Research On The Production Of Chenodeoxycholic Acid From Chicken Bile By Biological Enzyme

Chenodeoxycholic acid is synthesized from cholesterol in the liver and has been shown to modulate the lipid metabolism and play a key role in metabolic diseases,it is existed in animal bile in two conjugated forms,Glyco-CDCA and Tauro-CDCA.In the industry,bile acids are used in their deconjugated form.A hydrolysis reaction is needed to cleave the amide bond between the steroid core and the conjugated amino acid.Traditional deconjugation of bile acids applies high temperature,caustics and hazardous acids and many hours of hydrolysis.It is known that deconjugation is catalyzed by bile salt hydrolase（BSH）enzymes and several attempts have been made to prepare free bile acids by enzymatic hydrolysis.In this study,we screened and prepared a highly active bile salt hydrolase and applied the enzyme to the production of Chenodeoxycholic acid from chicken bile to establish a safer and eco-friendly enzymatic hydrolysis method.The main studies were as follows.The first was the screening and preparation of highly active bile salt hydrolases.Through qualitative and quantitative screening,highly active BSH strains were screened among several Bifidobacterium,then using E.coli to express a highly active bile salt hydrolase gene from Bifidobacterium to increase the expression level of BSH.The results showed that B infantis had the highest BSH activity with specific enzyme activities of 0.48±0.013 and 0.27±0.014 U/mg for GCDCA and TCDCA.Respectively,which were overexpressed by the E.coli expression system,and incubated under optimized conditions(IPTG 0.2 mmol/L,induction time 22h,OD₆₀₀ of 0.8before induction),The specific enzyme activities of recombinant enzymes for GCDCA and TCDCA were increased by 10.33 and 11.37 fold to 4.96±0.32 and 3.07±0.031 U/mg.In addition,the optimum temperature and p H of recombinant BSH were 37°C and 5.5,after holding at 37°C for 1h,This enzyme remained about 80%and 97%residual activity for GCDCA and TCDCA.After 3h incubation at p H 5.5,it remained 90%residual activity.Subsequently,a comparative analysis of two extraction methods,enzymatic and saponification,was carried out to investigate the process feasibility of the bioenzymatic extraction of chicken bile acids,and the enzymatic extraction conditions were optimized by single-factor test and Box-Behnken design,and the effects of enzyme addition,p H and enzymatic digestion temperature on the yield of CDCA were analyzed.The results showed that the enzymatic process yielded 4.1%（wet weight）of CDCA.Compared with the saponification method,the bioenzyme method was effective and had outstanding advantages in the extraction.The interaction terms of enzyme addition and p H,p H and enzymatic digestion temperature significantly affected the CDCA yield.The extraction was carried out at an enzyme addition of 8 mg/m L,p H 5.0 and enzymatic digestion temperature of 38°C.The maximum CDCA yield was 5.32%（wet weight）and the purity was 51.7%.Then,the separation and purification study of CDCA was carried out using an industrially applicable macroporous resin method.The most suitable macroporous resin for the purification of CDCA was screened by static adsorption and desorption tests,and the adsorption performance and adsorption mechanism of the resin on CDCA were investigated.The results showed that among the ten macroporous resins screened,AB-8 resin had the best overall performance.The adsorption behaviour of CDCA on AB-8 resin could be well fitted and explained by the Langmuir isotherm model,the adsorption was an exothermic process and the low temperature was favourable to the adsorption of CDCA.The purification process of CDCA was further optimized by dynamic adsorption and desorption tests.Finally,the recovery yield of CDCA with 87.8%,purity after pretreatment and macroporous resin treatment reached 91.4%.