Screening Of The Strain Producing β-galactosidase, Puirfication And Characterization Of The Enzyme And Enzymatic Synthesis Of Galactooligosaccharides

Galactooligosaccharides （GOS） are recognized as one of the most important functionaloligosaccharides and composed by2to10galactose units and1glucose unit. They can act asprebiotics for their selectively promoting the growth of beneficial intestinal microflora andstimulating the absorption of calcium and other minerals. Besides their prebiotic effects, lowcariogenecity, low caloric values and low sweetness are the most important characteristics ofGOS. Commercial GOS are mainly enzymaticly synthesized from lactose by the enzymeβ-galactosidase.β-galactosidase （EC3.2.1.23） is one kind of the hydrolytic enzyme. However, some ofthem have the transgalactosylation activity. When its acceptor is water, β-galactosidasehydrolyzes lactose to glucose and galactose; oligosaccharides are formed when the galactosylmoiety is accepted by another saccharide.Recently a novel strain of Shigella sonnei SK22.001was isolated by our laboratory fromthe soil of a dairy farm. The sugar constitutes of the reaction products were analyzed byHPLC and LC/MS. The results showed that GOS mixture contained trans-galactosylateddisaccharides, trisaccharides, tetrasccharides, glucose, galactose and unreacted lactose. Firstly,the constituents of the culture medium and fermentation conditions were investigated.Through single factor test, the optimum constituents of the culture medium and fermentationconditions were obtained. The optimum conditions were lactose10g/L as the carbon sourse,yeast extract10g/L and peptone form fish5g/L as the nitrogen source, fermentationtemperature30oC, the initial pH7.0, medium volume20%（v/v）, shaking speed200r/min,inoculum size3%（v/v） and fermentation time21h. Under these conditions, the hydrolyticactivity of β-galactosidase reached6.02UoNPG/mL fermentation broth, and thetransgalactosylation activity was0.51ULac/mL fermentation broth.Secondly, β-galactosidase from Shigella sonnei SK22.001was successively purified byammonium sulfate fractionation, heat treatment, DEAE ion-exchange and gel filtrationchromatography. The enzyme was purified to homogeneity level with24.31-fold purificationand a recovery of9.72%. The specific activity of purified β-galactosidase witho-Nitrophenyl-β-D-Galactopyranoside （oNPG） was184.24UoNPG/mg. The molecular weightof the enzyme was approximately120kDa as evaluated by SDS-PAGE and365kDa asdetermined by gel filtration. The subunit size and gel filtration molecular weight indicate thatthe enzyme forms a trimer.Thirdly, the characteristics of the enzyme were studied. The results showed that thepurified β-galactosidase exhibited the maximum hydrolytic activity at the optimal pH6.5andtemperature50oC, and obtained the maximum of the transgalactosylation activity at pH7.0 and temperature55oC. Both Na+and K+could partly inhibit the hydrolytic activity, but hadfew influence on the transgalactosylation activity. However, Cu2+could inactivate the enzyme.Lactose hydrolyzate glucose had a strong inhibitory effect on hydrolytic activity, whilegalactose had little effect on hydrolytic activity. Both Galactose and glucose could inhibit thetransgalactosylation activity. The Kmand Vmaxvalues for the substrates oNPG and lactosewere3.29mmol/L and0.227mol/L; and4.91μmol·min^-1·mg^-1and3.42μmol·min^-1·mg^-1,respectively.Finally, on the basic research of the enzymatic property, the conditions influencing theenzymatic synthesis of GOS were studied. The GOS yield was analyzed by HPLC and used asexperimental index. The influences of the type and concentration of the buffer solution, theinitial lactose concentration, enzyme dosage, the reaction temperature, pH and reaction timeon the yield of GOS were evaluated. The results showed that the optimal conditions for theenzymatic synthesis of GOS were:0.1mol/L, pH7.0Na2HPO4/NaH2PO4as buffer solution,the initial lactose concentration of45%（w/v）, enzyme dosage of20UoNPG/g lactose, thereaction temperature of55oC and reaction time of12h. Under these conditions, themaximum GOS yield finally achieved44.5%.