Cloning of a Thermomonospora fusca xylanase gene and its expression in Escherichia coli and Streptomyces lividans

Journal Article · Thu Jun 01 04:00:00 EDT 1989 · Journal of Bacteriology; (USA)

OSTI ID:7192165

Ghangas, G S; Hu, Y J; Wilson, D B ^[1]

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1. Cornell Univ., Ithaca, NY (USA)

Thermomonospora fusca chromosomal DNA was partially digested with EcoRI to obtain 4- to 14-kilobase fragments, which were used to construct a library of recombinant phage by ligation with EcoRI arms of {lambda}gtWES.{lambda}B. A recombinant phage coding for xylanase activity which contained a 14-kilobase insert was identified. The xylanase gene was localized to a 2.1-kilobase SalI fragment of the EcoRI insert by subcloning onto pBR322 and derivatives of pBR322 that can also replicate in Streptomyces lividans. The xylanase activity produced by S. lividans transformants was 10- to 20-fold higher than that produced by Escherichia coli transformants but only one-fourth the level produced by induced T. fusca. A 30-kilodalton peptide with activity against both Remazol brilliant blue xylan and xylan was produced in S. lividans transformants that carried the 2.1-kilobase SalI fragment of T. fusca DNA and was not produced by control transformants. T. fusca cultures were found to contain a xylanase of a similar size that was induced by growth on xylan or Solka Floc. Antiserum directed against supernatant proteins isolated from a Solka Floc-grown T. fusca culture inhibited the xylanase activity of S. lividans transformants. The cloned T. Fusca xylanase gene was expressed at about the same level in S. lividans grown in minimal medium containing either glucose, cellobiose, or xylan. The xylanase bound to and hydrolyzed insoluble xylan. The cloned xylanase appeared to be the same as the major protein in xylan-induced T.fusca culture supernatants, which also contained at least three additional minor proteins with xylanase activity and having apparent molecular masses of 43, 23, and 20 kilodaltons.

• DOE Contract Number: FG02-84ER13233
• OSTI ID: 7192165
• Journal Information: Journal of Bacteriology; (USA), Journal Name: Journal of Bacteriology; (USA) Vol. 171:6; ISSN JOBAA; ISSN 0021-9193
• Country of Publication: United States
• Language: English

Related Subjects

550200* -- Biochemistry
59 BASIC BIOLOGICAL SCIENCES
BACTERIA
BACTERIOPHAGES
CLONING
DNA
DNA HYBRIDIZATION
DNA-CLONING
ENZYMES
ESCHERICHIA COLI
GENE REGULATION
GLYCOSYL HYDROLASES
HYBRIDIZATION
HYDROLASES
MICROORGANISMS
NUCLEIC ACIDS
O-GLYCOSYL HYDROLASES
ORGANIC COMPOUNDS
PARASITES
RECOMBINANT DNA
STREPTOMYCES
VIRUSES
XYLANASE