Cloning, sequence analysis, and expression in Escherichia coli of a gene coding for a. beta. -mannanase from the extremely thermophilic bacterium Caldocellum saccharolyticum

Journal Article · Fri Mar 01 00:00:00 EST 1991 · Applied and Environmental Microbiology; (United States)

OSTI ID:5317399

Luethi, E; Jasmat, N B; Grayling, R A; Love, D R; Bergquist, P L ^[1]

---

1. Univ. of Auckland (New Zealand)

A {lambda} recombinant phage expressing {beta}-mannanase activity in Escherichia coli has been isolated from a genomic library of the extremely thermophilic anaerobe Caldocellum saccharolyticum. The gene was cloned into pBR322 on a 5-kb BamHI fragment, and its location was obtained by deletion analysis. The sequence of a 2.1-kb fragment containing the mannanase gene has been determined. One open reading frame was found which could code for a protein of M{sub r} 38,904. The mannanase gene (manA) was overexpressed in E. coli by cloning the gene downstream from the lacZ promoter of pUC18. The enzyme was most active at pH 6 and 80 C and degraded locust bean gum, guar gum, Pinus radiata glucomannan, and konjak glucomannan. The noncoding region downstream from the mannanase gene showed strong homology to celB, a gene coding for a cellulase from the same organism, suggesting that the manA gene might have been inserted into its present position on the C. saccharolyticum genome by homologous recombination.

• OSTI ID: 5317399
• Journal Information: Applied and Environmental Microbiology; (United States), Vol. 57:3; ISSN 0099-2240
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
BACTERIA
GENETIC ENGINEERING
GLYCOSYL HYDROLASES
GENES
DNA SEQUENCING
DNA-CLONING
ENZYME ACTIVITY
RECOMBINANT DNA
THERMOPHILIC CONDITIONS
BIOTECHNOLOGY
CLONING
DNA
DNA HYBRIDIZATION
ENZYMES
HYBRIDIZATION
HYDROLASES
MICROORGANISMS
NUCLEIC ACIDS
ORGANIC COMPOUNDS
STRUCTURAL CHEMICAL ANALYSIS
550200* - Biochemistry