Non-cellulose Degrading Property Of Erwinia Carotovora CXJZ95-198 And Cloning Of It's ManA Gene

Herbaceous fiber extracted biologically is the developmental trend which can reduce the heavy pollution, product cost arising from chemical method and upgrade the product quality meanwhile. Mannanase plays the key role in the degradation of mannan and lignin which are embedded in the fiber cell wall and can be widely used in pulping, textile, foodstuff, pharmaceutical and oil industries.E.carotovora CXJZ 95-19 is the most powerful strain reported for the bio-extraction of herbaceous materials within 6-8hr. The use of monosaccharides and the way non-cellulose degraded in the fermentation process were studied integratedly. Its manA gene was successfully cloned, analyzed and expressed.It's addicted to mannose. Mannanase is one of the key factors in the proficient extraction of herbaceous fibers. Non-cellulose in raw materials can be degraded by the chain reaction of "non-cellulose nourishing microorganism—microorganism secreting enzyme—enzyme decomposing non-cellulose" after inoculation with strains. Sugars, macromolecules and cast in the fermentation liquor increase and reach the peak 6-8hr after inoculation.8-11% of raw material can be degraded in the fermentation course, 90% of organics in the liquor are macromolecules and cast. The non-cellulose attached to the fibers can be easily removed in the coming treatment after fermentation.The fragments mainly sized 2-5 kb after optimized partial digestion with Sau3AI could be used directly for the genomic library construction. 8 positive clones were spotted from 15,000 transformants. One smallest recombined plasmid pT5 of 1844bp insert was sequenced. It was revealed that the left 7 clones harbored the same manA gene.It was determined that the 1.3kb sequence upstream of the 4,449,729 loci of E.carotovora is the location for manA gene. This is the first time to clarify the function of the genomic loci of E.carotovora.The 1137bp ORF for manA is of low similarity to other mannanase genes from different origins. It's a new manA gene. There is no typical promotor found upstream of the ORF, but a possible SD sequence existed on the location -8. The gene is registered at GenBank numbered DQ364440.The manA encodes for 387 AA of molecular weight of 41.9KD. A putative signal peptide exists from 1to 27 AA. The secondary structure is of 35% alpha helix and 51.5% random coil, and two domains including a catalytic one can be found. It's an extracellular enzyme classified as hydrolase 26.The Blast shows that some similarities exist between the current mannanases and those of other origins. The mannnanases from Bacillus sp. are of the highest similarities around 50-52% and the others less than 30%.Expressing vectors were constructed and the regulation factors were optimized. Signal peptide is of high importance to the secreting of the ManA.The most suitable IPTG concentration is 1mM and the most suitable temperature is 37 ℃. Two bands of MW of 42 and 47 KD of high density can be found for vectors with signal peptide and one targeted band can be found for vectors with no signal peptide.The optimal pH and temperature for the expressed ManA is 7.5 and 50℃ respectively.