Enzymatic Preparation Of Gluco-Mannooligosaccharide And Galactosidase

Functional oligosaccharides are low molecular weight carbohydrates, containingsugar moieties with degrees of polymerization (DP) between2and10. They possessimportantly physicochemical and physiological properties, promote a good balance ofintestinal probiotics and decrease gastrointestinal infections and serve as prebiotics.The best known functional oligosaccharides include gluco-manoligosaccharides(GMOS), galacto-ooligosaccharides (GOS) and so on.β-mannanase (β-1,4-D-mannan mannohydrolase EC3.2.1.78) is an importanthemicellulase, which can catalyze the hydrolysis of internal β-1,4-D-mannopyranosyllinkages in various β-mannan such as galactomannan, glucomannan,galactoglucomannan. In this study, response surface methodology (RSM) was appliedto optimize culture conditions for the production of β-mannanase. The finalcompositions of the optimized medium components in a shake-fask level weredetermined by PBD and CCD as follows (g/L): konjac34.68,(NH4)2SO44.69,Na2HPO412H2O4.00, KH2PO40.30, CaCO32.69, CaCl21.00, NaCO31.00,MgCl26H2O0.60. After optimizing, the maximum enzyme activity increased27.68%from120.42U/mL to153.75U/mL. Then, β-mannanase was produced in a7-Lfermentor and purified subsequently. The enzymatic properties showed that theoptimum pH and temperature of β-mannanase were6.0and60°C, respectively. It wasstable at pH5.0~8.0and below65°C. The enzyme activity was slightly stimulated bysome metal ions, such as Fe3+, K+, Fe2+, Ca2+and Na+. Finally, the purified β-mannanase was applied to the hydrolysis of konjac flour to prepare GMOS whichshowed excellent catalysis efficiency.β-galactosidase (β-D-galactosidase galactohydrolase, E.C.3.2.1.23) canhydrolyze lactose into glucose and galactose, and transfer the galactose unit to thehydroxyl group of water or another carbohydrate resulting in oligosaccharides with ahigher degree of polymerization.A β-galactosidase producing strain was identified as Kluyveromycs lactis by26SrDNA. The effects of lactose concentration and pH on K.lactis cultivation wereinvestigated. We also studied the activity of ethanol-permeabilized cells of K.lactis.After treatment at room temperature for1min using ethanol (final concentration40%),the permeability was changed, thus allowing the transit of low molecular weightcompounds in and out of the cells. β-galactosidase of ethanol-permeabilized cells were sTable at pH8.0and37oC, and slightly stimulated by some metal ions, such asFe2and Pb2+. GOS were preparated by ethanol-permeabilized cells of K.lactiscompared with a commercial β-galactosidase. The permeabilized cells exhibited betterhydrolysis and galactosyltransferase activity compared to commercial enzyme. Themaximum GOS concentration of95g/L with permeabilized cells was obtained wheninitial lactose concentration was500g/L while the maximum GOS concentration wasonly60g/L prepared by the commercial β-galactosidase.