Research For Screening Of Endophytic Bacteria Produceing ?–Mannanase From Coconut And Its Enzymatic Properties

In this study,the coconut was pretreated before the experiment.The method was followed by stripping the coconut skin,grinding the coconut with gauze,washing the sawdust with water,treating with 0.5% mercuric chloride for 10 min,75% ethanol for 3 min,Double the water to rinse the surface.The surface disinfection of the coconut tissue was determined to be 0.5% hg soaking for 9 min.The above-mentioned sterilization method was used to isolate and purify the treated coconut,and 56 strains of endophytic bacteria were obtained.The 56 isolates were mainly distributed in 5 orders,5 families,9 genera and 19 species.Among the 56 isolates,60 strains were Bacillus,accounting for 60.71% of the total strains,belonging to the dominant population of coconut endophytes,followed by 8 strains of Leclercia,Accounting for 14.29% of the total number of bacteria,the third is Stenotrophomonas maltophilia a total of 5 strains,accounting for 8.93% of the total number of strains.After isolation and purification of the isolates,the seedlings were incubated for 24 h,and the Congo red was screened.Among them,18 strains had obvious aperture,and the calculated ratio of clear circle and bacteria circle was calculated.Ten strains with high H/C ratio were selected The results showed that YZ-21 bacteria could be 20.67U/mL,so YZ-21 bacteria could be used as the research object.After the liquid activation of YZ-21 bacteria into the seed culture medium,determine the time of the bacteria do not listen to the absorbance,to draw the standard curve,and ultimately determine the YZ-21 bacteria in the 7-16 hours for the logarithmic period,to determine the best The seed culture time was 16 h.According to the observation of YZ-21,the culture conditions,physiological and biochemical indexes were consistent with the basic description of Bacillus subtilis in the Berger's Bacteria Identification Manual.The results of 16 S rDNA sequencing of YZ-21 were carried out by NCBI BLAST,Bacillus subtilis subspT similarity of 99.93%?Bacillus subtilis subsp.Subtilis subsp.Subtilis NCIB 3610 T similarity of 99.86%?Bacillus subtilis subsp.Spizizenii NRRL B-23049 T similarity of 99.93%?Bacillus tequilensis KCTC 13622 T was 99.79%.In this experiment,the enzyme was separated and purified by ethanol precipitation method,ammonium sulfate salting method and double aqueous phase.The experimental results showed that the two kinds of purification methods caused by ethanol precipitation and ammonium sulfate precipitation resulted in a large loss of enzyme activity.The results showed that the optimum salt content of the two aqueous phases was ammonium sulfate,and the optimum salt content of the aqueous solution was determined by optimizing the two-phase conditions.PEG 2000 and 18%(NH4)2SO4 double aqueous phase were the best,the highest extraction rate was 98.80%,and then the different concentration of NaCl was the best concentration of 18%,It was found that the 2% NaCl addition rate increased the enzyme extraction rate to 99.26%.The fermentation broth was centrifuged and the supernatant was measured to be 23.54 U/mL.The precipitated enzyme activity was 0.62U/mL after lyophilization using lysozyme.The ratio of extracellular and intracellular enzyme was 37.97,thus demonstrating that the enzyme belongs to the extracellular enzyme.The optimum temperature and pH of the enzyme were measured.The results showed that the optimum temperature of the enzyme was 40 ?,the optimum pH was 7.0,and the enzyme had poor tolerance to acidic environment.After the incubation at 55 ? for 2 h,the relative activity of the enzyme can still reach 65.7%.Under the condition of pH 5.0-8.0,the activity of the enzyme can still be kept above 80% after 30 min incubation.Mg2+,Ca2+,Co2+,Ba2+,K+ have the activation effect on the enzyme,and Ca2+,which is the strongest enzyme activation,can increase the activity of enzyme to the CK group by nearly 63.2% and the remaining five metals Cu2+,Zn2+,Mn2+,Na+ and EDTA showed the inhibitory effect on Cu2+,Zn2+,Mn2+.The recombinant plasmid pET-manA was cloned into Escherichia coli.BL21,and the recombinant plasmid was cloned into E.coli BL21.The recombinant plasmid pET-manA was transformed into E.coli BL21.The results showed that the activity of recombinant ?-mannanase was 372.86 U/mL.The expressed product was analyzed by SDS-PAGE gel electrophoresis,and the protein profile showed a specific band of about 56.07 KDa.The results showed that the optimal substrate for the recombinant ?-mannanase was melon gum,and its activity was 392.54 U/mL.The genomic DNA of the strain YZ-21 was amplified by PCR to obtain the mana gene of the production of ?-mannanase.The sequence analysis showed that the full length of the gene was 1127 bp,and the amino acid sequence of the enzyme was homologous to the same homologous ?-mannanase Can reach 97.56%,with ManA conserved domain,belonging to the glycoside hydrolase family of GH 26 members.The recombinant plasmid pET-manA was transformed into E.coli BL21,and the expression of the enzyme was induced.The results showed that the activity of recombinant ?-mannanase was 372.86 U/mL.The expressed product was analyzed by SDS-PAGE gel electrophoresis,and the protein profile showed a specific band of about 56.07 KDa.The optimum reaction temperature and pH were 40 ? and pH 7.0 respectively.The specificity of recombinant ?-mannanase substrate was tested,and the optimal concentration of recombinant ?-mannanase was determined.The species is melon gum and its activity is 392.54 U/mL.