Screening And Identification Of Biological Control Bacteria Producing β-galactosidase And Its Preliminary Fermentation Application Of Using Lactose Wastewater

β-galactosidase is a kind of important biological catalysts,with two important functions: catalyzing lactose hydrolysis and glycosidic.It is used to reduce the lactose content in food and wastewater treatment by hydrolysis activity,also widely used in food,medicine,immune,environment detection,gene diagnosis,gene therapy and other fields.In this paper,the strain producing β-galactosidase was screened from the bacillus strains that were originally isolated from honey and preserved in our laboratory.This research includes screening and identification of bacillus,its optimal fermention conditions for β-galactosidase production,purification and characterization of β-galactosidase,consumption of lactose and lactose wastewater by whole-cell catalysis,the inhibition on Fusarium oxysporum and growing indicators.It is preliminary for development of biological control agent in agricultural application,creating a good theoretical value and pertinent prospect.The main results were as follows:1)A β-galactosidase-producing strain was screened from Bacillus.sp isolated from honey.The results showed that the strain was able to inhibit Fusarium oxysporum,produce IAA and solubilize phosphate.The strain was identified as Bacillus licheniformis by its 16 S rRNA gene sequencing analysis,morphological observation,physiological and biochemical tests,therefore named after Bacillus licheniformis SYBC hb15.2)On the basis of single factor experiments,the effects of compositions of cultural medium and culture conditions were studied.Ultimately,the optimal fermentation medium and conditions were determined.The optimal carbon sources was lactose(25 g·L-1),the optimal nitrogen sources was yeast extract(5 g·L-1)and tryptone(10 g·L-1)and the metal ions promoting growth were NaCl(5 g·L-1),KCl(1 mmol·L-1),CaCl2(4 mmol·L-1)and FeCl3(2 mmol·L-1).The optimal initial pH,inoculums concentration,fermentation temperature,fermentation time and the shaking speed were 7.0,6%,30 ℃,45 h and 180 r·min-1,respectively.Under the optimal condition,β-galactosidase production can reach 349.11 U·L-1,which increased by 135.8% compared to that,148.47 U·L-1,in initial condition.3)Moreover,the enzyme was purified for enzymatic properties study with o-nitrophenyl-β-D-galactoside(ONPG)as substrate.The optimal temperature for the reaction of this enzyme was 60 ℃ and it had good thermal stability below 70 ℃.The residual activity was 65% after incubation at 70 ℃ for 60 min.The optimal reaction pH was 6 and enzyme activity was stable in the range of pH 6.0-9.0.In addition,when the ion concentration is 2 mmol·L-1,enzyme activity was markedly enhanced in the presence of K+,whereas completely inhibited by Co2+,Cu2+,Ca2+,Mn2+,and also inhibited by Mg2+,Ba2+,Zn2+ and Ni2+ to a less degree.Na+ has no effect on enzyme activity.Furthermore,monosaccharides of lactose hydrolyzate also have weaker inhibition to enzyme activity than that of the β-galactosidase from Talaromyces thermophilus.Therefore,β-galactosidase derived from B.licheniformis SYBC hb15 is more resistant to heat and inhibited weaklier by glucose of high levels,so it is a kind of thermotolerant and high glucose concentration resistant enzyme.4)With the function of hydrolyzing lactose,whole-cell fermentation was performed.Consumption of the lactose during the fermentation process was determined by HPLC,lactose consumption tended to be stable after 60 h,and eventually consumption was 70%.B.licheniformis SYBC hb15 can grow using lactose from waste water as a carbon source in fermentation.The consumption of lactose was 36% after 48 h,some substance in the fermented liquid can inhibit the growth of Fusarium oxysporum,laying a foundation for the research of biological control.Additionally,B.licheniformis SYBC hb15 produces auxin,up to 2.25 g·L-1 in the second day;with the phosphate-solubilizing ability,it can decompose phosphorus of phosphate rock.The concentration of PO43+ in fermented liquid reach the maximum stable at 102.3 mg·L-1 after 5 days.Therefore,We can realize the combination of environmental protection and agriculture through fermenting lactose wastewater and making biological agents.