Xylan-binding xylanase Xyl30 from Streptomyces avermitilis: cloning, characterization, and overproduction in solid-state fermentation.
A DNA fragment from the lignocellulolytic actinomycete Streptomyces avermitilis CECT 3339 was cloned using a DNA probe from the xylanase gene xysA of Streptomyces halstedii. The nucleotide sequence analysis revealed two potential ORFs, xyl30 and hd30, encoding a deduced multimodular F/10 xylanase with a binding domain and a secreted glycoxyl hydrolase, respectively. In Streptomyces lividans carrying the subcloned DNA fragment, two xylanase activity bands with estimated molecular masses of 42.8 and 35 kDa (named Xyl30 forms "h" and "l", respectively), were detected by zymograms and SDS-PAGE. The two xylanases had identical N-terminal sequences, suggesting that Xyl30 "l" derived from Xyl30 "h" by C-terminal processing in the culture supernatant. No transcripts of hd30 were detected by RT-PCR. Characterization of the partially purified Xyl30 "h" confirmed the presence of a modular endoxylanase containing a xylan-binding domain, which after processing in the culture supernatant loses the aforementioned domain and thus its capacity to bind xylan (Xyl30 "l"). Xyl30 "h" achieved maximal activity at pH 7.5 and 60 degrees C, retaining more than 50% of its activity from pH 3 to 9 and more than 40% after a 1-h incubation at 70 masculineC. Moreover, in the recombinant host strain up to 400 U xylanase/g medium (dry weight) was produced in solid-state fermentation (SSF) using cereal bran as substrate. The high production yields of this enzyme and its biochemical features make it a good candidate for use in industrial applications.
Hernández
Alberto
A
Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Alcalá, Alcalá de Henares, Spain.
López
José C
JC
Santamaría
Ramón
R
Díaz
Margarita
M
Fernández-Abalos
José M
JM
Copa-Patiño
José L
JL
Soliveri
Juan
J
D003001:Cloning, Molecular; D003470:Culture Media; D004269:DNA, Bacterial / Q000032:analysis; D004043:Dietary Fiber / Q000378:metabolism; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005285:Fermentation; D007218:Industrial Microbiology / Q000379:methods; D013302:Streptomyces / Q000145:classification / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D048170:Streptomyces lividans / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D014990:Xylans / Q000378:metabolism*
D003470:Culture Media; D004269:DNA, Bacterial; D004043:Dietary Fiber; D014990:Xylans; D043364:Endo-1,4-beta Xylanases