalytic domain was crucial for the oligomerization, catalytic activity, substrate binding and thermal stability of PxGal43. The substrate docking and site-directed mutagenesis proposed that Glu191, Gln244, Asp138 and Glu81 served as the catalytic acid, catalytic base, pKa modulator, and substrate identifier in PxGal43, respectively. These results provide a better understanding and optimization of multi-domain bacterial GH43 β-1,3-galactanase for the degradation of arabinogalactan.
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