Cloning, sequencing, and characterization of a membrane-associated Prevotella ruminicola B(1)4 beta-glucosidase with cellodextrinase and cyanoglycosidase activities.

Prevotella ruminicola B(1)4 is a gram-negative, anaerobic gastrointestinal bacterium. A 2.4-kbp chromosomal fragment from P. ruminicola encoding an 87-kDa aryl-glucosidase (CdxA) with cellodextrinase activity was cloned into Escherichia coli DH5 alpha and sequenced. CdxA activity was found predominantly in the membrane fraction of both P. ruminicola and E. coli, but P. ruminicola localized the protein extracellularly while E. coli did not. The hydrolase had the highest activity on cellodextrins (3.43 to 4.13 mumol of glucose released min-1 mg of protein-1) and p-nitrophenyl-beta-D-glucoside (3.54 mumol min-1 mg of protein-1). Significant activity (70% of p-nitrophenyl-beta-D-glucoside activity) was also detected on arbutin and prunasin. Less activity was obtained with cellobiose, amygdalin, or gentiobiose. CdxA attacks cellodextrins from the nonreducing end, releasing glucose units, and appears to be an exo-1,4-beta-glucosidase (EC 3.2.1.74) which also is able to attack beta-1,6 linkages. Comparison of the deduced amino acid sequence with other glycosyl-hydrolases suggests that this enzyme belongs to family 3 (B. Henrissat, Biochem. J. 280:309-316, 1991). On the basis of this sequence alignment, the catalytic residues are believed to be Asp-275 and Glu-265. This is the first report of a cloned ruminal bacterial enzyme which can cleave cyanogenic plant compounds and which may therefore contribute to cyanide toxicity in ruminants.     

Isolation and characterization of a 1,4-beta-D-glucan glucohydrolase from the yeast, Torulopsis wickerhamii

1,4-beta-D-Glucan glucohydrolase (exo-1,4-beta-D-glucosidase) (EC 3.2.1.74) was isolated from growth supernatants of Torulopsis wickerhamii and was subjected to hydrodynamic, optical (CD), and kinetic analysis after purification to homogeneity by ammonium sulfate precipitation, size exclusion chromatography, ion exchange chromatography, and isopycnic banding centrifugation in cesium chloride. The last step was found to separate the enzyme from strongly associating, high molecular weight polysaccharide. Enzyme homogeneity was established by isoelectric focusing, sodium dodecyl sulfate-gel electrophoresis, and analytical high performance size exclusion chromatography using dual detection. The native exo-1,4-beta-D-glucosidase was found to be a dimer of 151,000 +/- 21,100 daltons by high performance size exclusion chromatography and 143,600 +/- 1,800 daltons by sedimentation equilibrium. The enzyme has a 12% linked carbohydrate content (mostly mannose) and no essential metal ions. Hydrolysis of p-nitrophenyl-beta-D-glucopyranoside was found to be optimal at pH 4.25 and 50 degrees C. The enzyme was found to produce beta-D-glucose from cellodextrins (indicating retention of anomeric configuration during hydrolysis) and demonstrated depolymerization from the non-reducing polymer terminus. The enzyme followed competitive type inhibition with p-nitrophenyl-beta-D-glucopyranoside as substrate and demonstrated high values of Ki for D-glucose and D-cellobiose inhibition (190 and 230 mM, respectively). The exo-1,4-beta-D-glucosidase was found to hydrolyze cellotetraose more rapidly than D-cellobiose and aryl-beta-D-glycosides more rapidly than all other substrates. Low levels of activity were found for the polymeric substrates beta-glucan (yeast cell walls), Avicel, and Walseth cellulose. Although this enzyme demonstrates broad disaccharide substrate specificity, a characteristic common to beta-D-glucosidases from many sources, the ability to hydrolyze higher cellodextrins more rapidly than cellobiose renders this enzyme the first exo-1,4-beta-D-glucosidase purified from yeast.     

Purification and properties of the endoglucanase C of Clostridium thermocellum produced in Escherichia coli

The celC gene, which codes for a new endoglucanase of Clostridium thermocellum, termed endoglucanase C, was found to be expressed when cloned in Escherichia coli. The enzyme was purified to electrophoretic homogeneneity from E. coli and its biochemical properties were studied. It differs from the previously studied endoglucanases A and B. In particular, endoglucanase C displays features common to endo- and exoglucanases, since it had a high activity on carboxymethylcellulose and on p-nitrophenyl-beta-D-cellobioside where only the agluconic bond was split. In addition, the enzyme was able to release cellobiose units from G3, G4 and G5 cellodextrins. Endoglucanase C was characterized by Western blot in a culture supernatant from C. thermocellum grown on cellulose, using an antiserum raised against the enzyme produced by E. coli.     

