Screening Of Bacillus Producing β-galactosidase And Characterization Of The Enzymatic Reaction

β-galactosidase,also named Lactase,could hydrolysis lactose in the milk to glucose and galactose which were absorbed easily in the intestinal.Inadequate secretion or lack of the lactase in the intestinal resulted in symptoms of lactose malabsorption.At present,theβ-galactosidase utilized in dairy industry was produced by yeast,lactic acid bacteria or mould.However,they could not resistant to the high temperature of dairy process.If the dairy procese used mild temperature lactase,it could take long time for hydrolyzing the lactose to glucose and galactose,and the fresh milk is easily contaminated by bacteria.In this thesis,Bacillus were obtained from the heated soil.Eight strains with potentialβ-galactosidase activity were isolated on the plates containing x-gal which formed blue colonies.After detecting theβ-galactosidase activity of those isolates by ONPG method,one NH7 among eight isolates with highβ-galactosidase activity was screened.By morphological abservation, physiological and biochemical characteristics,and 16S rDNA sequence analysis,the strain NH7 was identified as Bacillus megaterium.The nitrogen sources,carbon source in the medium were optimized,the results showed that the optimium medium was 1.2%lactose,1.04%peptone, 0.63%beef extract and at pH 8.0.The effects of metal ions on theβ-galactosidase activity were detected.The resulted showed that Na⁺ have the ability of promoting theβ-galactosidase activity.The response surface methods was adopted to analysis the effect of main ingredient used in the medium.The results were further conformed.The growth curve and dynamics ofβ-galactosidase were measured,the maximum activity was obtained at 17 hours inoculation,the highest activity was increased from 1.19 to 5.49U/ml, increased by 4.62 times.Theβ-galactosidase properties produced by Bacillus megaterium NH7 have been charactized.The optimal hydrolysis temperature is at 50℃,the optimum pH is at 7.5,and thermal stability of this enzyme is at 50℃.85%of theβ-galactosidase activity was kept after 6h at 50℃,and 75%of the activity was left after 6h at 60℃.This enzyme at pH 6-8 is relatively stable.Metal ions could influence on theβ-galactosidase activity.K⁺,Na⁺ were not the main effective factors;Mg²⁺,Mn²⁺,Pb²⁺,Fe³⁺,Al³⁺,Fe²⁺ have great catalytic role. Morever,Fe²⁺ could promote theβ-galactosidase activity obviously;Cu²⁺ has a significant inhibitory effect.Km for this enzyme is 2.74mmol/L,Vm is 4.26μmol/min·ml.Chitosan was used to fix theβ-galactosidase.The optimum conditions were: 2%of acetic acid was adopted to dissolve the 3%of chitosan solution.The solution as above were droped into 20%NaOH,30%CH3OH gel with needle solution to form small balls.After deionized water to neutral cleansing repeatedly,0.6%glutaraldehyde cross-linking 80 minutes under 30℃,cleaning has been activated chitosan gel particles,the microspheres with the enzyme solution according to the quality of volume ratio must mixed immobilized 4℃for 6 hours,do deionized water-washed filter was immobilizedβ-galactosidase. Chitosan immobilizedβ-galactosidase enzyme optimum reaction temperature is 50℃,the optimum pH is 8.0,the compared to free enzyme at 50℃,60℃,70℃water bath 2 hours thermal stability.Continuous operation 9 times,enzyme activities were still kept 79%of the initial activity,the stability of immobilized enzyme to operate better.With sodium alginate as the carrier,the two fixed methods for embedding-cross-linked and cross-linking-embedded were choiced.The best conditions of embedded-fixed for crosslinking were:a certain amount enzyme was added into 1.2%of sodium alginate solution,then added 1.8%CaCl₂ solution,and one of sclerosis at 1 hour,remove the dry cleaning,of 0.8%glutaraldehyde cross-linked enzyme activity recovery rate of 2 hours maximum.The optimum reaction temperature of immobilized enzyme was 40℃,the optimum pH is 8. Cross-linking - the best conditions for a fixed embedding is:added enzyme into the 0.2%glutaraldehyde solution.After mixing,the mixture was droped into the 1%of CaCl₂ solution for small balls forming with 6 needles.For crosslinking- entrapped immobilized enzyme,optimum temperature was 50℃, the optimum pH was 7.0,the thermal stability and pH stability were similar to the free enzyme.However,its mechanical strength,thermal stability and pH stability are not as good as enzyme with chitosan.