Cloning, expression in Streptomyces lividans and biochemical characterization of a thermostable endo-β-1,4-xylanase of Thermomonospora alba UL JB1 with cellulose-binding ability

Abstract

Several thermophilic actinomycetes were isolated from urban solid waste. One of them, Thermomonospora alba ULJB1, showed a broad degradative activity on xylan, cellulose, starch and other polymers. Xylanase and cellulase activities were quantified and compared with those Thermomonospora fusca. Genes encoding two different endo-beta-1,4-xylanase were cloned from T. alba ULJB1. One of them, xylA, was sequenced, subcloned and overexpressed in Streptomyces lividans. It encodes a protein of 482 amino acids with a deduced molecular mass of 48,456 Da. The protein contains a 38-amino-acid leader peptide with six Arg+ residues in its amino-terminal end, a catalytic domain and a cellulose-binding domain connected by a linker region rich in proline and glycine. The XylA protein was purified to near homogeneity from S. lividans/XylA cultures. Two forms of the extracellular xylanase, of 48 kDa and 38 kDa, were produced that differed in their cellulose-binding ability. The 48-kDa protein showed a strong binding to cellulose whereas the 38-kDa form did not bind to this polymer, apparently because of the removal during processing of the cellulose-binding domain. Both forms were able to degrade xylans form different origins but not lichenam or carboxymethylcellulose. The major degradation product was xylobiose with traces of xylose. The xylanase activity was thermostable, showing a good activity up to 95 degrees C, and had broad pH stability in the range from pH 4.0 to pH 10.0.

Characterization and Cloning of CelR, a Transcriptional Regulator of Cellulase Genes from Thermomonospora fusca

“…The same palindrome was found in the regulatory regions of cellulase genes from different streptomycetes, in particular, the cenC gene from Streptomyces strain KSM9 (13), the celA genes from Streptomyces halstedii (14) and Streptomyces lividans (15), and also in the cel1 gene from Streptomyces reticuli (16), where it serves as the operator for a repressor protein (17). The same sequence was also found in a xylanase gene from Thermomonospora alba (18). The occurrence of this site in cellulase and xylanase genes from different species may indicate its special role in regulating carbohydrate catabolism in streptomycetes and other high GC Gram-positive bacteria.…”

Thermostability Improvement of a Streptomyces Xylanase by Introducing Proline and Glutamic Acid Residues

“…To obtain thermostable xylanases, one strategy is to search for novel xylanases from extremophiles. A number of thermostable GH10 xylanases have been reported for thermophilic Thermotoga maritima MSB8 (90°C) (10), Thermotoga petrophila RKU-1 (90°C) (11), and Thermobifida alba UL JB1 (80°C) (12) and for acidomesophilic Bispora sp. strain MEY-1 (85°C) (13).…”

Genome Sequence and Analysis of the Soil Cellulolytic ActinomyceteThermobifida fuscaYX

“…Using classical biochemical methods, six different cellulases have been identified: four endocellulase genes (7,12,15) and two exocellulases (18,53). In addition, an intracellular ␤-glucosidase that degrades cellobiose to glucose (46), an extracellular xyloglucanase (17), two secreted xylanases, and a GH family 81 ␤-1,3-glucanase (4,11,16,22,31) have been cloned and characterized. Secreted cellulases have great biotechnological promise for utilization in the degradation of agricultural products and waste to produce sugars that can be subsequently converted to ethanol.…”