| An component of xylanase was separated and purified by (NH4)2SO4 fractionation, Phenyl Sepharose CL-4B,Sephadex G-50 and DEAE Sepharose FF Ion exchange chromatography from the filtrate of solid state culture medium of Aspergillus usamii.Its recovery was 19.0 %,and purification fold was 26.9.Its specific activity was 2904.9 IU/mg protein.The purified Xynâ… exhibited a single protein band on SDS-PAGE and was shown to be homogeneous by HPLC.The molecular weight of Xynâ… was respectively estimated to be 20.7 kD and 19.4 kD by SDS-PAGE chromatography and gel filtration using Sephadex G-75,witch indicated that the xylanase was a monomer.The isoelectric point pI5.0 was determined by IEF-PAGE.Its carbohydrate content was 0.42%.Its temperature optimal for the activity and its optimum pH was 50℃and 5.0 respectively.It showed good stability below 50℃and was stable in pH 5.5 ~ 9.0.Its Km and Vmax was 3.5 mg/ml and 5000μmol/(min·mg) respectively with the xylan from beachwood as substrate.Based on the sequence of xylanase from Aspergillus niger on GenBank and 3'-terminal poly(A) of eukaryotic mRNA,PCR primers were designed and synthesized,mature peptide encoding region with 3'non-encoding region of xylanase cDNA from Aspergillus usamii was amplified with RT-PCR,and PCR product was cloned into the vector pUCm-T.Sequence analysis showed that mature protein of xylanase was composed of 188 amino acids.Amplification,cloning and sequencing of cDNA fragment from transcription start point of mRNA to xylanase encoding region were also carried out by SMARTTM PCR.From above two steps of PCR,an entire xylanase cDNA sequence from Aspergillus usamii was determined and analyzed.Based on having known the cDNA sequence ,a pair of primers was designed and synthesized near the start code and terminal code,respectively.The genemic DNA from Aspergillus usamii was acted as template,the DNA gene encoding xylanase was amplified and cloned with PCR method.Sequencing of xylanase DNA indicated that...
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