Study On The Properties And Application Of Antarctic Bacterial Esterase

Lipolytic enzymes belong to theα/βhydrolase superfamily,mainly including esterase（carboxylesterase）and lipase（triacylglycerol hydrolase）,and are widely used in the field of production and life.Lipolytic enzymes have a wide range of sources and are abundant in animals,plants,fungi,and bacteria,among which microorganisms are the main source.Moreover,lipolytic enzymes widely distribute in various environmental conditions,including extreme environments such as high/low temperature,high salt,and high alkali.Since microorganisms that can survive in extreme environments have evolved unique catalytic mechanisms to adapt to extreme conditions,lipolytic enzymes isolated from such environments generally have unique properties.With the development of bioinformatics,sequencing technology,and gene annotation technology,more and more novel lipolytic enzymes have been discovered from extreme environments.The unique climatic conditions of Antarctica facilitate the discovery of new lipolytic enzymes.In this study,we screened lipolytic enzyme genes in the genomes of bacteria isolated from Antarctic soil and obtained three genes encoding novel esterase,which encode Est19,Est33,and Est1404,respectively.The pure enzyme was obtained by heterologous expression and purification,and its bioinformatics analysis,enzymatic properties,and preliminary catalytic performance were studied.Esterase Est19 is a member of the HSL family.The enzyme possesses a GESAG motif containing an active serine（S）located within a highly conserved catalytic triad of Ser¹⁵⁵,Asp²⁵³,and His²⁸² residues.The optimum temperature of the enzyme is 40℃,the optimum substrate is p NP-C6,and the optimum p H is 8.0.The catalytic efficiency（kcat/Km）of Est19 for the p NP-C6 is 148.68 s^-1m M^-1.Replacing Glu¹⁵⁴ next to the key catalytic serine with Asp（E154→D）,the activity and catalytic efficiency of the enzyme decreased significantly,while the substrate affinity did not change much.The wild-type enzyme retained allmost whole activity over a temperature range of 10–60°C,while～50%of its activity was retained at 0°C.Bioinformatics analysis indicated that Est19 and its homologs represented a new subfamily of HSL family,which was designated as GESAG subfamily.The property of Est19 to maintain high catalytic capacity at a lower temperature,and the stereoselectivity suggest that it may be used for low-temperature catalysis or chiral catalysis.The catalytic triad of esterase Est33 consists of Ser⁹⁴,Asp²⁰⁵,and His²³³,and Ser⁹⁴is located in the conserved pentapeptide motif GVSWG.Est33 retains 25-100%activity from 10-30°C,and have optimal catalytic activity toward p NP-C2（30°C and p H7.5）.The serine modifying reagent phenylmethylsulfonyl fluoride（PMSF 10 m M）inhibited the activity of Est33 by 80%,while thiol reagents such as dithiol threitol（DTT）activated the enzyme by 3.8-fold.Metal chelating reagents（EDTA）had no effects.The results show that Est33 is cold-adaptive and can play a catalytic role at low temperatures.Phylogenetic analysis indicate that Est33 and its closely related homologs belonge to an independent group,which is different from other known esterase familiess.Combined with biochemical analysis,we identified Est33 as a novel esterase family（XXI）.Esterase Est1404 belongs to the HSL family.Est1404 prefers to catalyze esters with short acyl chains,and its optimal substrate is p NP-C4.Its optimum temperature and p H were 70°C and 8.5,respectively.When it was incubated at 70°C for 2h,the activity of Est1404 was almost unchanged,and when incubated at 95°C for 2h,the activity decreased by 20%.Although the activity of Est1404 is strongly inhibited by Zn²⁺,the response to other metal ions is not obvious,and it also exhibits good tolerance to organic reagents such as methanol and ethanol.The resultssuggest that the enzyme may have the potential for catalytic applications in high-temperature conditions or organic reagents.The three esterases obtained in this study exhibit completely different biochemical characteristics,unique catalytic properties and even extreme adaptability.The discovery and characterization of three esterases expanded the family of esterases/lipases,which is of great significance to obtain novel ester hydrolases with higher industrial application value from the Antarctica.