Cloning, Expression And Characterization Of Xylosidase And Xylanase From The Deep-sea Kocuria Sp.Mn22

Xylanase are widely used in various industries.For instance,in animal feed industry, xylanases are used to increase digestability and nutritive value of poorly degradable feeds. In brewing,xylanases can increase wort filterability and reduce haze in the final products. Xylanase can hydrolyze xylan to produce pharmacologically active polysaccharides for use as antimicrobial agents.In pulp and paper industry,the xylanases are employed in the prebleaching process to reduce the use of the toxic chlorine chemicals,and so on.The marine microorganisms that live in extreme conditions remain the largest resource from which to isolate novel xylanase genes.We isolated a deep-sea bacterium Kocuria sp. Mn22 produced xylanase from the marine bacteria supplied by Third institute of Oceanography State Oceanic Administration.We constructed the genomic DNA library of Kocuria sp.Mn22 by shotgun method.And finally we cloned,expressed and characterized the endo-β-1,4-xylanase andβ-xylosidase,respectively.This is the first time to report endo-β-1,4-xylanase andβ-xylosidase gene from Kocuria.The genomic DNA of Kocuria sp.Mn22 was partly digested by Sau3AI and ligated to pUC18 vector to construct a DNA library.We isolated a xylanase-positive clone by the Congo red method.The clone was named as pUC-kxyn.The insert fragment contained four open reading frames（ORFs）.The first truncated ORF encoded a putative permease component of the ABC-type sugar transport system,the second ORF encodedβ-xylosidase,defined as kxyn3,the third ORF encoded endo-β-1,4-xylanase,defined as kxyn10,and the last truncated ORF encoded a putative xylose repressor.Kxyn3 gene consists of 2358bp and encodes a 786-residue polypeptide with a calculated mass of 82,358Da.It was GC-rich,and the overall GC content was 74%. Kxyn3 shows the highest identity（56%） with the xylosidase from Streptomyces thermoviolaceus.The mature protein was expressed in Escherichia coli BL21（DE3） and the purified Kxyn3 displayed the maximum activity at 55℃and at pH 6.5.The activity of Kxyn3 was not affected by K⁺、Zn²⁺、Co²⁺、Mg²⁺、Ca²⁺、Na⁺ or EDTA,but it was strongly inhibited by Hg²⁺ and Cu²⁺.DTT could increase the activity of Kxyn3 by 10%. Using pNPX as substrate,the K_m,V_max and k_cat of Kxyn3 was 1.04 mmol/L,150μmol/min mg and 41.2/s,respectively.Kxyn3 was a bifunctional enzyme with xylosidase and arabinofuranosidase activity. Kxyn10 gene consists of 1,170 bp and encodes a protein of 390 amino acids that shows the highest identity（63%） with a xylanase from Thermobifida fusca YX.A putative signal peptide of 30 amino acid residues was located at the N terminus.After processing of the signal peptide,the mature polypeptide was predicted to have a mass of 40,825 Da.The mature protein was expressed in Escherichia coli BL21（DE3）. Recombinant Kxyn10 displayed the maximum activity at 55℃and at pH 8.5.Kxyn10 was an alkalic xylanase.It was stable over a wide pH range,remaining more than 80% activity after overnight incubation at pH 7.5-12.After incubation at 55℃for an hour, Kxyn10 still had 70%of the highest activity.The K_m,V_max and k_cat values of Kxyn10 for birch wood xylan were 5.4 mg/mL,272μmol/min·mg and 185.1/s,respectively.Kxyn10 hydrolyzed birch wood xylan to produce xylobiose and xylotriose as the predominant products.And the shortest xylooligosaccharide which could be hydrolyzed by Kxyn10 was xylotetraose.The activity of Kxyn was not affected by Ca²⁺,Mg²⁺,Na⁺,K⁺,β-mercaptoethanol,or DTT,but it was strongly inhibited by Hg²⁺,Cu²⁺,Zn²⁺ and Pb²⁺. Kxyn10 was tolerant to SDS,showing 90%residual activity after incubation with 5 mmol/L SDS.Kxyn was a cellulase-free xylanase.These properties make it a candidate for various industrial applications,especially the biobleaching of paper pulp.