The Heterologous Expression And Enzymatic Properties Analyses Of Novel Marine Lipolytic Enzymes E13 And E41

Lipolytic enzymes,including lipases and esterases,catalyze the hydrolysis and synthesis of ester bonds,and are widely found in plants,animals and microorganisms.Due to its broad substrate specificity,excellent enantioselectivity and regioselectivity,no cofactors required for the reaction,and good stability in organic reagents,lipolitic enzymes are widely used in biocatalyst production,detergents production,food industry,agricultural fertilizer production,biological treatment and other actual production and life.Due to the influence of its native environment,the lipolytic enzymes derived from marine microorganisms have excellent characteristics such as low temperature resistance,salt tolerance and unique substrate selectivity,and thus have broad application prospects in biotechnology and industry.With the rapid development of genomic sequencing technology,sequence screening of sequenced genomic data of marine microorganisms can rapidly obtain new families/subfamilies of multiple lipolytic enzymes sequences,greatly accelerating the pace of mining new genes and new enzymes.In this thesis,a gene encoding a novel monoacylglyceride lipase?E13?and a no-vel esterase gene?E41?encoding an Abhydrolase-3 domain were obtained by sequence screening of the genomes of the marine bacteria Glaciecola nitratireducens and Glaciecola lipolytica.Subsequently,we heterologously expressed and isolated these two genes,and characterized their catalytic properties.Our research on lypolytic enzymes E13 and E41 enriches the existing marine lypolytic enzymes resource data.1.Study on the catalytic properties of monoacylglyceride lipase E13.A gene encoding a monoacylglyceride lipase,E13,was screened from the genome sequence of the marine bacterium G.nitratireducens.The biochemical properties of none of the homologous proteins with a similarity of more than 30%to the E13 sequence were characterized,indicating that E13 is a novel lipase.Phylogenetic analysis showed that E1 3 belongs to the monoglyceride lipaselysophospholipase family.In the phylogenetic tree,E13 and other bacterial-derived homologous sequences,fungal-derived homologous sequences,and invertebrate/vertebrate-derived homologous sequences clustered to form three independent branches,indicating that the Monoglyceridelipase_lysophospholipase family of enzymes proteins are likely to differentiate in bacteria,fungi,and invertebrates/vertebrates.Subsequently,we heterologously expressed and isolated E13,and analyzed its basic enzymatic properties.Substrate specificity analysis showed that E13 did not have lysophospholipase activity.E13 could efficiently degrade saturated and unsaturated monoacylglycerol substrates,and there is no significant difference in the degradation activity of saturated and unsaturated monoacylglycerides,which is different from the reported eukaryotic-derived monoacylglyceride lipase.As a monoacylglyceride lipase,E13 may play an important role in the lipid catabolism of its source G.nitratireducens.The optimum reaction temperature for E13 was 30?and also retains 30%of enzyme activity at 0?,indicating that E13 is a low temperature enzyme.The optimum reaction pH of E13 was 9,and it had a better tolerance in a wide pH range?5-11?buffer system.The enzyme activity of E1 3 was not affected by 0-3.5 M NaCl,and after an incubation of 24 hours at 4.5 M NaCl,the residual activity was still as high as 70%,indicating that E13 is a salt tolerant enzyme.E13 was tolerant to most of the metal ions tested.The enzyme activity of E13 was substantially unaffected by the inhibitors urea and thiourea,the reducing agent?-mercaptoethanol and DTT,the denaturants Tween 20 and Triton X-100,and the metal ion chelating agent EDTA.In addition,E13 was resistant to organic solvents such as DMSO.These features provide the possibility for E13 in industrial applications.To date,E13 is the only reported monoacylglyceride lipase from the bacterial origin of the Monoglyceridelipase_lysophospholipase family.2.Study on the catalytic properties of esterase E41.A gene encoding the esterase containing Abhydrolase 3 domain,E41,was screened from the genome of the marine bacterium G.lipolytica.Among the homologous proteins with a similarity of more than 30%with the E41 sequence,none of the biochemical properties of the protein was characterized,indicating that E41 is a novel esterase.Phylogenetic analysis of lypolytic enzymes known to contain the Abhydrolase 3 domain revealed that E41 and its homologous proteins form an independent branch,suggesting that E41 and its homologue may belong to a new lypolytic enzyme group.Multiple sequence alignment analysis showed that the catalytic triad of E41 consists of Ser148,Asp230 and His253,where the Ser catalytic residue is located on the GHSAG motif.Subsequently,we performed heterologous expression and purification in Escherichia coli BL21?DE3?,and analyzed the basic enzymatic properties of E41.Esterase E41 and its homologous proteins gE40 and E42 were only able to efficiently degrade the substrate pNPC2,and it is difficult to degrade pNPC4 or larger substrates.This is different from the reported Family ? and Family XV lypolytic enzymes.Esterases E41,gE40 and E42 exhibited similar substrate selectivity and both prefer to degrade small ester substrate molecules?pNPC2?.Compared with the Family ? and Family XV lypolytic enzymes,E41 and its homologous proteins may have smaller substrate binding cavities and can only bind smaller substrates.The optimum reaction temperature of E41 was 40?,and it could stably exist at 0-30?.The optimal reaction pH of E41 was 8 and it had a better tolerance to in a wide pH range?6-10?buffer system.The enzyme activity and stability of E41 were not substantially affected by high salt?4.5 M NaCl?,indicating that E41 is a salt-tolerant enzyme,reflecting its adaptation to the salt-containing marine environment.Among the tested metal ions,Zn2+,Cu2+,and F2+significantly inhibited the enzyme activity of E41 and was the potential inhibitors of E41.In summary,E41 and its homologous proteins differ in sequence,substrate selectivity,and possible structure from the already reported lypolytic enzymes Family IV and Family XV.E41 and its homologous proteins represent a new family of lypolytic enzymes with the Abhydrolase₃ domain.