Expression and characterization of beta-1,4-galactosyltransferase from Neisseria meningitidis and Neisseria gonorrhoeae

The lgtB genes that encode beta-1,4-galactosyltransferases from Neisseria meningitidis ATCC 13102 and gonorrhoeae ATCC 31151 were isolated by a polymerase chain reaction using the pfu DNA polymerase. They were expressed under the control of lac and T7 promoters in Escherichia coli M15 and BL21 (DE3). Although the genes were efficiently expressed in E. coli M15 at 37 degrees C (33 kDa), most of the beta-1,4-galactosyltransferases that were produced were insoluble and proteolysed into enzymatically inactive polypeptides that lacked C-terminal residues (29.5 kDa and 28 kDa) during the purification steps. When the temperature of the cell growth was lowered to 25 degrees C, however, the solubility of the beta-1,4-galactosyltransferases increased substantially. A stable N-terminal his-tagged recombinant enzyme preparation could be achieved with E. coli BL21 (DE3) that expressed lgtB. Therefore, the cloned beta-1,4-galactosyltransferases were expressed under the control of the T7 promoter in E. coli BL21 (DE3), mostly to the soluble form at 25 degrees C. The proteins were easily purified to homogeneity by column chromatography using Ni-NTA resin, and were found to be active. The galactosyltransferases exhibited pH optimum at 6.5-7.0, and had an essential requirement for the Mn(+2) ions for its action. The Mg(+2) and Ca(+2) ions showed about half of the galactosyltransferase activities with the Mn(+2) ion. In the presence of the Fe(+2) ion, partial activation was observed with the beta-1,4-galactosyltransferase from N. meningitidis (64% of the enzyme activity with the Mn(+2) ion), but not from N. gonorrhoeae. On the other hand, the N(+2), Zn(+2), and Cu(+2) ions could not activate the beta-1,4- galactosyltransferase activity. The inhibited enzyme activity with the Ni(+2) ion was partially recovered with the Mn(+2) ion, but in the presence of the Fe(+2), Zn(+2), and Cu(+2) ions, the Mn(+2) ion could not activate the enzyme activities. Also, the beta-1,4-galactosyltransferase activity was 1.5-fold stimulated with the non-ionic detergent Triton X-100 (0.1-5 percent).     

The action on cellulose and its derivatives of a purified 1,4-beta-glucanase from Trichoderma koningii.

The specific properties have been examined of the 1,4-beta-glucanase component of Trichoderma koningii that participates in an early and effective stage of random breakdown of native cellulose to short fibres. The enzyme was purified and freed from associated components of the cellulase complex (particularly beta-glucosidase) that interfere with, and complicate interpretation of, the action of such enzymes. Purification increased the specific activity 25-fold over culture filtrates; the enzyme hydrolysed CM-cellulose faster than the purified beta-glucosidase from the same organism hydrolysed any of its substrates (cellobiose or cellodextrins). The specificity of the glucanase was directed towards soluble derivatives of cellulose, CM-cellulose and cellodextrins, and not to insoluble cellulose or alpha-linked polymers. The approximate Km was 2.5 mg of CM-cellulose . ml-1 at 37 degrees C at the optimum pH, 5.5, where enzymic activity was maximal with 6--7 mg of CM-cellulose . ml-1 and inhibited by higher concentrations. The temperature optimum was 60 degrees C. The glucanase attacked larger cellodextrins (cellohexaose to cellotetraose, in that order) much more readily than smaller dextrins (cellobiose and cellotriose) and released a mixture of products, glucose up to cellopentaose, which was quantitatively determined after chromatography on charcoal. Similar examination of hydrolysates of the reduced cellodextrins showed clearly the high specificity of the enzyme for the central bond of its natural substrates (the cellodextrins), whatever their chain length, and indicated the nature of the enzyme as an endoglucanase. Outer bonds shared a weaker, but similar, susceptibility to enzymic cleavage. Transferase activity was absent and no larger dextrins than the initial substrate were formed.     

