Expression,Enzymatic Properties And Stabilization Of Cold-adapted ?-galactosidase From Deep-sea Microorganism

The deep sea is an ecological environment with darkness,low temperature,high pressure and poor nutrition.The marine enzymes isolated from deep-sea microorganism with special enzymatic properties such as low temperature catalytic high efficiency,salt tolerance and wide pH tolerance,so they have great potential applications.In this study,the?-galactosidase gene Bgal was cloned from the deep-sea bacteria Alteromonas sp.ML117 in the Mariana Trench,and transformed into E.coli by constructing an expression vector for exogenous expression.Purification and enzymatic properties of the Bgal were studied.the optimal formulation of enzyme protective agent was screened and optimized by single factor test and Box-Behnken experiment.The recombinant Bgal belong to the glycoside hydrolase 2 family by analyzing the amino acid conserved sequence.SDS-PAGE and gel filtration chromatography results revealed that the native Bgal protein is tetramer.The electrophoresis-purified Bgal was obtained using Ni²⁺affinity chromatography column,and the fold purification was 2.1.Bgal can specifically hydrolyze the natural substrate lactose and o-nitrophenyl-?-D-galactopyranoside?ONPG?.When lactose was used as substrate,the optimum reaction temperature of Bgal was 30^oC,and the optimum pH was 8.0.With ONPG as substrate,the optimum reaction temperature of Bgal was 35^oC,and the optimum pH was 8.0.ONPG and lactose were used as substrates for the reaction temperature of 10^oC,and the K_m values of Bgal were 3.8 mM and 1.6 mM,respectively.In the metal ion experiment,K⁺and Mn²⁺had obviously promoting effect on Bgal activity,Na⁺and Mg²⁺had a slight promoting effect on Bgal,and Al³⁺,Cu²⁺and Zn²⁺showed complete inhibition.The thiol compounds,glutathione,cysteine,and mercaptoethanol had no significant effect on the enzyme activity of Bgal.In the reaction solution containing 4 M NaCl,Bgal still retained more than 50%of relative activity,indicating that recombinant Bgal was well tolerated by NaCl.In the effect of ethanol on the activity of Bgal,20%ethanol can increase the activity of Bgal to 130%.Glucose and galactose had different degrees of inhibition on Bgal.With ONPG as substrate,Bgal retains 83%relative enzyme activity when glucose concentration is 5 mM.while galactose concentration increases to 100 mM,relative activity of Bgal was 92%.In addition,the 1 U recombinant Bgal can hydrolyze 86%of lactose in 1 mL of milk after 24 h at 10°C.This result indicates that the recombinant Bgal can be used in the dairy industry.As a cold-adapted enzyme,determining that storage stability is unstable.The additives affecting the stability of the recombinant Bgal were examined by accelerated temperature rising experiment,and mycose,glycerin,and sorbitol were preferably used as protective additives for complex enzyme solution.After optimization by Box-Behnken experiment,the optimal formula of the compositive protectant is:trehalose 2.5%,glycerin 23%,sorbitol 25%.The Bgal added with the stabilizer still retained more than 90%of the relative activity after being left at 25°C for 12 days,while the control group was completely inactivated,which greatly improved the stability of cold-adapted Bgal.According to the results of the kinetic model,the optimized enzyme protective agent can keep the liquid Bgal solution more than 90%of relative activity under 4^oC for 90 days.