Screening Of α-Galactosidase With Transglycosylation And Enzymatic Synthesis Of Globotruiose

α-Galacto-oligosaccharides widely exist in nature and have very important biological functions and application value in functional food and medicine industry. For examples, there are many kinds of non-digestible α-galacto-oligosaccharides in soymilk, and their structural analogues, usually containing Galα1-6 linkages, can be used as prebiotics for selective stimulation of the beneficial gut microbiota. α-Gal epitope oligosaccharides bearing the Galal-3 linkages are present as the major carbohydrate antigens in non-primate mammals, prosimians and New World monkeys, resulting in the immune processes when it is recognized by the nature antibody anti-Gal in human. Thus the artificial α-Gal epitope oligosaccharides and their derivatives can be used in xenotransplantation to decrease immunological rejection by neutralizing antibody. Besides, the α-Gal epitope oligosaccharides can be used in vaccine development for cancer therapy to enhance immunogenicity. Another important α-galacto-oligosaccharide, globotriose （Galα1-4Galβ1-4Glc）, exists on human cell surface. It acts as the receptor for binding the Shiga toxins produced by Shigella dysenteriae or enterohemorrhagic E. coli. The resulting invasion of Shiga toxins is dangerous and could finally result in lethal hemolytic uremic syndrome （HUS）. The artificially synthesized globotriose and its derivatives can mimic the receptor structure and then bind the toxins, thus can be used as an affinity inhibitors for the toxins in the prevention and treatment of related diseases. Globotriose is also the core structure of Globo H and SSEA4 existed in many cancer cells such as brain, breast, lung, ovary, stomach, and small-cell lung tumor cells, thus it can be used as a building block in the synthesis of the carbohydrate-related cancer antigens for anticancer vaccine development. Additionally, recent reports showed that cellular or soluble Pk/Gb3 histo-blood group antigen containing galabiose （Galal-4Gal） terminal structure and its analogues can resist HIV-1 infection by probablely inhibiting the fusion of the HIV-1 envelope to the cell target membrane. These results suggested that globotriose and its derivatives might be developed as a novel therapeutic approach for the prevention of HIV/AIDS.Oligosaccharides or their analogues have potential application value in functional food and medicine industry, but the large-scale production of these biomolecules is still remains difficult because of their complicated structures. Oligosaccharide synthesis methods include chemosynthesis and enzymatic synthesis. Chemosynthesis of oligosaccharides need tedious work including extensive protection and deprotection manipulation and has low yields. Enzymatic synthesis of oligosaccharides and glycosides has been a highly attractive approach as it possesses the advantages of high stereo/regioselectivity which can be achieved only through many protecting group manipulations in chemical synthesis. Glycosyltransferases and glycosidases are two classes of enzyme which have been applied for oligosaccharide synthesis in one-step reactions. Glycosyltransferases, the natural enzymes responsible for the synthesis of oligo- and polysaccharides, catalyze stereo/regioselective reactions effectively, but their use has been hampered by the requirement of costly nucleotide diphosphate glycosyl donors. Glycosidases, the enzymes that normally hydrolyze carbohydrates, can catalyze reverse hydrolysis or transglycosylation reactions for glycoside formation using simple sugar donors. Glycosyl donors of glycosidase include monosaccharides, oligosaccharides and glycoside compounds, these properties make glycosidases attract worldwide attentions for low cost, large-scale synthesis of oligosaccharides.a-Galactosidases （EC 3.2.1.22） are an important class of glycosidases that catalyze the hydrolysis of terminal galactose from various oligosaccharides and glycoconjugates. Based on the protein sequence similarities, a-galactosidases have been classified into glycosyl hydrolase families GH4, GH27, GH36, GH57, GH97, and GH110 （http://www.cazy.org/）. Transglycosylation activities of some α-galactosidases have been investigated and applied in the enzymatic synthesis of important a-galacto-oligosaccharides or α-galactosides. Most of the known a-galactosidases form Galal-3 or Galal-6 linkages. To the best of our knowledge, only very few enzymes are able to form Gala 1-4 linkage, including thea-galactosidase from Stachys affinis, Lactobacillus reuteri 100-16/100-23 and Bifidobacterium breve 203. There is only one a-galactosidase from B. breve 203 which could synthesize globotriose derivative using methyl β-lactoside as glycosyl acceptor, but two isomeric products have been synthesized due to relaxed regioselectivity of the enzyme. Screening a-galactosidase with transglycosylation and strict regioselectivity would be significant for enzymatic synthesis of oligosaccharides and glycosides.First of all, screening of a-galactosidase with transglycosylation was carried out in order to obtain a-galactosidase capable of synthesizing Galα1-4 linkage using lactose as glycosyl acceptor in this work. Nine a-galactosidase genes from four different kinds of microorganism were cloned by normal PCR and TAIL-PCR. These genes included agaLc from Lactobacillus casei JCM1134（2295 bp）, agaLb737 from Lactobacillus breve JCM 1059（2214 bp）, CPF₀491 gene from Clostridium perfringens ATCC13124（2205 bp） and genes of BF1418, BF4189, BF0233, BF0498, BF0803, BF0227 from Bacteroides fragilis NCTC 9343（1503 bp,1491 bp,2160 bp, 2169 bp,076 bp,1593 bp）All of the a-galactosidase genes were cloned into the expression vector pBAD/His A respectively. The resulting recombinant plasmid was subsequently transformed into E. coli LMG194. All of the 9 recombinant a-galactosidase could hydrolyze