The Study On Screening Of Producing Glycyrrhetic Acid 3-O-mono-beta-D-glucuronide Strains And The Enzyme Catalytic Properties

Glycyrrhetic acid 3-O-mono-beta-D-glucuronide has a higher bioavailability when compared with glycyrrhizin.Due to its strong anticancer and anti-allergic characteristics,glycyrrhetic acid 3-O-mono-beta-D-glucuronide has a potential and application prospect in the biological medicine industry while the preparation of this substance is very difficult.Accordingly,how to screening of highly cell growth performance,safety strains center on the production of glycyrrhetic acid 3-O-mono-beta-D-glucuronide is becoming a potential research.As a glycoside hydrolase,?-D-glucuronidase is more competent in hydrolyzing glycosides containing glucuronic acid,so it is of great application value in various fields of food,biomedicine and chemical industry.Owing to the drawbacks of lower expression output and difficulty in industrialized application of ?-D-glucuronidase secreted by microorganisms,it is of vital significance to separate enzyme-active ?-D-glucuronidase and to apply it in the production of bioactive glycosidens.The major research findings in this paper are as follows:?1?Soil samples were collected at random from the licorice land in Northwest China.After a series of screening,we got a bacterial strain called A-32,which could producing glycyrrhetic acid 3-O-mono-beta-D-glucuronide.One strain of enzyme-active ?-D-glucuronidase was effectively obtained after the mutagenetic treatment of plasma at proper temperature and quantitative analysis on enzyme activity.Its colonial morphology,microscope observation and ITS analysis made it clear that the strain belongs to talaromyces.So it was named Talaromyces sp.02,whose fermentation enzyme activity reaches 83.84 U/m L,about 70% higher than that of the wild counterpart,verified that it has stable hereditary features after ten passages.?2?Conditions of producing fermentation enzyme were optimized through single-factor experiment.Eventually,the ratio of best culture medium was determined: glycyrrhizic acid-5 g/L,sodium nitrate-3 g/L,Tween80-0.16%,magnesium sulfate-0.5 g/L and dipotassium phosphate-2.2 g/L;and the best culture condition was: initial pH of the fermentation broth-5.5,fermentation temperature-30 oC,liquid volumn-50/250 mL,inoculum size-10%,shaking speed-160r/min and fermentation period-144 h.The yield of glycyrrhetic acid 3-O-mono-beta-D-glucuronide reached 89.88%.And the enzyme activity of glucuronidase broth reaches 226.80 U/mL,about 2.7 times of that of the initial activity.?3?Extracellular crude enzyme liquid collected from the fermentation culture was treated through 70% ethanol precipitation,Q Sepharose FF-anion exchange chromatography and Superdex G-200 gel filtration chromatography to purify the ?-D-glucuronidase and then its enzyme was also tested and analyzed through HPLC?High Performance Liquid Chromatography?and polyacrylamide gel electrophoresis respectively.Finally,?-D-glucuronidase was purified,whose relative molecular mass is about 76.44 kDa.The enzyme was purified to homogeneity level with 24.24-fold,and the specific enzyme activity raised from 15.18 U/mg to 367.89 U/mg.?4?Enzymatic properties of ?-D-glucuronidase were also investigated.The optimal pH of ?-D-glucuronidase is 5.0,The optimal temperature is 45?.and its activity is more stable in the range of 25⁵5?,remaining over 70% even after keeping warm for 240 min.10 mmol/L Ca²⁺?Mn²⁺ and Ba²⁺ can stimulate the activity of ?-D-glucuronidase differently while Fe3+ exerts the most noticeable inhibiting effect on the activity,Al3+?Na+?K+?Cu²⁺?Mg²⁺ and Zn²⁺ have little influence on its activity,5 mmol/L mental ions had no obvious influence on the enzyme activity.