Production Suitable Screening Of The Cold Xylanase Strains, Purification, Cloning And Expression, And Enzymatic Properties

Xylan is a major component of hemicellulose, and the second most abundant renewable organic carbon on the earth. Endo-l,4-P-xylanase (EC 3.2.1.8) can hydrolyze xylan to produce xylooligosaccharide and xylose. Xylanases have extensive applications in many industries such as bioconversion, food, paper and pulp, animal feed and medicine, showing the broad exploitation prospect. Cold-active xylanases, of which the optimal reaction temperature is between 20-30℃, are generally produced by microorganisms from polar regions and sea ice, where the temperature is very low. At present, cold-active xylanases have not been widely studied.In this study, we screened two cold-active xylanases-producing bacterial strains from the strains preserved in our lab and a total of 147 strains from arctic sea ice which had been isolated and identified by Polar Reseach Institute of China. These two strains were the sea-ice bacterium Pseudoalteromonas elyakovii Bsi20429 donated by Polar Reseach Institute of China and the bacterium Glaciecola mesophila KMM 241^T purchased from German Collection of Microorganisms and Cell Cultures (DSMZ). The deposited number of G. mesophila KMM 241 is 15026^T.We purifed the xylanse produced by P. elyakovii Bsi20429 through ammonium sulfate precipitation and cation exchange chromatography. The molecular weight of the purified xylanase was approximately 45 kDa. The temperatue optimum of this enzyme was 30℃and pH optimum was 8.0. The N-terminal amino acid sequence of the enzyme showed very high identity with the xylanase pXyl from Pseudoalteromonas haloplanktis TAH3a, and then most of the gene sequence (1201 bp) of this enzyme was amplified by PCR from the genome DNA of G. mesophila KMM 241 using the gene sequence of pXyl as primers. Blast analysis showed that this xylanase was a family 8 xylanase, having 97% amino acid identity with pXyl.A family 10 xylanase gene xynA was cloned from G mesophila KMM 241 by PCR using the sequence of a hypothetical xylanase identified from the genome of Pseudoalteromonas atlantica T6c. xynA was 1272 bp in length, encoding a protein of 423 amino acid residues which was composed of a catalytic domain and a N-terminal peptide sequence and was predicted to be a family 10 xylanase (named XynA). XynA showed 97% identity with the xylanase from P. atlantica T6c and among characterized xylanases, it showed the higest identity with Xyn10 from Flavobacterium sp. MSY2 (46%). xynA was ligated to pET-22b(+) and expressed in E. coli BL21(DE3). The medium supernatant and cell lysate showed xylanase activity of 1.2 U/ml and 0.7 U/ml, respectively. Through ammonium sulfate precipitation and Ni-affinity chromatography, we purified the mature recombinant XynA. Ananlysis of the N-terminal sequence of XynA showed that it contains 376 amino acids with a molecular weight of 42 kDa.We studied the characters of recombinant XynA in detail. The optimum temperature of XynA was 30℃, and the optimum pH was 7.0. XynA was thermoliable, showing a half life of 20 min at 30℃, and the melting temperature assayed was 44.5℃. XynA could hydrolyze xylan from beechwood, birchwood and oat spelt wood efficiently, but it could not hydrolyze cellulose, starch, chitosin, laminarin and mannan. Kinetic studies showed that at 30℃, the K_m, V_max, and k_cat of XynA towards beechwood xylan were 1.40 mg/ml, 103.2μmol min^-1 mg^-1 and 72 s^-1, respectively, while at 4℃, they were 0.77 mg/ml, 17.33μmol min^-1 mg^-1 and 12 s^-1, respectively. Ca²⁺, EDTA, Li²⁺ and K⁺ increased the activity of XynA while Zn²⁺, Cu²⁺, Ni⁺, Sn²⁺, Co²⁺, Mn²⁺, Fe³⁺ and SDS had evident inhibitory effect on XynA, and the effect was stronger at 10 mM than at 1 mM. XynA had high tolerance to NaCl, retaining nearly 50% of its highest activity when the NaCl concentration was 4.0 M. Hydrolysis products analysis showed that XynA could effectively hydrolyze xylooligosaccharides larger than xylotriose and beechwood xylan. The products from xylan hydrolysis were xylobiose and xylotriose without xylose. These results suggest that XynA may have good application in xylooligosaccharide production and flour modification.