A-galactosidase From Bifidobacterium Breve:Molecular Evolution,Transglycosylation And Crystallization

Glycosidases (EC 3.2.1) have been of tremendous utility in the enzymatic synthesis of oligosaccharides. They are advantageous for low cost substrates, availability and stability. These enzymes form glycoside linkage by one-step reaction from glycosyl donors and acceptors. However, the glycoside product yields are usually moderate due to the secondary hydrolysis of products catalyzed by the glycosidases themselves. In recent years, molecular evolution of the glycosidases has greatly improved the transglycosylation efficiency and made them become the truly powerful tool for the oligosaccharides synthesis. a-Galactosidase (EC 3.2.1.22) are among the most important glycosidase and are known to catalyze both hydrolytic and transgalactosylation reactions. The hydrolytic activity has been applied in the food,paper, and medical field for decades. The transgalactosylation activity could be used to synthesize galacto-oligosaccharides and galacto-containing chemicals, such as alkyl-glycosides and the like. These enzymes mainly synthesized Gala 1-6 and Gal?1-3 linkages. Only two a-galactosidases from Bifidobacterium breve 203 and Stachys affinis could generate the Gal?1-4 linkages. The Gal?1-4 linkages are natural component of the oligosaccharides part of some important glycoconjugates on the cell surface,such as the receptor of Shiga toxin,globotriose,and tumor-associate glycan,Globo-H. Thus ?-galactosidases producing the Gal?1-4 linkages are of valuable applications.The transglycosylation catalyzed by a-galactosidases proceeds through the same reaction mechanism as general glycosidases, rusutlint in moderate product yields.Four examples of site-mutation of a-galactosidases based on structure guidance had generated mutants. Nevertheless, there was no report on engineering a-galactosidases through random mutagenesis. Although two a-galactosidases was fount to have the ability to synthesize Galal-4 linkage, these enzymes stil has not been applied for the production of globotriose or its derivatives to date. The regioselectivity of glycosidases is usually influenced by the structures of glycosyl acceptors. The molecular mechanism of the flexible regioselectivity towards different transglycosylation reactions is unclear, since the reports on determining the a-galactosidase structures still focused on the explanation of the hydrolysis reaction mechanism. In this work, directed evolution of the a-galactosidase from Bifidobacterium breve 203 by random mutagenesis and subsequently by site-directed mutagenesis was carried out to enhance the transglycosylation activity and obtained an effective transgalactosidase. The muant enzyme was applied to synthesize the globotriose derivative and studied the regioselectivity of a-galactosidase from B.breve 203. The crystallization of this enzyme was also explored and expected to determine the crystal structure for the explanation of the improved transglycosylation efficiency and the molecular mechanism of the flexible regioselectivity.In our previous report, a ?-galactosidase (designated as Aga2) with transglycosylation activity was purified from B. breve 203 and its N-terminal sequence was A-I-M-D-F-H-G. Its gene aga2 was cloned by Southern hybridization and contained an ORF of 2226-bp nucleotides encoding 741 amino acids with a calculated molecular mass of about 81.5 kDa. The nucleotide sequence of aga2 has been submitted to GenBank under accession number DQ267828. The recombinant enzyme Aga2 was heterogeneously expressed and purified and its characteristics were consistent with the enzyme purified from B. breve 203. The subunit molecular mass of recombinant Aga2 was about 80.5 kDa, consistent with the predicted molecular mass deduced from the nucleotide sequence. The molecular mass of the native recombinant Aga2 was about 152 kDa, which indicated it was a homodimer. Besides the hydrolysis activity, the recombinant Aga2 also catalyzed the transglycosylation reaction and synthesized a trisaccharide (Gal?1-4Gal?1-6Glc) with melibiose as substrate.In this work, the Aga2 enzyme was evolved by random mutagenesis for the first time. The ?