Improving Enzyme Stability Of 3α-hydroxysteroid Dehydrogenase From Comamonas Testosteroni

3α-hydroxysteroid dehydrogenase(3α-HSD; EC.1.1.1.50), is a kind of enzyme for diagnosis. 3α-HSD could be used to determine the content of serum total bile acids(TBA). The TBA level in serum is a common indicator in the diagnosis of various liver disease. However, The poor instability and high cost of 3α-HSD limit its routine use in clinical applicaion. The gene hsd A of 3α-HSD was chosen as template from Comamonas testosteroni ATCC11996. The recombinant bacteria, E. coli BL21/p ET28a- hsd, was constructed and expressed. In the research, 3α-HSD was efficiently purified by affinity purification. Then the strategies of protective agents and chemical modification were also to enhance the stabilty of 3α-HSD. The main results are as follows:(1) N i2+-IDA sepharose was chosen as affinity medium, crude enzyme sample was eluted by 100、200、300、400 mmol/L different concentration imidazole. 300 mmol/L imidazole is the best eluent concentration. The 3α-HSD with high activity was finally separated and purified. After Ni2+-Sepharose column affinity purification, The yield of the expressed enzyme was 17.5% of crude extract proteins; the recovery yield of 3α-HSD was 69.3%; the specific activity was 3.6 times. 3α-HSD was revealed as a non-covalent homodimer with molecular mass of ~56 k Da by 15.0% SDS-PAGE analysis and SE-HPLC analysis.(2) The effects of seven protective agents such as trehalose, mannose, glucose, sucrose, glycerol, 1,2-propylene glycol, sorbitol on the stability of 3α-HSD were researched. According to the concentrations and screening of additives, 0.15 mol/L trehalose was the stablest protective agent. Compared to the control enzyme solutions, The thermostability, p H stability and storage stability of 3α-HSD were improved in different levels after adding protective agents. When incubated in 55°C water bath for 30 min, The residual activity of the enzyme solution with trehalose stable protective agent was 12.1%, while the residual activity of control enzyme was almost zero. The protective advantage of trehalose was obvious in high temperature.(3) 3α-HSD was modified by phthalic anhydride(PA). After the optimization of chemical modification conditions, the modification effects were characterized by trinitrobenzene sulfonic(TN BS), 15% SDS-PAGE, determination of kinetics parameters, circular dichroism(CD) spectra and fluorescence spectroscopy. The changes of stability, catalytic activity, conformation of 3α-HSD were determined after phthalic anhydride modification. The experimental results are as follows: The degree of modification of amino groups was determined as 78% by trinitrobenzene sulfonic(TNBS); 15% SDS-PAGE revealed that the modifier has been connected to the 3α-HSD enzyme. The average molecular weight of modified 3α-HSD was about 30 k Da. The thermostability, p H stability and storage stability of 3α-HSD were obviously improved after phthalic anhydride modification. The measurement results of kinetics parameters were that the enzyme specificity of substrate and catalytic efficiency were both improved. These results of circular dichroism spectra and fluorescence spectrum indicatied that modification of lysine residues did not significantly affect the conformation of 3α-HSD enzyme.