Cloning, sequence analysis, and expression in Escherichia coli of the gene encoding monovalent cation-activated levodione reductase from Corynebacterium aquaticum M-13

The gene encoding (6R)-2,2,6-trimethyl-1,4-cyclohexanedione (levodione) reductase was cloned from the genomic DNA of the soil isolate bacterium Corynebacterium aquaticum M-13. The gene contained an open reading frame consisting of 801 nucleotides corresponding to 267 amino acid residues. The deduced amino acid sequence showed approximately 35% identity with other short chain alcohol dehydrogenase/reductase (SDR) superfamily enzymes. The probable NADH-binding site and three catalytic residues (Ser-Tyr-Lys) were conserved. The enzyme was sufficiently produced in recombinant Escherichia coli cells using an expression vector pKK223-3, and purified to homogeneity by two-column chromatography steps. The enzyme purified from E. coli catalyzed stereo- and regio-selective reduction of levodione, and was strongly activated by monovalent cations, such as K+, Na+, and NH4+, as was the case of that from C. aquaticum M-13. To our knowledge, this is the first sequencing report of a monovalent cation-activated SDR enzyme.     

Cloning and overproduction of biodegradative threonine deaminase from Escherichia coli W strain.

We have cloned the structural gene (tdcB) of biodegradative threonine deaminase from Escherichia coli W strain by utilizing the polymerase chain reaction. The JM109/pUCTDA strain, which was obtained by transforming E. coli JM109 with a vector plasmid (pUCTDA) containing the cloned tdcB gene, produced a large amount of the enzyme corresponding to more than 5% of the total soluble protein. Amino acid sequence analysis of this recombinant enzyme showed that the amino acid sequence is identical to the nucleotide-deduced sequence of biodegradative threonine deaminase from E. coli K-12.     

Purification and properties of a 1,3-1,4-beta-D-glucanase (lichenase, 1,3-1,4-beta-D-glucan 4-glucanohydrolase, EC 3.2.1.73) from Bacteroides succinogenes cloned in Escherichia coli.

A 1,3-1,4-beta-D-glucanase (lichenase, 1,3-1,4-beta-D-glucan 4-glucanohydrolase, EC 3.2.1.73) from Bacteroides succinogenes cloned in Escherichia coli was purified 600-fold by chromatography on Q-Sepharose and hydroxyapatite. The cloned enzyme hydrolysed lichenin and oat beta-D-glucan but not starch, CM(carboxymethyl)-cellulose, CM-pachyman, laminarin or xylan. The enzyme had a broad pH optimum with maximum activity at approx. pH 6.0 and a temperature optimum of 50 degrees C. The pH of elution from a chromatofocusing column for the cloned enzyme was 4.7 (purified) and 4.9 (crude) compared with 4.8 for the mixed-linkage beta-D-glucanase activity in B. succinogenes. The Mr of the cloned enzyme was estimated to be 37,200 by gel filtration and 35,200 by electrophoresis. The Km values estimated for lichenin and oat beta-D-glucan were 0.35 and 0.71 mg/ml respectively. The major hydrolytic products with lichenin as substrate were a trisaccharide (82%) and a pentasaccharide (9.5%). Hydrolysis of oat beta-D-glucan yielded a trisaccharide (63.5%) and a tetrasaccharide (29.6%) as the major products. The chromatographic patterns of the products from the cloned enzyme appear to be similar to those reported for the mixed-linkage beta-D-glucanase isolated from Bacillus subtilis. The data presented illustrate the similarity in properties of the cloned mixed-linkage enzyme and the 1,3-1,4-beta-D-glucanase from B. subtilis and the similarity with the 1,4-beta-glucanase in B. succinogenes.     

Properties of the exo-1,4-beta-xylosidase of Aspergillus niger 15

Properties of exo-1,4-beta-xylosidase from the fungus Aspergillus niger 15 were investigated. The enzyme was homogeneous during gel filtration, electrophoresis in polyacrylamide gel in the presence and absence of Na dodecyl sulfate, ultracentrifugation and isoelectric focusing. The enzyme had a temperature optimum at 70 degrees, pH optimum 3.8-4.0 for p-nitrophenyl-beta-D-xylopyranoside (p-NPX), was stable at pH 3-8, retained its 100% activity for 1 hour at 50 degrees and 42% activity at 60 degrees. Km was 0.23 mM for p-NPX and 0.67 mM for xylobiose. Xylose was a competitive inhibitor of exo-1,4-beta-xylodidase with Ki = 2.9 mM. The enzyme showed a transglycosilase activity. The aminoacid analysis of exo-1,4-beta-xylosidase showed that the enzyme molecule contained predominantly dicarboxylic and hydrophobic amino acids as well as serine. The enzyme contained no carbohydrates. Its activity was inhibited by p-chloromercury benzoate.     