-galactosidase gene aga2 was subjected to random mutagenesis by error-prone PCR. After the blue-white spot screening with 5-bromo-4-chloro-3-indolyl-?-D-galactopyranoside (X-a-Gal) and the detection of transglycosylation activity with melibiose as the substrate, two mutants RM70 and RM103 that exhibited elevated transglycosylation activity compared to Aga2 were selected from about 4000 clones. The transglycosylation efficiencies of RM70 and RM103 with melibiose as substrate were found to be 47% and 45%, which were 21%and 19% higher than that of the Aga2 (26%),respectively. Both RM70 ? RM103 could be effectively expressed as soluble protein, which indicated that mutations existing in mutants RM70 and RM103 did not affect the correct folding of the protein.Sequencing of these two mutants revealed the presence of three mutations (G218S,D457A and H729R) in RM70 and two mutations (V564E and H573L) in RM103.Five independent mutants G218S, D457A, V564E, H573L and H729R were constructed via site-directed mutagenesis. After the comparison of their transglycosylation efficiencies towards melibiose with that of Aga2, residues V564,H573 and H729 seemed to weigh more importance for the elevated transglycosylation efficiency of mutants RM70 and RM103. Therefore, these three 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. Residue V564 exhibited a greater impact on improving the transglycosylation efficiency of Aga2 after the analysis of the transglycosylation efficiencies towards melibiose. Especially, the mutant V564N gave the highest transglycosylation efficiency of 58%, being 32%higher than that of Aga2. The model structure of mutant V564N showed a significant difference from Aga2, the catalytic cavity of V564N became shallow and wide. This alteration might be responsible for the improved transglycosylation efficiency as it led to easier access or separation of the enzyme from substrate or product. The model structures alignment of Aga2 and H573N, Aga2 and H729S exhibited that no obvious structure change were made by mutants H573N and H729S. For position 573, the substitution of non-polar histidine by polar asparagine could trap the water molecules through the H-bonds, which were not available for hydrolysis and favored transglycosylation reaction. The reason for position 729 to improve the transglycosylation activity of Aga2 was not clear,since this site was located in the C-terminal domain far away from the catalytic center.The effective mutants mentioned above were tested for the globotriose or its derivative synthesis using pNPGal as donor and lactose or methyl ?-lactoside as acceptor. For the reaction with lactose, three products were synthesized and they were identified as Gal?1-3Gal?1-4Glc, Galpl-4(Galal-6)Glca and Gal?1-6Gal?1-4Glc.The target globotriose was not detected and another important medicine oligosaccharide appared. While for the methyl ?-lactoside,a one-step reaction for the simultaneous synthesis of two important kinds of medicine oligosaccharides,?-Gal epitope and globotriose derivatives, was achieved. The Aga2 and all the mutants (RM70, RM103, V564N, H573N and H729S) gave the same product pattern,namely, forming two products from pNPGal and methyl ?-lactoside substrates.Among them, the mutant RM70 gave the same products yield (28%) as Aga2, while the mutants RM103, H573N and H729S reached 3%-5% higher yields than that of Aga2. For mutant V564N, the maximal total product yield was 38%, which was 10%higher than that of Aga2. As mutant V564N displayed the highest yield, it was further chosen for the oligosaccharide synthesis in a preparative scale. The impacts of temperature and pH on products synthesis were slight at 25?-40? and pH 6.5-8.5.The product yield obtained by V564N was stable after reaching the maximum at 10 min, and there was hardly any hydrolysis of the products during the testing time (6 h).These results indicated that V564N acted as a transgalactosidase without apparent hydrolysis of the transglycosylation products. The transglycosylation products were purified and identified as Gal?1-3Gal?1-4Glc?OMe and Gal?1-4Gal?1-4Glc?OMe by MS and NMR analysis. In this reaction, ?