| Urate oxidase is an important enzyme in the metabolic pathway of indole.It can degrade uric acid to form allantoin.The solubility of allantoin in water is 5-10 times that of uric acid,which can greatly reduce the content of uric acid in the body.In most organisms and microorganisms,uric acid can be further oxidized by urate oxidase.However,the urate oxidase gene of animals such as humans and other primates has been mutated during evolution.The body lacks the biologically active urate oxidase gene.Uric acid is the end product of purine metabolism in humans.However,if the level of uric acid in the body is over high for a long time,it will cause hyperuricemia in the human body and aggravate many diseases such as gout,cardiovascular disease and kidney disease.Urate oxidase could be applied in the clinic detection of uric acid and therapy for its ability of degradation of uric acid.However,the urate oxidase used in pharmacectic production and Medical examination have a lot of inadaptability.People also through various methods to improve urate oxidase enzyme activity or raise the enzymology characteristics of it.The results of this research were summarized as follows.1.How to screen for strains with thermostable urate oxidase and optimize enzyme production capacity,the biggest difficulty is the extraction and detection of intracellular enzymes.First,the rapid extraction method of yeast intracellular enzymes,using liquid nitrogen freeze-thaw,the cells are frozen and then thawed,increasing cell permeability.This method has a fast extraction speed and good effect,but the extraction amount is small,which is in line with the requirement of less samples in the rapid screening.The extraction time of ultrasonication was 30 min,and the extraction time of liquid nitrogen freeze-thaw method was 1 min,and the extraction time was greatly shortened.Second,the rapid detection of urate oxidase by display method.The principle of measuring blood uric acid content by uric acid oxidase-peroxidase coupling method was used for rapid screening.The method measures the enzyme activity by generating the amount of red quinone imine,and the color depth reflects the concentration of uric acid,which is more intuitive.At the same time,in order to verify the effectiveness of the rapid extraction method and the rapid detection method for screening,the relative enzyme activity was verified.2.The primary screening was carried out by the transparent circle method,and the uric acid oxidase having heat resistance was screened by high-throughput screening.In the first round,strain CU1 producing heat-resistant urate oxidase was screened from 132 yeasts.This strain was used as a starting strain,and the strain was induced to the strain CUM09 from ultraviolet rays using 254 nm.The starting strain was treated with 70?for 30 min,and retained 86.01%of the enzyme activity.The strain numbered CUM09 retained 88.89%of the enzyme activity after being treated at70?for 30 min.Under the heat treatment conditions,CUM09 uric acid oxidase increased by 2.88%compared with the original strain.3.In the experiment,the fermentation conditions of Candida utilis(CUM09)were optimized,optimum schemes were established by a series of single-factor experiments and orthogonal test.In order to determine the optimal Candida utilis growth and enzyme production condition,experiments were at temperature 28?,pH8.5,quantity 2.5%and 16 h cultivation.The optimum carbon nitrogen source was malt extract.The enzyme activity was 1249.50 U/mL,which was 56.58%higher than the initial conditions.4.Enzymatic characteristics were also studied.The enzymatic properties of urate oxidase were researched.The temperature and pH value of urate oxidase the enzyme activity was 40?and 8.5,K_m is 36.1?mol/L.Under 70?heat preservation 30 min,enzyme activity to keep more than 80%. |
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