Cellulolytic Activity of Thermomonospora curvata: Nutritional Requirements for Cellulase Production

The use of a minimal medium for cellulase (C(1) and C(x)) production by Thermomonospora curvata increased extracellular C(1) activity (measured by rate of cotton fiber hydrolysis) 11-fold compared with the previously used yeast extract medium. Ground cotton fibers supported the highest cellulase production when compared to other soluble and insoluble carbohydrate sources. Maximal cellulase production occurred at 45 C, slightly less at 55 C, and was insignificant at 65 C (the highest temperature at which cellulase activity appeared stable). At a temperature of 55 C, an optimal pH of 8.0, and a cotton fiber concentration of 8 mg/ml, shake cultures of T. curvata degraded about 75% of the cellulose during the 10-day period.     

Cellulase biosynthesis in a catabolite repression-resistant mutant of Thermomonospora curvata.

A catabolite repression-resistant mutant of the thermophilic actinomycete Thermomonospora curvata was obtained by treatment with ethyl methanesulfonate and UV light. Cellulase biosynthesis was undiminished by glucose, 2-deoxyglucose, or alpha-methyl glucoside, which are potent repressors in the wild type. Intracellular cyclic AMP levels were higher in the mutant in both the absence and the presence of repressors.     

Cellulase biosynthesis during degradation of cellulose derivatives by Thermomonospora curvata

A variety of commercially used cellulose derivatives were compared with crystalline cellulose as substrates for induction of cellulase biosynthesis in the actinomycete Thermomonospora curvata. Cellulase induction during growth on uncoated cellophane was as rapid as that on crystalline cellulose, but on coated cellophanes, induction was delayed. Susceptibility to enzymatic attack determined the inductive potential of the substrate. Cellulose acetate was a poor substrate because of its extreme recalcitrance to attack. With other cellulose derivatives, soluble sugar accumulation caused a transient repression of cellulase biosynthesis, but the ratio of cellobiose (a cellulase inducer) to glucose (a cellulase repressor) was not a controlling factor. Crystalline cellulose yielded the lowest inducer/repressor sugar ratio (1.1:1 compared to 3.8–4.0:1 for cellulose derivatives), but supported the highest cellulase production. Glucose could not repress cellulase biosynthesis in the presence of cellobiose due to the strong preference for uptake of the disaccharide even by glucose-grown cells.     

Cellulase biosynthesis in a catabolite repression-resistant mutant of Thermomonospora curvata

A catabolite repression-resistant mutant of the thermophilic actinomycete Thermomonospora curvata was obtained by treatment with ethyl methanesulfonate and UV light. Cellulase biosynthesis was undiminished by glucose, 2-deoxyglucose, or alpha-methyl glucoside, which are potent repressors in the wild type. Intracellular cyclic AMP levels were higher in the mutant in both the absence and the presence of repressors.     

Purification and characterization of the major β-1,4-endoglucanase from Thermomonospora curvata

The major β-1,4-endoglucanase (EG) of the thermophilic actinomycete, Thermomonospora curvata , contributed over 80% of the total EG activity recovered from cell-free culture fluid after growth on cellulose. The enzyme was purified to electrophoretic homogeneity by ammonium sulphate precipitation, ion-exchange chromatography and size exclusion HPLC. This monomeric enzyme had a specific activity of 750 IU mg⁻¹ when assayed with 2.5% (w/v) carboxymethyl cellulose (CMC) at 70°C, pH 6.0. Highest activity was observed on CMC with a degree of polymerization of 3200. The EG was stable for 48 h at 60°C, pH 6.0 and had a half-life of 30 min at 80°C; temperature and pH optima were 70–73°C and 6.0–6.5, respectively. The mol. wt was 100000 and the pI was 4.0. The K _m and V _max values were 7.33 mg ml⁻¹ and 833 μmol min⁻¹, respectively. EG activity was inhibited by Fe^{2 +}, Hg^{2 +}, Ag⁺ and Pb^{2 +}, and enhanced by dithiothreitol and Zn^{2 +}. The first 12 amino acid residues at the N -terminus were: Asp-Glu-Val-Asp-Glu-Ile-Arg-Asn-Gly-Asp-Phe-Ser. Glutamic and aspartic acid constituted 24% of the total amino acid composition; no amino sugar was found.     

Production, purification, and characterization of alpha-amylase from Thermomonospora curvata.

