Purification?Cloning And Expression Of An Endoxylanase Gene From Streptomyces Sp.H33 In E.coli

Endoxylanase(?-1,4-endoxylanase, Endoxylanase/Endo-?-xylanase, EC3.2.1.8) is the most important enzyme for xylanase system, which only randomly hydrolyzes internal?-1,4-glycossidic bonds to produce xylose and xylo-oligosaccharides(XOs). According to the optimum pH for the enzymatic reaction, endoxylanase could be classified into three types:acidic endoxylanase(optimal pH3.0?5.0), neutral endoxylanase(optimal pH6.0?8.0) and alkaline endoxylanase(optimal pH8.0?10.0). In contrast to acidic endoxylanase, neutral endoxylanase and alkaline endoxylanase which are mainly produced by bacteria and actinomyces, have wider pH for catalysis, good thermostability and short fermentation time,etc. Therefore, it could be well applied in paper-making?food and feed industry. In this paper,we describe the screening of neutral endoxylanase-producing bacteria, the purification and characterization of the neutral endoxylanase from the cultrue supernatant of Streptomyces sp.H33, and the cloning and express of the neutral endoxylanase gene in E.coli BL21(DE3). The main contents of this paper are as follows:(1)A relatively high neutral endoxylanase-producing strain Streptomyces sp. H33 was isolated from soda lakes in Inner Mongolia,P.R.China. Using the Congo-red staining method,seven endoxylanase-producing strains were isolated from soda lakes in Inner Mongolia,P.R.China. Streptomyces sp. H33 could produce the highest enzymatic acitivity and its endoxylanase activity reached as high as 51.4 U/mL after liquid fermental cultivation. It was subsequently identified as Streptomyces sp. H33 based on its morphological characters and16 S rDNA sequence. Compared With the current industrial strains, Streptomyces sp. H33 could be cultured in high alkaline condition(pH 9.0), in this way bacteria pollution could be effectively suppressed, and becoming more favorable for large-scale fermentation production.This paper focuses on the research of Streptomyces sp. H33.(2)The neutral endoxylanase was purified from the culture supernatant of Streptomyces sp. H33 with cation-exchange chromatography, and the native protien had molecular mass of approximately 42 kDa. Purified enzyme showed an optimum activity at temperature 55? andpH 7.0, and it retained more than 80% activity after incubation at pH 10.0 and 60? for 60 min,respectively. Furthermore, it was resistant to most metal ions and reagents(SDS?EDTA)examined, and had good thermostability and pH stability. This neutral endoxylanase with superior stability characteristics coud be a suitable candidate in paper industry.(3)A new endoxylanase gene which had an open-reading frame(ORF) of 1380 bp and coded for 459 amino acids was successfully cloned from genome DNA of Streptomyces sp.H33 through PCR method. Among the 459 amino acids,the first 22 amino acids were the signal peptide and the mature protein started from Leu23, amounting to 437 amino acids, and its isoelectric point(pI) was 8.18. BLAST analysis of the H33 sequence in NCBI database showed that it had 78% highest similarity to the ?-1,4-endoxylanase from Streptomyces pristinaespiralis ATCC 25486 which belonged to family GH10. Based on the BLAST results and the differences in molecular mass, pI and enzymatic properties, it could be inferred that the gene H33 was a novel endoxylanase gene.(4)The neutral endoxylanase gene from Streptomyces sp. H33 was successfully overexpressed in E.coli BL21(DE3). By the method of PCR, the endoxylanase gene was cloned from genome DNA of Streptomyces sp. H33 and was ligated with the expressing vector pET-28a(+) through restriction endonuclease EcoR I and Xho I, generating a recombinant plasmid pET-28a-H33 which was directly transformed into E.coli B121(DE3).The most efficient recombinant E.coli strain, which was designated as E.coli H33, expressed large amount of endoxylanase when cultivated in IPTG containing medium. The recombinant endoxylanase was purified to homogeneity from the cultrue supernatant with a Ni-Sepharose column-chromatographic procedure, and the purified recombinant endoxylanase XYN-H33 exhibited high specific activity of 62 U/mL. The properties of the recombinant endoxylanase XYN-H33 was verified to be identical to the native endoxylanase in corresponding aspects,indicating that the neutral endoxylanase gene had been successfully expressed.