Preliminary Research On Characterization,Immobilization And Application Of SGNH Esterase Est882

Esterase,such as carboxylesterase and lipase,catalyze the formation and dissolution of ester bonds.Esterase is a type of biological enzyme that is commonly used in indust rial manufacturing,including pharmaceutical synthesis,environmental remediation,food processing,and biodiesel manufacturing.Natural esterase,on the other hand,has low thermal stability and activity,making it challenging to employ in industrial manufacturing.As a result,new enzymes with superior properties must be produced as soon as possible.Bioinformatics was used to evaluate the esterase gene est882 obtained from the previous laboratory’s metagenomic library.The esterase gene’s complete length of 882 bp encodes 293 amino acids.Est882 and its homologues may form a novel esterase family,according to amino acid sequence alignment and phylogenetic analyses.Est882 has the typical SGNH family esterase structural features,such are the preserved Gly⁷⁷-Asp⁷⁸-Ser⁷⁹-Xxx motif and the Ser⁷⁹-Asp²⁶⁹-His²⁷⁵ catalytic triad.The enzyme’s amino acid sequence was deposited in Gen Bank with the accession number MZ429070.To achieve heterologous expression,the gene est882 was cloned into the expression system p ET-28a（+）and injected into E.coli BL21（DE3）.The recombinant strain’s expression conditions were optimized,and the optimal expression conditions were cultivated at 30°C until the starting cell concentration OD₆₀₀ was 1.0,and then 0.08 m M IPTG was induced for 12 h.Est882 favored to hydrolyze short-chain esters due to its enzymatic characteristics.Km and Vmax were 0.76 m M and 0.023 m M·min^-1,respectively.Est882’s optimum reaction temperature was 40°C,and its optimum p H was 9.0.The enzyme was extremely stable in the p H range of 7.0-10.0,notably after 25h incubation period at p H 8.0 and p H 9.0,where nearly 80%of the residual activity was retained.Furthermore,Est882 was found to be resistant to the majority of the metal cations and organic solvents used in the experiment.The recombinant strain’s fermentation conditions were optimized using a single factor experiment,and the optimum fermentation conditions were as follows:glycerol tributyrate 12 g·L^-1,tryptone 8 g·L^-1,yeast powder 2 g·L^-1,Mg SO₄ 2 g·L^-1,liquid volume 45 m L and inoculum 1.5%.Then through the Plackett-Burman experimental design,it was determined that the three significant factors affecting the results of enzyme production were yeast powder,magnesium sulfate and liquid volume.Combined with Box-Behnken experiment,the best ratio of culture medium was further optimized.After optimization,the enzyme activity was 2279.24 U·L^-1,which was 1.39 times greater than before optimization.Est882 was immobilized using chitosan,sodium alginate,and mesoporous silica SBA-15 as carriers.Under the optimum immobilization conditions(immobilization p H 5.0,time 1.5 h,enzyme concentration 0.8 mg·m L^-1,chitosan-glutaraldehyde solution cross-linking amount 0.1 m L),the recovery rate of enzyme activity was 86.5%using SBA-15 as the immobilization carrier,and the immobilization effect was the best.The catalytic performance of the immobilized enzyme Est882@SBA-15 was examined after it was extensively described.The enzyme was successfully immobilized in the highly porous channel,and the morphology of the carrier did not change,according to TEM,SEM and FTIR measurements.The optimum reaction temperature of Est882@SBA-15 was 40°C,and the optimum reaction p H was elevated to 10.0,which considerably enhanced the tolerance of various metal ions,organic solvents,and detergents when compared to the free enzyme.Furthermore,Est882@SBA-15 has a high reuse rate,with 52.6%of its initial activity remaining after 9 recycling cycles.The breakdown of several pyrethroids before and after Est882immobilization was investigated using fenpropathrin,cyhalothrin,permethrin,cypermethrin,and esfenvalerate as substrates.At 37°C,five pyrethroids degraded at rates of 86.2%,90.3%,84.9%,87.6%,and 88.3%,respectively.Est882@SBA-15 degraded five pyrethroids at rates of 78.4%,83.0%,76.2%,81.6%,and 79.1%,respectively,and the pyrethroid combination degraded at a rate of 54.9%after seven times of recycling.The results revealed that the enzyme had a broad substrate specificity and excellent pyrethroid pesticide degradation ability,and that the immobilized pyrethroids had good reusability in the pyrethroid degradation process.We were able to achieve heterologous expression of the SGNH family esterase Est882,as well as the characterization of enzyme characteristics,optimization of the fermentation process,immobilization,and prospective application in pyrethroid degradation,in this study.It adds to the SGNH family’s esterase gene resources and provides an early experimental foundation for its use in environmental cleanup,drug development,and detection.