Thermomonospora curvata produces an extracellular alpha-amylase. Maximal amylase production by cultures in a starch-mineral salts medium occurred at pH 7.5 and 53 degrees C. The crude enzyme was unstable to heating (65 degrees C) at pH 4 to 6, and was activated when heated at pH 8. The enzyme was purified 66-fold with a 9% yield and appeared homogeneous on discontinuous gel electrophoresis. The pH and temperature optima for activity of the purified enzyme were 5.5 to 6.0 and 65 degrees C. The molecular weight was calculated to be 62,000. The Km for starch was 0.39 mg/ml. The amylolytic pattern consisted of a mixture of maltotetraose and maltopentaose.     

Production, purification, and characterization of alpha-amylase from Thermomonospora curvata.

Thermomonospora curvata produces an extracellular alpha-amylase. Maximal amylase production by cultures in a starch-mineral salts medium occurred at pH 7.5 and 53 degrees C. The crude enzyme was unstable to heating (65 degrees C) at pH 4 to 6, and was activated when heated at pH 8. The enzyme was purified 66-fold with a 9% yield and appeared homogeneous on discontinuous gel electrophoresis. The pH and temperature optima for activity of the purified enzyme were 5.5 to 6.0 and 65 degrees C. The molecular weight was calculated to be 62,000. The Km for starch was 0.39 mg/ml. The amylolytic pattern consisted of a mixture of maltotetraose and maltopentaose.     

Effects of the detergent Tween 80 on Thermomonospora curvata

Tween 80 (0.1%, v/v) added to Thermomonospora curvata growing in minimal medium caused a transient lowering of the dry cell mass, decreased the optimal growth temperature of the thermophile from 62 to 54°C, and increased extracellular esterase activity. Cells grown in the presence of Tween 80 had decreased concentrations of branched chain fatty acids and increased concentrations of oleic acid. The detergent removed surface protuberances from mycelia and increased the liberation of enzymes active against crystalline cellulose, but did not stimulate liberation of enzymes active against carboxymethylcellulose, starch or pectin.     

Extracellular and cell-associated forms of beta-glucosidase in Thermomonospora curvata

The thermophilic actinomycete, Thermomonospora curvata , released 16-times the beta-glucosidase when grown on protein-extracted lucerne fibre compared with growth on cellobiose or purified cellulose. The intracellular and extracellular betaglucosidases had the same mol. wts (66 kD), but the extracellular enzyme had higher affinities for both p -nitrophenyl glucoside and cellobiose and was more resistant to thermal inactivation.     

Simple Dye Release Assay for Determining Cellulolytic Activity of Fungi

A colorimetric assay, based on the release of dye from a paper substrate, is described as a simple and rapid means of detecting cellulolytic activity. It is readily adaptable to rapidly screening in situ large numbers of fungal liquid cultures. Since it is a nondestructive assay, samples can be recovered for later use.     

Purification and Cooperative Activity of Enzymes Constituting the Xylan-Degrading System of Thermomonospora fusca

The thermophilic actinomycete Thermomonospora fusca produced endoxylanase, α-arabinofuranosidase, β-xylosidase, and acetyl esterase activities maximally during growth on xylan. Growth yields on glucose, xylose, or arabinose were comparable, but production of endoxylanase and β-xylosidase was not induced on these substrates. The crude xylanase activity was thermostable and relatively resistant to end product inhibition by xylobiose and xylan hydrolysis products. Six proteins with xylanase activity were identified by zymogram analysis of isoelectric focusing gels, but only a 32-kDa protein exhibiting three isomeric forms could be purified by fast protein liquid chromatography. Endoglucanases were also identified in carboxymethylcellulose-grown cultures, and their distinction from endoxylanases was confirmed. α-Arabinofuranosidase activity was due to a single dimeric protein of 92 kDa, which was particularly resistant to end product inhibition by arabinose. Three bands of acetyl esterase activity were detected by zymogram analysis, and there was evidence that these mainly consisted of an intracellular 80-kDa protein secreted to yield active 40-kDa subunits in the culture supernatant. The acetyl esterases were found to be responsible for acetyl xylan esterase activity in T. fusca, in contrast to the distinction proposed in some other systems. The addition of purified βxylosidase to endoxylanase increased the hydrolysis of xylan, probably by relieving end product inhibition. The enhanced saccharification of wheat straw caused by the addition of purified α-arabinofuranosidase to T. fusca endoxylanase suggested a truly synergistic relationship, in agreement with proposals that arabinose side groups on the xylan chain participate in cross-linking within the plant cell wall structure.     