-Gal epitope and globotriose derivatives were synthesized simultaneously through enzymatic approach for the first time,and also it was the first discovery on the ability of synthesis of globotriose derivative by glycosidase.?-Galactosidase usually have broad substrate specificity and flexible regioselectivity towards different substrates. Mutant V564N had the same regioselectivity as Aga2 in the transglycosylation reaction and showed better property for the oligosaccharide synthesis. Thus,it was applied to study the regioselectivity of the a-galactosidase from B. breve 203 towards different glycosyl acceptors. This enzyme generated transglycosylation products in all the reactions with pNPGal as donor and trehalose (Glc?1-1Glc?), kojibiose (Glcal-2Glc), nigerose (Glc?1-3Glc),maltose (Glc?1 -4Glc), isomaltose (Glcal-6Glc), sophorose (Glc?1-2Glc),laminaribiose (Glc?1-3Glc), cellbiose (Glcpl-4Glc) or gentiobiose (Glcpl-6Glc) as acceptor. V564N produced single product with specific regioselectivity when using large steric hindrance substrates, such as gentiobiose and disaccharides bearing a-linkage between two glucose residues, whereas it showed various regioselectivity and generated isomers from small steric hindrance substrates, such as disaccharides bearing a-linkage between two glucose residues. These transglycosylation products were purified and identified as Galc?1-6Glc?1-1Glca, Glc?1-2(Gal?1-6)Glc?,Gal?1-6GIc?1-3Glc, Gal?1-6Glc?1-4Glc, Gal?1-6Glc?1-6Glc?,Gal?1-6Glc?1-2Glc?, Gal?1-6Glc?1-3Glc?,Galal-6Glc?1-4Glc? and Galal-6Glc?1-6Glc. These results indicated that V564N catalyzed the synthesis of linear Gal?1-6 oligosaccharides as only or main products with trehalose, nigerose,maltose, isomaltose, sophorose, laminaribiose, cellbiose or gentiobiose as acceptor,while with kojibiose as acceptor, only the branched Gal?1-6 product was synthesized.The selectivity towards these substrates of V564N was completely different from that towards melibiose and methyl ?-lactoside. The molecular mechanism of the different regioselectivity was unclear.The crystallization of Aga2 was explored with the aim to elucidate the molecular mechanism of the efficient mutants for transglycosylation as well as the flexible regioselectivity of the enzyme toward various acceptors. Aga2 was purified to homogenization through nickel affinity chromatography, Source 15Q exchange chromatography and Superdex 200 gel filtration chromatography.The initial screening for crystallization was performed by fourteen commercially kits from Hampton Research and Emerald Bio. A buffer condition from PEGRx-233 yielded crystals of target Aga2. This condition was 4% 2-propanol, 0.1 M Bis-Tris pH 9.0 and 20% PEG monomethyl ethero 5000. The obtained crystals were too small to X-ray diffraction studies. Based on this condition, we further optimized the condition through matrix method, changing the growing temperature, seeding and using the Additive and Detergent kits. Better crystals of Aga2 (9 mg/ml in 10 mM Tris and 0.1 M NaCl, pH 8.0) were obtained using the vapor diffusion method from a solution containing 4% v/v 2-propanol, 0.1 M Bis-Tris propane (pH 8.95) and 21% PEG 5000 at a temperature of 4? with the detergent of ANAPOE(?)-X-1 14 and a 1:1 ratio of protein to reservoir solution.The crystal was diffracted routinely to 3.2A resolution and had a space group of P212121 Because of the low identify of homologous of Aga2 in PDB database, the structure of Aga2 was not determined by the method of molecular replacement.Thus we prepared the Selenium Aga2 protein and expected to determine the structure of Aga2 through the method of Muti-wavelength anomalous dispersion.The crystal of Se-Aga2 (9 mg/ml in 10 mM Tris and 0.1 M NaCl, pH 8.0) were yielded using the vapor diffusion method from a solution containing 4% v/v 2-propanol, 0.1 M Bis-Tris propane (pH 8.9) and 23% PEG 5000 at a temperature of 4? with a 1:1 ratio of protein to reservoir solution. This crystal was diffracted routinely to 2.8A resolution. As the lattice orientation of the Se-Aga2 became incorrect, we still could not determine the structure of Aga2. The approaches via modifying Aga2 to obtain high quality crystals were in progress.