Increase In Enzymatic Activity Of Firefly Luciferase By Site-directed Mutagenesis

The luminescent reaction catalyzed by luciferase is a double substrate reaction.The basic biochemical reaction process is that the luciferase catalyzes the oxidation of D-luciferin to emit the visible light in the presence of oxygen and Mg²⁺.The luminescence intensity of the reaction is positively correlated with the concentration of ATP up to certain concentration range.ATP widely and stably exists in living cells,and its content can indicate the number of microorganisms in food product and processing environment.Therefore,the luciferase can be used for the rapid detection of food production process and products.In addition,luciferase has been applied to cell metabolism research,biosensor,drug screening and so on.Therefore,obtaining luciferase with stable and excellent luminescent properties is of great importance for improving the food safety and cell visualization.The target of this thesis is to construct gene engineering bacteria mutants to express luciferase with high enzyme activity,to compare the enzymatic properties of mutants with wild type and to explore the effect of stabilizers on enzymes.Meanwhile,the study tries to express luciferase in Bacillus subtilis.Firstly,to construct the recombinant bacteria named E.coli BL21?DE3,pET30a-luc1.1?expressing luciferase mutant Luc1.1,amino acid residue of Ile423,Asp436 and Leu530 in wild-type luciferase Luc were mutated to Leu,Gly and Arg by site-directed mutate.The enzyme activity of mutant Luc1.1 was 11.62 times higher than that of wild.The maximum emission wavelength?MEW?of the light catalyzed by type Luc1.1 is red-shifted by 6 nm comparing with the wild-type luciferase Luc.The influence factors of luciferase showed that the enzyme activity was highest at pH 7.5.The MEX of the light catalyzed by wild type Luc had small fluctuation in different pH.The MEW of mutant Luc1.1 is red shifted when the system is neutral or partial alkaline.With the increasing of temperature,the mutant Luc1.1enzyme decreased slowly when the temperature is higher than 30?.Thus it shows better heat resistance.The effect of increasing temperature on the enzyme activity was studied.The result showed that the mutant Luc1.1 enzyme activity decreased slowly after 30°C indicating better thermal stability.Secondly,the luciferase gene after amplified with plasmid pET30a-luc and pET30a-luc1.1 connected with the plasmid pMA0911 to construct recombinant Bacillus subtilis named B.subtilis WB800?p MA0911-luc?and B.subtilis WB800?pMA0911-luc1.1?,which expressed Luc and Luc1.1,respectively.The results showed that the expression of luciferase in Bacillus subtilis decreased by about 55%,and the activity of the enzyme was basically the same as that expressed in Escherichia coli.Thirdly,the mutant luciferase Luc1.1 was performed by a single point saturate mutation.The mutation were at site 423,site 436 and site 530 where 19 residues amino acids replacing the original amino acid type beside Luc1.1.A total of 57 luciferase mutants were obtained.Each mutant enzyme was expressed,purified and quantified.The enzyme activity was compared with the activity of the wild-type Luc enzyme.The results showed that the activity of Luc could be increased by replacing the amino acid residues at Leu423,Gly436 and Arg530 with amino acids with non-polar side chains,amino acids with side chains of small steric hindrance and amino acids with positive charges,respectively.A total of 8 strains with10 times enzyme activity over Luc were obtained.Among them,when the mutants were obtained by replacing Gly436 of Luc 1.1 with Ala,Cys or Ser,the enzyme activity could reach 22.60 times,21.37 times and 19.96 times compared with Luc,respectively.The experiment also compared the high enzyme activity luciferase mutant with the marketed products.The results showed that the catalytic activity of the enzyme mutant was higher than that of the domestic luciferase.Finally,the effects of four reagents on the activity and dynamic character of luciferase Luc1.2 that constructed by replacing amino acid at position 436 of Luc1.1 with Ala were studied.The results showed that the optimum concentration of coenzyme A?CoA?,potassium pyrophosphate?PPiK?,sodium tripolyphosphate?STPP?and inosine 5?-triphosphate sodium salt?ITP?was 1.0 mmol/L,0.08 mmol/L,1.0 mmol/L,and 0.8 mmol/L for reaction system of Luc1.2,respectively.Adding the optimal concentration of CoA to the mutant Luc1.2 catalytic system can increase the relative luminescence intensity to about 47.2 times that of the wild-type Luc enhancer-free reaction system.Adding the optimal concentration of STPP can enhance the relative luminescence of the Luc1.2 reaction system to about 31.6 times that of the wild type system without the enhancer.PPiK and ITP can also enhance the relative luminescence intensity to some extend.To study the dynamic characteristics of the luminescent system,the optimum concentration of reagents was added.The best stabilizer of the system is CoA which can maintain the 80%of light intensity for 15 s after the reaction begins.The other three substances STPP,PPiK and ITP can make the luminous intensity keep80%in 5 s.