Production and activity of Thermomonospora curvata cellulases on protein-extracted lucerne fibers

Summary Protein-extracted lucerne fibre (PELF) was evaluated as a carbon/energy source for cellulase production by Thermomonospora curvata and as a substrate for enzymatic conversion to soluble sugars. In shake cultures grown at 53°C, pH 8, maximal cellulase (50 endoglucanase IU and 0.7 filter paper IU/ml) was attained at a PELF concentration of 1.3% (w/v) in a vitamin-mineral salts medium buffered with 0.05 M concentrations of phosphate and N-2-hydroxyethylpiperazine-N-2-ethanesulfonate. Multiple endoglucanase enzymes were produced. The major form had an apparent molecular weight of 22 000 and K _m values of 0.23% and 0.56% for carboxymethylcellulose and PELF respectively. NaOH treatment of PELF increased its susceptibility to enzymatic hydrolysis. During enzymatic hydrolysis of NaOH-treated PELF (60°C, pH 6.1, 24h) two-thirds of total fibre carbohydrate was solubilized as cellobiose, melibiose, cellopentaose, cellotetraose, xylose and glucose in descending order of concentration.     

Assay,Purification, and Partial Characterization of Choline Monooxygenase from Spinach

The osmoprotectant glycine betaine is synthesized via the path-way choline -> betaine aldehyde -> glycine betaine. In spinach (Spinacia oleracea), the first step is catalyzed by choline monooxygenase (CMO), and the second is catalyzed by betaine aldehyde dehydrogenase. Because betaine aldehyde is unstable and not easily detected, we developed a coupled radiometric assay for CMO. [14C]Choline is used as substrate; NAD+ and betaine aldehyde dehydrogenase prepared from Escherichia coli are added to oxidize [14C]betaine aldehyde to [14C]glycine betaine, which is isolated by ion exchange. The assay was used in the purification of CMO from leaves of salinized spinach. The 10-step procedure included polyethylene glycol precipitation, polyethyleneimine precipitation, hydrophobic interaction, anion exchange on choline-Sepharose, dimethyldiethanolamine-Sepharose, and Mono Q, hydroxyapatite, gel filtration, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Following gel filtration, overall purification was about 600-fold and recovery of activity was 0.5%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a polypeptide with a molecular mass of 45 kD. Taken with the value of 98 kD estimated for native CMO (R. Brouquisse, P. Weigel, D. Rhodes, C.F. Yocum, A.D. Hanson [1989] Plant Physiol 90: 322-329), this indicates that CMO is a homodimer. CMO preparations were red-brown, showed absorption maxima at 329 and 459 nm, and lost color upon dithionite addition, suggesting that CMO is an iron-sulfur protein.     

Effect of carbon source on growth temperature and fatty-acid composition in Thermomonospora curvata

The growth-temperature range of the actinomycete, Thermomonospora curvata, was influenced by the nature of the soluble carbon sources used, which were derived from cellulose, pectin, starch and xylan. This thermophile had the broadest (38 to 65°C) and narrowest (42 to 59°C) temperature range during growth on cellobiose (from cellulose) and 4-deoxy-Lxxx-threo-t-hexoseulose uronic acid (from pectin), respectively. This substrate-temperature interaction was accompanied by changes in cellular fatty acids: uronic-acid-grown cells had relatively low amounts of branched chain fatty acids (particularly iso-16:0) and high amounts of monounsaturated fatty acids (particularly cis-18:1) compared with cells grown on any other substrate. Moreover, uronic-acid-grown cells could not respond to increased growth temperature by altering the ratio of branched chain fatty acids to straight chain fatty acids.F.J. Stutzenberger is with the Department of Microbiology, Clemson University, Clemson, SC 29634-1909, USA; T.C. Jenkins is with the Department of Animal, Dairy and Veterinary Sciences at the same university.     

