| Lactulose is a non-digestible disaccharide widely used in food and pharmaceutical industries as prebiotic food additives and medicines against chronic constipation and hepatic encephalopathy. Currently, commercially available lactulose is produced mainly through the chemical isomerization of lactose under alkaline media, which requires a considerable amount of chemical catalysts and involves complicated separation and purification steps to remove catalysts and high level of undesirable by-products. In the last decade, particular attention has been dedicated to the use of β-galactosidases(EC 3.2.1.23) and cellobiose 2-epimerases(CEs, EC5.1.3.11) as biocatalysts for lactulose production. The enzymatic synthesis of lactulose catalyzed by Kluyveromyces lactis β-galactosidase(KlβGal) and Caldicellulosiruptor saccharolyticus cellobiose 2-epimerase(Cs CE) were investigated in this thesis.The enzymatic transgalactosylation of lactose in the presence of fructose using KlβGal leading to the formation of oligosaccharides was investigated in detail. The reaction mixture was analyzed by high performance liquid chromatography with differential refraction detector(HPLC-RI) and two main transgalactosylation products were discovered. To elucidate their overall structures, the products were isolated and purified using preparative liquid chromatography and analyzed by LC/MS, one-dimensional(1D) and two-dimensional(2D) NMR studies. 1-Lactulose(β-D-galactopyranosyl-(1â†'1)-D-fructose) with two main isomers in D2 O, β-D-galactopyranosyl-(1â†'1)-β-D-fructopyranose and β-D-galactopyranosyl-(1â†'1)-β-D-fructofuranose, was identified to be the major transgalactosylation product. Lactulose(β-D-galactopyranosyl-(1â†'4)-D-fructose) turned out to be the minor one. These results indicated that KlβGal was regioselective with respect to the primary C-1 hydroxyl group of fructose.The alginate-gelatin-calcium phosphate(AGCa P) hybrid carrier, which was the alginate capsule covered with gelatin film and calcified shell, was successfully prepared through a facile biomimetic mineralization process, and further utilized to immobilize KlβGal. Gelatin was used to inspire and template the assembly of calcium phosphate for enzyme immobilization at ambient temperature and neutral p H. The surface morphology, swelling performance and mechanical stability of calcium alginate(Alg) capsules and AGCa P hybrid capsules were investigated. Experimental results indicated that the assembled calcium phosphate shell significantly enhanced the mechanical stability and lowered the swelling degree of the capsules, additionally improved the immobilization efficiency and dramatically inhibited the leakage of KlβGal. Thus, the relative activity of KlβGal immobilized in AGCa P capsules was more than 60% after the 20 th cycle and about 90% after 30 d storage. Meanwhile, AGCa P immobilized in hybrid capsules, in comparison to free enzyme and enzyme immobilized in Alg capsules, exhibited the broadest temperature and p H ranges. At 38 °C, p H 6.8 with 250 mg/m L lactose and fructose, the immobilized KlβGal in AGCa P capsules produced the two transgalactosylation products lactulose at 26.1 mg/m L and 1-lactulose at 74.2 mg/m L after 3 h. The yield of both lactulose and 1-lactulose were approximately 40%.Cs CE belongs to N-acyl-D-glucosamine 2-epimerase(AGE) superfamily, and usually catalyzes epimerization at the C-2 position of the glucose moiety at the reducing end of cellobiose and lactose to generate 4-O-β-D-glucopyranosyl-D-glucose and epilactose(4-O-β-D-galactopyranosyl-D-mannose), respectively. However, Cs CE additionally covert lactose into lactulose(4-O-β-D-galactopyranosyl-D-fructose). Cs CE was cloned, expressed in Escherichia coli, purified by anion exchange, hydrophobic interaction, and size exclusion chromatography. At 80 °C, p H 7.5, the specific activity and half-life of thermal inactivation of the purified Cs CE for lactulose synthesis was 10.8 U/mg and 141.4 min. Cs CE requires no metal ion, coenzymes or nucleotides for catalysis. According to the catalysis properties, it was proposed that Cs CE was a aldose-aldose epimerase with aldose-ketose isomerase activity and could catalyze the direct isomerization