| α-Galactosidase(α-D-Galactoside galactohydrolase; EC 3.2.1.22), also called melibiase, is an exoglycosidase.α-Galactosidase from coffee beans could transfer the galactosyl residue directly to the cyclodextrin ring and synthesize theα-D-galactosyl-cyclodextrin. And thisα-D-galactosyl-cyclodextrin with wide application in various fields is one kind of branched cyclodextrins. In the present study, oneα-galactosidase was extracted and purified from germinating coffee beans, and its properties were studied in detail. Theα-D-galactosyl-cyclodextrin was synthesized by thisα-galactosidase and its chemical composition and structure were identified by some analysis methods.The germinating coffee beans with the germination for 35d were in refrigeratory(-40°C) for 24h, then they were ground and washed in the extraction(0.9% sodium chloride, 0.1mmol/L ethylene diamine tetraacetic acid, pH4.8). Other conditions were: ratio of material to liquid 1:5, time 30min, pH4.8, stir 100r/min. Under these conditions, total activity was 132.60nkat, and specific activity was 6.44nkat/mg. Then, these condition parameters were optimized by response surface method in the next process. The optimum parameters were: ratio of material to liquid 1:5, time 35min, pH5.0, stir 150r/min. Under these optimum conditions, total activity was 148.00nkat, and specific activity was 7.16nkat/mg. Total and specific activities were observably increased. Plant polyphenols interfered and impact the extraction ofα-D-galactosidase from the germinating coffee beans. Acetone (50%, V/V) was used to remove plant polyphenols in this extraction process and the 76.4 percent of plant polyphenols were removed. Total and specific activities ofα-galactosidase were 129.05nkat and 8.26nkat/mg. Specific activity was improved by removal of plant polyphenols.α-Galactosidase was purified by ammonium sulfate precipitation, DEAE Sepharose ion exchange chromatograph, and Sephacryl S-200-HR chromatograph. The final purifiedα-galactosidase obtained a specific activity of 191.50nkat/mg and there was a 23.18-fold increase in specific activity when compared with the crude extract.α-Galactosidase was purified to homogeneity by SDS-PAGE analysis. The single clear band representedα-galactosidase. The analysis allowed an estimated of molecular mass of purifiedα-galactosidase and was determined to be 38.8 kDa.With p-nitrophenyl-α-D-galactopyranoside as substrate, the optimum reaction temperature ofα-galactosidase was 45°C, and it was stable within the temperature range 20-50°C. The optimum reaction pH ofα-galactosidase was 6.0, and it was stable within the pH range 5.0-6.5.α-Galactosidase had a Km=0.556mmol/L and Vmax=1.19μmol/min. The metal ions had the different level influence on the purifiedα-galactosidase. Its activity was completely inhibited by Hg2+, and no obviously effect by Ba2+, Na+, K+, Mg2+and Cu2+, while Zn2+ had an activator effect. And it was stable within the ion (sodium chloride) concentration range 0-0.25 mol/L. The chemical modification of NBS resulted in a completely loss ofα-galactosidase activity, suggesting that Trp was an essential amino acid to the enzyme active site. -SH,-COOH and His were involved in it. And Arg, Lys and -S-S- were not involved in it. The number of the essential Trp was estimated as one.The purifiedα-galactosidase was applied to synthesize branched cyclodextrins using melibiose as donor andβ-cyclodextrin as acceptor. The reaction conditions were:β-cyclodextrin 0.4mol/L, melibiose 0.8mol/L,α-galactosidase 50nkat, 2mL acetate buffer (50mmol/L) pH 6.5, reaction temperature 30°C, reaction time 36h, oscillatory speed 100r/min. It showed that some relatives ofβ-cyclodextrin were synthesized by thisα-galactosidase. The new products were isolated with using preparative high-performance liquid chromatography (HPLC). The structure of one branchedβ-cyclodextrin was analyzed and elucidated by HPLC, fourier transform infrared spectrometer (FTIR), electro spraying ionization-mass spectrometry (ESIMS), and nuclear magnetic resonance (NMR). The results strongly demonstrate that the synthesized product is a mono-6-O-α-D-galactopyranosyl-β-cyclodextrin.The parameters affecting yield ofα-galactosyl-β-cyclodextrin were systematically studied by using experimental designs including Plackett-Burman design, steepest ascent method and Box-Behnken design. The conditions were optimized as follows: enzyme concentration 73nkat, reaction time 28h and pH6.3. The other parameters were: reaction temperature 40°C, oscillatory speed 75r/min,β-cyclodextrin 0.4mol/L, melibiose 0.8mol/L and 2mL acetate buffer (50mmol/L). Under these conditions, the yield ofα-galactosyl-β- cyclodextrin was 28.4%. Theα-galactosyl-β-cyclodextrin yield was increased by 41.3% as compared to that under unoptimized reaction conditions.
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