Molecular characterization of the Thermomonospora curvata aglA gene encoding a thermotolerant alpha-1,4-glucosidase

The cloning, sequencing and structural characterization of a gene encoding a thermostable alpha-1,4-glucosidase from Thermomonospora curvata is described. DNA sequence analysis revealed four open reading frames designated aglA, aglR, aglE and aglF. The aglA gene encodes a thermostable alpha-1,4-glucosidase from T. curvata and is situated between two genes, aglR and aglE. Genes aglA, aglE and aglF are transcribed in the same direction, while aglR is transcribed in the opposite direction. By comparing the amino acid sequence of the alpha-1,4-glucosidase from T. curvata with other alpha-glucanases, it appears that the enzyme is a member of the alpha-amylase family. The proteins of this family have an (alpha/beta)8 barrel super secondary structure. The topology of the alpha-1,4-glucosidase was predicted by computer-assisted analysis. The topology of the secondary structures of the alpha-1,4-glucosidase resembles the structure of barley alpha-amylase, but the primary structure resembles most closely the oligo-1,6-glucosidase from Bacillus cereus. Putative catalytic residues (D221, E281 and D343) and calcium binding residues (N116, E179, D191, H224 or G225) are proposed.     

分选酶A(SrtA)的GST融合表达、纯化及活性测定

构建GST/金黄色葡萄球菌分选酶A (SrtA)的原核表达载体,在大肠杆菌中表达、纯化分选酶,并利用展示在酵母表面的底物检测分选酶的活性.以pMD20-SrtA为模板,PCR扩增得到SrlA△N59基因,经BamH I和Xho I双酶切,连接到原核表达栽体pGEX-4T-1中,构建重组表达栽体pGEX-SrtA△N59,转化大肠杆菌BL21( DE3),IPTG诱导表达,GST亲和层析分离纯化得到SrtA△N59,与展示在酵母表面的底物序列QALPETGEE-linker-EGFP作用,产生游离的EGFP,通过酶标仪检测EGFP荧光强度确定分选酶的活性.结果显示,重组表达栽体pGEX-SrtA△N59经IPTG诱导,表达出相对分子质量约为42 kD的融合蛋白,SDS-PAGE分析,该融合蛋白是以可溶形式表达.分离纯化得到的分选酶与底物作用,其荧光强度由568.66±12.14增加至921.43±13.02.以上结果表明,成功构建了重组表达裁体pGEX-SrtA△N59,并在大肠杆菌中获得了可溶表达的有活性的分选酶.     

Fusion of Carbohydrate Binding Modules to Bifunctional Cellulase to Enhance Binding Affinity and Cellulolytic Activity

Bifunctional cellulase (glycoside hydrolase 5, GH5) from Bacillus sp. D04 having both endo- and exoglucanase activities was fused with two types of carbohydrate binding modules (CBMs). CBM3 from Bacillus sp. D04 and CBM9 from Thermotoga maritima Xyn10A were added to GH5 to hydrolyze microcrystalline cellulose (Avicel) as well as water-soluble cellulose (carboxymethyl cellulose, CMC). The optimum temperature of GH5 was 50oC, while it increased to 60oC for the fusion GH5-CBM3 and GH5-CBM9, indicating that addition of CBM increased the thermostability of the enzyme. Addition of CBM3 and CBM9 enhanced the GH5 affinity (K_M), for which K_M decreased from 104 to 33.9 ~ 35.1 mg/mL for CMC, and from 115 to 55.5 ~ 80.3 mg/mL for Avicel, respectively. The catalytic efficiency (k_cat/K_M) also increased from 4.80 to 5.36 ~ 6.46 (mL/mg)/sec for CMC, and from 1.77 to 2.40 ~ 4.45 (mL/mg)/sec for Avicel, respectively, by addition of CBM3 and CBM9.     

Component-specific stimulation of cellulase secretion in Thermomonospora curvata by the surfactant Tween 80

The non-ionic surfactant, Tween 80, stimulated the secretion of extracellular proteins by 35–140% in Thermomonospora curvata during growth on a variety of substrates. Cellulase secretion was also stimulated but fractionation of extracellular proteins by ion-exchange high performance liquid chromatography showed that this stimulation was largely confined to a single enzymatic component (or group of closely related components) active against crystalline cellulose. The surfactant's effect was more pronounced during growth on cellobiose octaacetate than on the soluble sugar, cellobiose, or on crystalline cellulose.     

Cloning of a thermostable alpha-amylase gene from Thermomonospora curvata and its expression in Streptomyces lividans

The gene from Thermomonospora curvata CCM 3312 coding for thermostable alpha-amylase (tam) has been cloned in Streptomyces lividans TK 24 and localized to a 2.6 kb HindIII-BamHI fragment of DNA. The data presented here show that the tam gene is expressed at a high level in S. lividans and that the protein is efficiently excreted.