| Hemicell?Lose as a renewable resources with great application potential,its main composition is xylan.The completely degradation of xylan need composition complex xylan degradation enzyme system.?-xylosidase is the key enzyme of xylan degradation enzyme system,enzyme catalytic function is the hydrolysis of?1?4?-?-D-xylans,to remove successive D-xylose residues from the non-reducing termini of xylooligosaccharide and xylobiose.The study of ?-xylosidase for the hemicell?Lose degradation is of great significance.This paper reports the selection of high-yielding ?-xylosidase strain from forest soil,the highest enzyme activity and the corresponding specific activity produced by this strain were 1.29 U/m L and 1.05 U/mg respectively in fermentation.Through the determination of extracell?Lar fluid and intracell?Lar fluid,the res?Lts determine that the ?-xylosidase produced by this strain is inside the cell.By 16 S r DNA phylogenetic tree analysis,morphological observation and physiological and biochemical experiment,the strain was identified to be an Enterobacter cloacae and named LY-62.Based on the initial fermentation culture conditions,the preliminary optimization was operated on the fermentation conditions for enzyme production of Enterobacter cloacae LY –62.That different carbon source and its concentration,different nitrogen source and its concentration,medium initial p H and temperature effects on fermentation of enzyme production was investigated.After optimization of fermentation conditions,the highest level of enzyme activity and the corresponding specific activity produced by Enterobacter cloacae LY – 62 can achieve2.2 U/m L and 2.87 U/mg respectively,enzyme activity up to 170% compared with the former.The research of enzyme production curve and growth curve under the optimization of fermentation conditions demonstrated that the Enterobacter cloacae LY – 62 with the characteristics of tachyauxesis,high utilization efficiency of xylan,enzyme production quickly and high enzyme activity.For further research on ?-xylosidase of Enterobacter cloacae LY – 62,I have designed primers to successful clone and expresse its ?-xylosidase gene,and performed molecularidentification on zymologic function of this gene.On the basis of the enzyme gene sequence and its amino acid sequence,the bioinformatics methods were performed to obtain its protein amino acid composition,hydrophobicity,relative molecular weight and other relevant information of primary structure.The secondary structure components and protein tertiary structure were also predicted.Subsequently,the?-xylosidase of Enterobacter cloacae LY –62 was expressed,and the recombinant protein His-Xyl62 with N-terminal His-tagged fusion were stadied.The optimum p H of ?-xylosidase is p H 7.0,and this enzyme keep comparable stability under weak acid or week alkaline solution.The optimum temperature of ?-xylosidase with relatively poor thermal stability is 40?.The Km,Vmaxand Kcat values of His-Xyl62 when the PNPX as substrate were 1.55 mmo L/L,38.61umol/?min·mg?and 8.51s-1. |
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