p-nitrophenyl-a-D-galactopyranoside. Except the recombinant BF0803, all the other 8 recombinant a-galactosidases displayed transglycosylation property using pNPaGal as the glycosyl donor and lactose as glycosyl acceptor. The transglycosylation products synthesized by CPF₀491、BF0233 and BF0227 had the same migration distance with the standard globotriose on the TLC plate. The result of HPAEC analysis also displayed the peak of transglycosylation products （named as PLac） synthesized by BF0227 shared identical retention time to the globotriose standard. The gCOSY result of acetylated modified PLac showed that this product was Galαl-4Lac. BF0227 was the first a-galactosidase that which could form Gala 1-4 linage using lactose as glycosyl acceptor. Thus, the further research would be related to BF0227.Further study showed that recombinant a-galactosidase BF0227 could not purified by nickel affinity chromatography. The gene of a-galactosidase BF0227 was 1593 bp, encoding a 58.3kDa protein which was classified into glycosyl hydrolase families GH27. According to analysis of SignalP 4.1, BF0227 had putative signal sequence of 24 amino acids at N-terminus. The gene encoding the truncated protein devoid of signal sequence was amplified by PCR and named as agaBf3S. The E. coli LMG194/pBAD/His A-agaBf3S had been constructed. The recombinant enzyme AgaBf3S was expressed and purified to electrophoretic purity. The subunit molecular mass as determined by SDS-PAGE was about 59.2 kDa, consistent with the predicted molecular mass deduced from its nucleotide sequence. AgaBf3S was highly active at pH 4.5-5.0 and stable at pH 4.0-11.0. The optimal temperature for enzyme activity was 40 ℃, and it was stable below 40 ℃. Hg2+ and Ag+ completely inactivated the enzyme activity, and EDTA slightly affected the enzyme activity. AgaBf3S was highly active toward pNPaGal. It displayed 37.6% of the pNPaGal activity when oNPaGal was used as the substrate, but it showed no activity towards other nitrophenyl glycosides. The Km, kcat values of AgaBf3S for p NPaGal, melibiose, and globotriose were determined to be 1.27 mM and 172.97 S-1,62.76 mM and 17.74 S-1,4.62 mM and 388.45 S-1,respectively. Thus the kcat/Km values of AgaBf3S for pNPαGal, melibiose, and globotriose were 135.88 mM-1S-1,0.28 mM-1S-1,84.12 mM-S-1, respectively. Thus, the enzyme would prefer pNPaGal to globotriose for hydrolysis.The influences of various factor on the yields of transglycosylation product PLac formed by AgaBf3S were further evaluated, including substrate concentration, pH, reaction time and temperature. the optimal conditions for transglycosylation product synthesis by AgaBf3S were an initial concentration of 20 mM pNPaGal and 500 mM lactose, at pH 4.5 and incubation at 40℃ for 30 min. As a whole, the highest yield of PLac achieved 32.4% at these conditions. The 1H NMR and 1H,13C-HSQC spectra of the product were fully in agreement with those of the standard globotriose. Combining these results with the results of TLC and HPAEC analyses, the transglycosylation product formed by AgaBf3S was completely confirmed as globotriose. AgaBf3S produced globotriose as a single transglycosylation product in high efficiency using pNPaGal and lactose as substrates. This enzyme was the first glycosidase that showed strict α1-4 regioselectivity toward lactose. Its use in the synthesis of globotriose would be of practical meaning as it has advantages of low cost and short reaction time when compared to the current synthetic methods.Transglycosylation products could be formed by AgaBf3S in the reactions with pNPaGal as donor and cellbiose （Glcβ1-4Glc）, maltose （Glcαl-4Glc）, melibiose （Glcal-6Glc）, galactose, glucose, mannose or arabinose as acceptor, but no transglycosylation product was detected when the acceptor was ketose, sugar alcohol, xylose or rhamnose. These results showed that the structure of saccharide ring, position of hydroxyl, especially hydroxyl bond types of C-4 were important for acceptor selectivity of AgaBf3S.Mutant enzymes with improved transglycosylation efficiency had been obtained by the combination of random mutagenesis and site-directed mutagenesis. Meanwhile, amino acid site involved in acceptor selectivity of AgaBf3S was studied. The transglycosylation catalyzed by a-galactosidases proceeds through the same reaction mechanism as general glycosidases, resulting in moderate product yields. Thus, the a-galactosidase gene agaBf3S was subjected to random mutagenesis by error-prone PCR in order to improve the transglycosylation product yield. After the comparison of their transglycosylation efficiencies, residues A141 and H426 seemed to weigh more importance for the elevated transglycosylation efficiency. Therefore, these two residues were chosen for the further directed evolution through site-directed mutagenesis, and different kinds of amino acid residues were used to probe these sites. Finally, the highest transglycosylation yield of the mutant A141W and H426S were 36.06% and 38.25%. Compared with wild-type enzyme, transglycosylation yield of the mutant enzyme increased 11% and 18%, respectively. The homology modeling of recombinant AgaBf3S was conducted by Phyre2 （http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index, Arap27A as a template）. The model structure of AgaBf3S showed A141 at the side of the catalytic cavity, the substitution of alanine by non-polar tryptophan could reduce water activity, which tends to favor the transglycosylation reaction over the hydrolysis reaction. The alignment result showed D135 was the nucleophile residue, D191 was the acid/base residue, D48, K133, C170, R187 surrounded the donor, Y101, W172, P194 surrounded the putative aglycon subsite. The mutant W172F formed autocondensation products with pNPaGal as substrate, whereas the AgaBf3S could not synthesize autocondensation products. It was confirmed that the replacement of W172 changed acceptor selectivity of AgaBf3S.