Extracellular Enzyme Activities during Lignocellulose Degradation by Streptomyces spp.: A Comparative Study of Wild-Type and Genetically Manipulated Strains

Abstract

The wild-type ligninolytic actinomycete Streptomyces viridosporus T7A and two genetically manipulated strains with enhanced abilities to produce a water-soluble lignin degradation intermediate, an acid-precipitable polymeric lignin (APPL), were grown on lignocellulose in solid-state fermentation cultures. Culture filtrates were periodically collected, analyzed for APPL, and assayed for extracellular lignocellulose-catabolizing enzyme activities. Isoenzymes were analyzed by polyacrylamide gel electrophoresis and activity staining on the gels. Two APPL-overproducing strains, UV irradiation mutant T7A-81 and protoplast fusion recombinant SR-10, had higher and longer persisting peroxidase, esterase, and endoglucanase activities than did the wild-type strain T7A. Results implicated one or more of these enzymes in lignin solubilization. Only mutant T7A-81 had higher xylanase activity than the wild type. The peroxidase was induced by both lignocellulose and APPL. This extracellular enzyme has some similarities to previously described ligninases in fungi. This is the first report of such an enzyme in Streptomyces spp. Four peroxidase isozymes were present, and all catalyzed the oxidation of 3,4-dihydroxyphenylalanine, while one also catalyzed hydrogen peroxide-dependent oxidation of homoprotocatechuic acid and caffeic acid. Three constitutive esterase isozymes were produced which differed in substrate specificity toward ot-naphthyl acetate and 4x-naphthyl butyrate. Three endoglucanase bands, which also exhibited a low level of xylanase activity, were identified on polyacrylamide gels as was one xylanasespecific band. There were no major differences in the isoenzymes produced by the different strains. The probable role of each enzyme in lignocellulose degradation is discussed.

SIMULATED ATMOSPHERIC NO3− DEPOSITION INCREASES SOIL ORGANIC MATTER BY SLOWING DECOMPOSITION

“…Because slow-growing white-rot basidiomycetes are poor competitors for labile organic substrates, a reduction in their ability to metabolize lignin could decrease their abundance or activity. Such a response would reduce lignolytic enzyme activity in the forest floor because both brown-rot basidiomycetes and actinomycetes produce smaller amounts of these extracellular enzymes than do white-rot basidiomycetes (Ramachandra et al 1987, D'Souza et al 1996. The shift in microbial community composition and activity described above is consistent with the decline in phenol oxidase and peroxidase activity, the increase in forest floor mass, and the greater production of phenolic DOC induced by experimental NO 3 À (DeForest et al 2004, Smemo et al 2007).…”

Enhanced Expression and Hydrogen Peroxide Dependence of Lignin Peroxidase from Streptomyces viridosporus T7A

“…S. viridosporus T7A secretes four isoforms of lignin peroxidase (Ramachandra et al, 1987). Of the four, isoform ALiP‐P3 has the widest known substrate range (Ramachandra et al, 1987).…”

“…S. viridosporus T7A secretes four isoforms of lignin peroxidase (Ramachandra et al, 1987). Of the four, isoform ALiP‐P3 has the widest known substrate range (Ramachandra et al, 1987). S. viridosporus T7A lignin peroxidase was previously obtained from a yeast‐extract‐based medium, and concentrating LiP from this medium using ultrafiltration (Lodha et al, 1991) is relatively easy because all of the medium components are soluble in water.…”

“…The Gram‐positive bacterium, Streptomyces viridosporus T7A, also produces a variety of enzymes which play a role in lignin degradation. These extracellular enzymes include various peroxidases, cellulases, esterases, and endoglucanases (Adhi et al, 1989; Borgmeyer and Crawford, 1985; Crawford et al, 1983; Ramachandra et al, 1987). S. viridosporus T7A produces four extracellular lignin peroxidase isoforms, and although all require hydrogen peroxide as an electron donor, each isoform has a different substrate range (Ramachandra et al, 1988).…”

Molecular biology of xylan degradation

“…Six proteins exhibiting xylanase activity were identified and found to be different from enzymes with endoglucanase activity. Thus in T. fusca, endoxylanases are distinct from endoglucanases, unlike enzymes from some other actinomycetes and thermophilic bacteria [71,72].…”