Cloning of the Thermomonospora fusca Endoglucanase E2 gene in Streptomyces lividans: Affinity purification and functional domains of the cloned gene product

Journal Article · Sat Oct 01 00:00:00 EDT 1988 · Applied and Environmental Microbiology; (USA)

OSTI ID:5035982

Ghangas, G S; Wilson, D B ^[1]

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

Thermomonospora fusca YX grown in the presence of cellulose produces a number of {beta}-1-4-endoglucanases, some of which bind to microcrystalline cellulose. By using a multicopy plasmid, pIJ702, a gene coding for one of these enzymes (E2) was cloned into Streptomyces lividans and then mobilized into both Escherichia coli and Streptomyces albus. The gene was localized to a 1.6-kilobase PvuII-ClaI segment of the originally cloned 3.0-kilobase SstI fragment of Thermomonospora DNA. The culture supernatants of Streptomyces transformants contain a major endoglucanase that cross-reacts with antibody against Thermomonospora cellulase E2 and has the same molecular weight (43,000) as T. fusca E2. This protein binds quickly and tightly to Avicel. It also binds to filter paper but at a slower rate than to Avicel. Several large proteolytic degradation products of this enzyme generated in vivo lose the ability to bind to Avicel and have higher activity on carboxymethyl cellulose than the native enzyme. Other smaller products bind to Avicel but lack activity. A weak cellobiose-binding site not observed in the native enzyme was present in one of the degradation products. In E. coli, the cloned gene produced a cellulase that also binds tightly to Avicel but appeared to be slightly larger than T. fusca E2. The activity of intact E2 from all organisms can be inactivated by Hg{sup 2+} ions. Dithiothreitol protected against Hg{sup 2+} inactivation and reactivated both unbound and Avicel-bound Hg{sub 2+}-inhibited E2, but at different rates.

• DOE Contract Number: FG02-84ER13233
• OSTI ID: 5035982
• Journal Information: Applied and Environmental Microbiology; (USA), Vol. 54:10; ISSN 0099-2240
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
BACTERIA
ENZYME ACTIVITY
CELLULASE
DNA-CLONING
AFFINITY
CELLULOLYTIC ACTIVITY
CELLULOSE
ENZYME REACTIVATION
INACTIVATION
MERCURY COMPOUNDS
PLASMIDS
PROTEOLYSIS
PURIFICATION
CARBOHYDRATES
CELL CONSTITUENTS
CHEMICAL REACTIONS
CLONING
DECOMPOSITION
DNA HYBRIDIZATION
ENZYMES
GLYCOSYL HYDROLASES
HYBRIDIZATION
HYDROLASES
MICROORGANISMS
O-GLYCOSYL HYDROLASES
ORGANIC COMPOUNDS
POLYSACCHARIDES
SACCHARIDES
550400* - Genetics