| The development of diagnostic kits has become a focal point in clinical medicine and bioassays,with enzymes serving as the core component.Xanthine oxidase and long-chain fatty acyl-co A synthetase are important enzyme preparations in gout kits and long-chain fatty acid detection kits,respectively.Currently,there are deficiencies such as single source,high price and poor stability.This study investigates Xanthine oxidase and Long-chain fatty acyl-coenzyme A synthetase,delving into subunit gene reconstruction,optimized expression,and enzymatic property characterization to enhance enzyme stability for future diagnostic kit development.Initially,this research examined the three subunit genes of xanthine oxidase/dehydrogenase derived from Cellulosimicrobium cellulans ATCC21606,focusing on heterologous expression and activity assembly.Two research strategies were devised: 1)designing translation coupling for the three subunit genes,aiming for their linked translation in genetically engineered bacteria,and inserting synthesized genes into the p ET-20 b vector using molecular biology techniques for Escherichia coli expression;2)connecting the three subunit genes via a flexible linker gene and inserting them into the p ET-28 a vector,hoping to achieve fusion expression of the three subunits.Results indicated that in the first research strategy,subunits A and C are expressed in soluble form,while subunits B are expressed in inclusion body form.Despite attempts at expression condition exploration,optimization,and denaturation-refolding assembly,no active recombinant protein was obtained.In the second strategy,soluble coexpression of the three subunits in E.coli was achieved through host screening and optimization of expression conditions.Activity analysis showed the recombinant protein exhibited oxidase activity,with an optimal reaction temperature of 50 °C and p H of 8.0.The specific activity reached1.16 U/mgSubsequently,to acquire a more stable long-chain fatty acyl-COA synthetase,the enzyme gene was obtained from the acid-and heat-tolerant bacterium A.cellulolyticus 11 B ATCC43068.An expression vector was constructed and introduced into E.coli,with expression conditions explored and optimized to yield the soluble recombinant protein LACS-0976.A comparison of basic enzymatic properties was conducted between LACS-0976 and the previously constructed superimposed mutant ACSLX-6s.Results revealed optimal temperatures of 60 °C and 50 °C for LACS-0976 and ACS-LX-6s,respectively,with LACS-0976 having a halflife of approximately 120 minutes at 65 °C.The optimal p H for both enzymes was 8.0,under which ACS-LX-6s exhibited a specific activity approximately 1.12 times that of LACS-0976.This study investigated the heterologous assembly and expression of xanthine oxidase subunit genes,obtaining an active recombinant protein and laying the foundation for subsequent diagnostic kit development.A more stable long-chain fatty acyl-COA synthetase gene was obtained from thermophilic bacteria,and corresponding genetically engineered bacteria were constructed,the recombinant protein showed higher thermal stability. |
