Purification, Gene Cloning And Expression Of Xylanases From Streptomyces Olivaceoviridis A1

Xylanases can hydrolyze xylans into xylooligosaccharides and D-xylose. They havewide commercial aPplications in industrial processes, such as feed, paPer and pulp,foodstuff and energy industry. Stroptomyces olivaceovirch Al from soil produce twoextracellular xylanase XYNA of the molecular weigh 43kD and XYNB of the molecularweight 23kD. Optimal pH Value and temperature of XYNA fOr its activity were 5.6 and 60OC, respectively. The Km Values and Vm. of XYNA for 4- O - Me - D - gl ucurono -D - xyl anunder 55C were 3.32g/Kg and l5.72pmol/ml. min, respectively. The activity of XYNAcould be decreased slighly by SDS and EDTA. Optimal pH Value and temperatUre ofXYNB for its activity were 5.2 and 60aC, respectively. The Km Values and Vm. of XYNBfor 4- O -Me-D-glucurono-D-xylan under 55C were 22.lg/Kg and l05.26pmoUml' min,respectively. Mg'+ and Cr3+ can increased the activity of XYNB slighly. Both XYNA andXYNB didn't display cellulase activity Both XYNA and XYNB were treated by pepsinor trypsin fOr 30 min under 37C, their activity weren't decreased. The N-terminal aminoacids sequences of XYNB and XYNA were determined by axnino acids sequence analysis.The result show that the ten N-terminal amino acids sequences of XYNA are same as thoseof FXYN published by Atsushi. According to the published data, three primers had beendesighed and synthesized, xyndj-s including siginal sequence and xynA!-wnon-including siginal sequence had been cloned by PCR. According to the determinedN-terminal amino acids sequence of XYNB and the C-terminal conservative amino acidssequence of G/ll family xylanase. Two primers were desighed and synthesized. XynBjcoding nature protein of XYNB have been cloned by PCR. Then xynd1-s nynA]-w andxynB1 were all cloned into the plasmid vector pET-22b(+), and were all expressed in E coli.rynAI-w and xynB1 were all cloned into the plasmid vector pPIC9 Q,and were alloverexpressed in pichia pastoris GS l 1 5.The nucleic acids sequence and amino acids sequence of xynBi were homologouslyanalysed with the sequences coding fOr xylanases published in GeneBank. The resultindicated that xynB] was the highest similarity to the gene xyli from sm ptomyces sP.Strain S38, up to 86%. And the properties of XYLl was different in those of XYNB. So,itproves that xynBi is a new gene encoding xylanase. The sequence of xynB] has beenaccepted by EMBL(Accession number is AJ2923 l7).