of lactose into lactulose via a cis-enediol intermediate. The recombinant Cs CE converted lactose(500 mg/m L) to 56.9% lactulose and 16.3% epilactose.The industrial application of Cs CE for lactulose synthesis is limited by low enzyme activity and the formation of by-product epilactose. A microplate screening strategy was designed for the efficient screening directed evolution libraries of Cs CE. After four sequential rounds of random mutagenesis and screening, an optimum mutant G4-C5 was obtained. Compared to wild type enzyme, it has a 2.8-fold increase in specific activity and a 3.0-fold increase in kcat/Km for lactose isomerization without compromising thermostability, and its epimerization activity was completely inhibited under conditions for maximal isomerization activity. DNA sequencing of mutant G4-C5 revealed five amino acid substitutions, R5 M, A12 S,I52V, K328 I and F231 L. The yield of lactulose catalyzed by mutant G4-C5 increased to approximately 76% with very little epilactose detected, indicating that mutant G4-C5 was much more suitable for the production of lactulose than the wild type enzyme.Information about the structural bases of the cellobiose 2-epimerase catalysis of lactose isomerization is lacking. Single crystals of wild type Cs CE(WT-Cs CE) and mutant G4-C5 were obtained by the hanging drop vapor diffusion method. Under the crystallization condition of 0.1 M sodium citrate p H 5.6, 10% v/v 2-propyl alcohol, 10% w/v polyethylene glycol(PEG) 4,000, the crystals of WT-Cs CE were obtained. And the condition for mutant G4-C5 crystallization was 0.2 M magnesium chloride, 0.1 M HEPES buffer p H 7.5, 30% w/v PEG 400. The best WT-Cs CE and mutant G4-C5 crystals diffracted to 1.54 ? and 1.67 ? resolution, respectively. The structure demonstrates an(α/α)6 barrel fold, which shows significant structural homology with other enzymes from AGE superfamily. The(α/α)6 barrel structure consists of 6 outer helices running in roughly the same direction and 6 inner helices oriented in the opposite direction and the formed deep cleft is considered to be a putative active site. Important residues were identified by amino acid sequence alignment and tertiary structure superposition with enzymes from the same superfamily. Site-directed mutagenesis of these residues showed that Arg56, His188 and His377 were strictly required for activity and Glu191, Glu312, Arg380, Trp308 and Trp372 were also essential for high activity. Based on the catalysis properties and structural information, Cs CE likely perform a deprotonation/protonation mechanism through a cis-enediol intermediate in a metalindependent manner and His377 and His188 may serve as the acid/base catalytic sites. Structure of mutant G4-C5 showed that three mutations A12 S, R5 W and K328 I were in the different α-helixes at the surface region and far from the putative catalytic center. Aminio acid substitution, F231 L, was also at the surface of the protein, but it was close to the entrance of the cleft. Mutation I52 V located in an inner α-helix and was close to the conserved residues Arg56, Tyr114 and Trp372.Based on the determined crystal structure of Cs CE, semi-rational and computational design were applied to enhance the thermostability of the enzyme. A total of eight single-site mutants were designed, including S99 P, T110 G, S180 P, S304 G, S351 G, E94 G, E161 D and N365 P. Five mutants, S180 P, S351 G, E94 G, E161 D and N365 P, showed prolonged half-life of inactivation at 80 °C. Combinational mutations were subsequently introduced, and the best one was the double mutant E161D/N365 P. Its half-life was approximately 4-fold of that of the wild type enzyme, reaction temperature for maximum activity increased from 80 °C to 87.5 °C, and catalytic efficiency(kcat/Km) for lactulose production increased by about 29%. Mutant E161D/N365 P was also more stable against chemical denaturation and showed a broader p H profile. The second most stable variant was mutant E161D/S180P/S351 G with a 3.27-fold increase in half-life. These results provided new insights into the thermostability of Cs CE and suggested further potential industrial applications. |
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