Screening, Gene Cloning And Enzymatic Properties Study Of Cold-adapted β-galactosidase Isolated From Marine Microorganisms

Marine is a special ecosystem with high-salt, high-pressure, low-temperature andtemperature-changing-slowly, which differs greatly from terrestrial ecosystem. Because ofthis, enzymes isolated from marine microorganisms may have special properties, such aslow optimum temperature and tolerance to most metal ions. One of these enzymes,β-galactosidase, is worthy of its potential application in the food industry. The purpose ofthis research is to study the cold-adapted β-galactosidase from marine microorganisms.The methods and results are as follows:Based on above conditions,1. Screening the β-galactosidases producing bacteriaUsing the modified Zobell2216E solid culture medium (5g peptone,1g yeast extract,0.1g FePO4,0.004%X-Gal and2%lactose) to screen the β-galactosidase from the marinemicroorganisms that we had conserved before,31strains were positive. Using TLC andHPLC for secondary screening, Halomonas sp. S62was chosen for further study.2. Purification of the β-galactosidase produced by Halomonas sp. S62andthe recombinant enzyme obtainedFollowing by fermentation, ultrasonication, the crude β-galactosidase produced byHalomonas sp. S62was purified through PABTG affinity chromatography,(NH4)2SO4salting-out, Capto DEAE ion exchange chromatography, Capto Q ion exchangechromatography and Sephacryl S200gel filtration. The BGalH was electrophoreticallypure and the fold of purification was1007.7and the activity yield was1.389%. It isstrongly suggested the bioactive BGalH was a dimeric protein because SDS-PAGE showedthe molecular weight of BGalH was48kDa, however, Native-PAGE showed the weightwas90kDa.The β-galactosidase gene sequence of Halomonas sp. S62was obtained through thegenomic sequencing and bioinformatic analysis. The BGalH gene was amplified andidentified by PCR and the nucleotide sequence of BGalH from Halomonas sp. S62was submitted to Genbank. The accession No is JQ337961, the Open Reading Frame (ORF) ofBGalH is1170bp and the most similarity of BGalH to other genes in Genbank is67%(GenBank Accession No. CP002881.1).Using plasmid pET32a(+) and pSP72as vectors, the recombinant moleculepET32a(+)-BGalH and pSP72-BGalH were constructed respectively. After identified theBGalH’s bioactivity by pSP72-BGalH, the recombinant plasmid pET32a(+)-BGalH wastransformed into E.coli BL21cells and then fermented to harvest. The recombinantenzyme was purified by Ni2+-NTA affinity column and Capto DEAE ion-exchange column.The fold of purification was146.24, and the activity yield was53.23%. SDS-PAGE andNative-PAGE results suggested the rBGalH was also a dimeric protein, which is as same asthe natural enzyme.3. Enzymatic properties studies of BGalH and rBGalHThe optimum reaction conditions and enzymatic constants of BGalH and rBGalHwere determined by ONPG and lactose respectively. Using ONPG as a substrate, theoptimum pH and temperature for the natural β-galactosidase activity were8.0and40°C,respectively. The Kmand Kcatwere11.17mM and1097.1s-1, respectively. Using lactose asa substrate, the optimum pH and temperature for the natural β-galactosidase activity were7.5and35°C, respectively. The Kmand Kcatwere204.6mM and2.3s-1,respectively.Using ONPG as a substrate, the factors influencing on enzyme stabilities wereassayed. BGalH was stable at a range of pH7.0-9.5after1h incubation, retaining morethan80%of its maximum activity. And60%of its maximum activity was remained after1h incubation at20°C. The activity of BGalH was not inhibited by various metal ions andFe2+hastened the catalytic capacity of BGalH remarkably (190.1%). Ethanol with aconcentration of40%(v/v) and below could accelerate the activity of BGalH.To rBGalH, we tested the optimum pH, optimum temperature and enzyme kineticparameters using ONPG and lactose as the substrates. The thermostability and pH stabilityof BGalH were also detected using ONPG as substrate. Also, the reaction with pure milkwas tested. Using ONPG as a substrate, the optimum pH and temperature for rBGalH activitywere7.0and45°C, respectively. The Kmand Kcatwere2.9mM and390.3s-1, respectively.Using lactose as a substrate, the optimum pH and temperature for rBGalH were determinedto be7.5and35°C, respectively. The Kmand Kcatwere32.06mM and269.5s-1,respectively. rBGalH was stable at a wide range of pH5.5-9.5after1h incubation,retaining more than80%of its maximum activity. Thermostability of rBGalH wasincreased against natural BGalH. After1h incubation at50°C,55%of the maximumactivity of rBGalH was remained. Like the natural enzyme, the activity of rBGalH washastened by Fe2+remarkably（218.82%）and Cu2+(50.84%) significantly inhibited theactivity of BGalH.rBGalH could hydrolyze lactose at low temperature (7°C) and room temperature(28°C). At7°C, hydrolysis products of lactose were detected after the reaction began12h.When reaction temperature rose to28°C, lactose was hydrolyzed completely in24h.In conclusion, a novel cold-adapted β-galactosidase gene was cloned from Halomonassp. S62and the natural BGalH and recombinant rBGalH protein were obtained. BGalH andrBGalH had wide pH tolerance range, good thermostability, high activity at lowtemperature and tolerance to many metal ions and suitable to hydrolyze lactose in complexsystem. They could used as new resources of exogenous β-galactosidase in the foodprocessing industry after further researches in the future.