Isolation And Preliminary Characterization Of Psychrotrophs Purifying Domestic Sewage

In terms of winter or northern areas in China,low temperature is the mainspring leading to poor efficiency of domestic sewage treatment.A fundamental measure to address the sewage problem could be screening special microorganisms which show high activity even in low temperature.This paper narrowed the objects to specific constituents of domestic sewage,such as protein,starch and pectin.Respectively,we studied psychrotolerant bacteria which can secret proteases,amylases or pectinases.Cold-active enzymes also have been studied,to throw a light over enzyme engineering.(1)Using various methods such as cryogenic liquid enrichment,dilution coated plate,streaking plate with selection medium,temperature screening method and detection of enzymatic activity,we isolated psychrotrophic bacteria producing proteases,amylases or pectinases from the soil of Daqing Oilfield,a non-extremely natural environment.It seemed that there were no psychrotrophic bacteria with high yield of amylases or pectinases.In contrast,we successfully obtained two psychrotrophs with pleasant ability to produce proteases,one stenopsychrotroph,named as LS3,which belongs to genus Pseudomonas,and one eurypsychrotroph,named as LS4,which belongs to genus Exiguobacterium.(2)These two psychrotrophs both grow and produce enzymes well in low temperature,with different carbon and nitrogen sources affecting their enzyme-producing abilities.Glucose,maltose and soluble starch all cannot be utilized for enzyme production.What's more,LS4 can only assimilate casein as the nitrogen source to secret protease,which may act as induced enzyme;while the enzyme activity of protease produced by LS3 could reach 52.46 U/mL in the absence of urea.These properties provide mighty instructions when it comes to practical application.(3)We tried to immobilize psychrotrophs taking advantage of entrapping method with calcium alginate.Gel beads at micro-size were successfully achieved without any help of automatic device like encapsulator.Psychrotrophs encapsulated in micro-beads grow and produce as well as naked ones.However,entrapping with sole material like calcium alginate cannot ensure ideal protection for bacteria from relatively extreme pH or temperature.(4)Extracellular proteases produced by LS3 and LS4 were purified rudimentarily using method of ammonium sulphate precipitation,and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE).It seemed that LS3 produced more kinds of proteases when cultivated in media containing urea.Comparing different SDS-PAGE results under different culture conditions,we could tell that LS3 at least produces one kind of extracellular proteases around 50 KDa,while LS4 seems to have the capability producing three kinds of extracellular proteases.(5)Enzymes produced by LS3 and LS4 strains,all sensitive to heat,sustain good enzymatic activity at low temperature,and present optimum activity at 40 ? and 30 ?,respectively,with the optimum pH both at 8.These enzymes are confirmed as alkaline cold-adapted proteases.Enzyme produced by LS3 could be suppressed by metal ions to some degree,and only enzyme produced by LS4 is strongly sensitive to EDTA,indicating that it may be metalloproteinase.Contrarily,tween 80,as a stabilizer,activates enzymes.Understanding Enzymological properties has instructive meanings.At present,isolation research relating to cold-adaptive microorganisms mainly refers to extreme environment such as deep oceans,polar region,glaciers and frozen soil.Innovatively,here we screened psychrotrophic bacteria from the soil of Daqing Oilfield,a non-extremely natural environment,building up more resource to psychrotrophs applied in environment,industrial food area and so on,as well as providing reference data to cold-adapted mechanisms,and research and development of cold-adapted proteases.