Isolation And Identification Of Halophilic And Basophilic Bacteria From Rhizosphere Of Suaeda Salsa And Study On Amylase-producing Strains

PurposeThe bacteria in the rhizosphere of Suaeda salsa were isolated and identified for preliminary genetic analysis;the bacteria producing amylase in the rhizosphere of Suaeda salsa were screened;the strains with the highest amylase activity were optimized for fermentation conditions and the exploration of enzymatic properties.MethodsThe soil of Suaeda salsa growing area under Linghai bridge in Linghai District,Jinzhou City,Liaoning Province was collected,and the suitable experimental conditions were screened by different pH and NaCl concentration(w/v);the number of bacteria in the rhizosphere of Suaeda salsa and the adjacent mud ditch was compared;the bacteria were screened and purified by dilution coating plate method,and the morphology and microscopic examination of the bacteria were carried out to eliminate the phase The same strain.The genomic DNA of the obtained strains was extracted,the extracted DNA was amplified by PCR and the gene sequence of the bacteria was detected,and the result of sequencing was compared with blast on NCBI to determine the species with the highest similarity;the phylogenetic analysis was carried out by mega-6,and the preliminary genetic analysis was carried out for the rhizosphere bacteria of Suaeda salsa.The culture medium containing 1%soluble starch was used to screen the amylase producing strains.The transparent circle method was used in the initial screening,and the DNS method was used to detect the amylase activity in the secondary screening.The strains with the highest activity of amylase were selected to optimize the fermentation conditions and explore the best fermentation conditions;the enzyme properties of the obtained amylase were studied preliminarily;the growth and amylase activity of the bacteria with the highest activity of amylase were measured and the growth curve and enzyme activity curve were drawn.ResultsThe most suitable condition was p H 9.0 and Na Cl concentration(w/v)10%.It was found that the number of bacteria in the rhizosphere of Suaeda salsa was more than that in the adjacent mud ditch.Through the isolation,purification and weight removal of bacteria,61 strains of bacteria were obtained.Through16Sr DNA sequencing and blast comparison,it was found that the 61 strains belonged to 17 genera,of which Halomonas denitificans was the dominant genus,accounting for 24.59%.Two strains of bacteria No.5 and No.41 with clear ring were obtained by primary screening of amylase.The enzyme activity of bacteria No.5 was 78.51 U/ml and that of bacteria No.41 was 76.61 U/ml.The amylase activity was 6.09 times higher than the initial enzyme activity.Through the study on the properties of the amylase produced by bacterial No.5,it was found that the optimum temperature of this kind of amylase was 70?,the enzyme activity was still more than 60%between 20?and 90?,and the enzyme activity was still more than 70%after being kept at 90?for one hour,which indicated that this kind of enzyme had good temperature stability.The optimum p H is p H 9.0,and it can keep more than 50%activity after being kept between p H 8.0 and p H 10.0 for one hour,which indicates that this kind of amylase can keep high activity under alkaline condition.Ten metal ions,Cu²⁺,Mg²⁺,Zn²⁺,Co²⁺,Fe²⁺,Mn²⁺,Ni²⁺,Ca²⁺,K⁺and Ba²⁺were added to the medium respectively,and the activity of the enzyme was determined.It was found that Fe²⁺had the most obvious activation effect on amylase,and the stability was the best when Mn²⁺was added.Through the analysis of the growth curve and enzyme activity curve of bacteria No.5,it was found that bacteria No.5 was in a slow period in the first 10 hours,a logarithmic growth period between 10 hours and 22 hours,a relatively stable period between 22 hours and 34 hours,and a decay period after 34 hours;the enzyme activity was the highest at 24 hours after fermentation.ConclusionsIn this experiment,61 strains of bacteria were isolated from the rhizosphere of Suaeda salsa,which belong to 17 genera.The phylogenetic tree of these 61strains was constructed and found that the diversity of bacteria in the rhizosphere of Suaeda salsa is relatively good.By screening the amylase production ability of the 61 strains of bacteria,it was determined that the amylase activity of bacterial No.5 was the highest;by response surface optimization,the enzyme activity of bacterial No.5 was increased to 6.09 times of the initial enzyme activity;through the study of the enzyme properties of this amylase,it was found that this amylase had good stability under high temperature and high pH,and had good prospects for industrial enzyme development.