Screening Of Thermo-Stable Bacteria And Characterization Of Inulinase

As people increase the awareness of health care,inulin becomes the focus all over the world.Scientists have paid more attention to inulinases and microorganisms related,which is also the aim of this paper.In this paper,two thermophilic inulinase producing strains,which grow optimally at 50℃,were isolated from soil samples with medium containing inulin as a sole carbon source. They were identified as Bacillus smithii by analysis of 16s.rDNA and blast in Genbank.They were defined as Bacillus smithii T4 and Bacillus smithii T7,genbank accession numbers are EU652724 and EU628681 respectively.Through the study on different carbon sources,nitrogen sources,mineral ions,surfants, temperature,pH,rotation and incubation time,optimal fermentatal conditions of Bacillus smithii T4 are as follows:inulin 3.0%,(NH₄)H₂PO₄ 0.5%,yeast extract 0.5%,CaCl₂ 0.5mM, Brij-35,0.006%(w/v),50℃,pH 5.5,1.50rpm and 72h incubation.Maximum inulinase yield of it was 134.4 IU/ml.As for Bacillus smithii T7,Maximum inulinase yield of 136.5 IU/ml was achieved with medium pH7.0,containing inulin 2.0%,(NH₄)H₂PO₄ 0.5%,yeast extract 0.5%,at 50℃200rpm shaker for 72h incubation.Under the optimal fermental conditions,the fermental liquid of Bacillus smithii T7 was centrifugated at 4℃10000rpm for 15 min.Then the supernatant was collected as crude enzyme. The purified inulinase was achieved through 10kD ultra-filtration,ammonium sulphate precipitation,10kD dialysis,DEAE-Sepharose CL-6B and Superdex 75 chromatography.The inulinase was identified as a single band,subunit MW 47kD by SDS-PAGE.The product of hydrolyzation shows endo-inulinolytic activity by the detection of TLC. The optimum pH for this endo- inulinase is 4.5 and stable at pH range of.4.5-6.5.The optimum temperature for enzyme activity was 70℃,and the half life of the endo-inulinase is 9h and 2.5h at 70℃and 80℃respectively.Fe²⁺ enhanced the endo-inulinase activity immensely,however,Cu²⁺,Ba²⁺,K⁺ and Na⁺ decrease it.Moreover,comparatively lower Km (4.17 mM) and higher Vmax(833 IU/mg protein) demonstrate the endo-inulinase's greater affinity for inulin substrate.In all,the thermophilic bacteria Bacillus smithii T7 can produce heat-stable,great affinity endo-inulinase,which is significant potential for the application and development of Jerusalem artichoke or inulin.