Three‐Enzyme Phosphorylase Cascade for Integrated Production of Short‐Chain Cellodextrins

Cellodextrins are linear β‐1,4‐gluco‐oligosaccharides that are soluble in water up to a degree of polymerization (DP) of ≈6. Soluble cellodextrins have promising applications as nutritional ingredients. A DP‐controlled, bottom‐up synthesis from expedient substrates is desired for their bulk production. Here, a three‐enzyme glycoside phosphorylase cascade is developed for the conversion of sucrose and glucose into short‐chain (soluble) cellodextrins (DP range 3–6). The cascade reaction involves iterative β‐1,4‐glucosylation of glucose from α‐glucose 1‐phosphate (αGlc1‐P) donor that is formed in situ from sucrose and phosphate. With final concentration and yield of the soluble cellodextrins set as targets for biocatalytic synthesis, three major factors of reaction efficiency are identified and partly optimized: the ratio of enzyme activity, the ratio of sucrose and glucose, and the phosphate concentration used. The efficient use of the phosphate/αGlc1‐P shuttle for cellodextrin production is demonstrated and the soluble product at 40 g L^?1 is obtained under near‐complete utilization of the donor substrate offered (88 mol% from 200 mm sucrose). The productivity is 16 g (L h)^?1. Through a simple two‐step route, the soluble cellodextrins are recovered from the reaction mixture in ≥95% purity and ≈92% yield. Overall, this study provides the basis for their integrated production.     

Cloning,Sequence Analysis,and Expression in Escherichia coli of the Gene Encoding Monovalent Cation-activated Levodione Reductase from Corynebacterium aquaticum M-13

The gene encoding (6R)-2,2,6-trimethyl-1,4-cyclohexanedione (levodione) reductase was cloned from the genomic DNA of the soil isolate bacterium Corynebacterium aquaticum M-13. The gene contained an open reading frame consisting of 801 nucleotides corresponding to 267 amino acid residues. The deduced amino acid sequence showed approximately 35% identity with other short chain alcohol dehydrogenase/reductase (SDR) superfamily enzymes. The probable NADH-binding site and three catalytic residues (Ser-Tyr-Lys) were conserved. The enzyme was sufficiently produced in recombinant Escherichia coli cells using an expression vector pKK223-3, and purified to homogeneity by two-column chromatography steps. The enzyme purified from E. coli catalyzed stereo- and regio-selective reduction of levodione, and was strongly activated by monovalent cations, such as K⁺, Na⁺, and NH₄ ⁺, as was the case of that from C. aquaticum M-13. To our knowledge, this is the first sequencing report of a monovalent cation-activated SDR enzyme.     

一种新的乙酰高丝氨酸内酯水解酶基因的克隆和表达

目的 获得新的降解革兰阴性细菌数量阈值感应信号分子乙酰高丝氨酸内酯类化合物(AHL)的水解酶基因。方法 选择性富集和培养土壤中耐热细菌,抽取细菌总DNA作为模板,特异性聚合酶链反应扩增乙酰高丝氨酸内酯水解酶基因,进行克隆和DNA序列分析及原核表达。结果 得到1个新的AHL水解酶基因,该基因与已知基因的核苷酸序列和对应的氨基酸序列同源性最高分别为87％和94％。该基因在原核表达系统中表达,得到了与预期相对分子质鲢(Mr)一致的蛋白质。结论 证实乙酰高丝氨酸内酯水解酶广泛存在于环境微生物中。为进一步研究提供条件。     

Purification, cloning, and expression of an alpha/beta-galactoside alpha-2,3-sialyltransferase from a luminous marine bacterium, Photobacterium phosphoreum

A novel sialyltransferase, alpha/beta-galactoside alpha-2,3-sialyltransferase, was purified from the cell lysate of a luminous marine bacterium, Photobacterium phosphoreum JT-ISH-467, isolated from the Japanese common squid (Todarodes pacificus). The gene encoding the enzyme was cloned from the genomic library of the bacterium using probes derived from the NH(2)-terminal and internal amino acid sequences. An open reading frame of 409 amino acids was identified, and the sequence had 32% identity with that of beta-galactoside alpha-2,6-sialyltrasferase in Photobacterium damselae JT0160. DNA fragments that encoded the full-length protein and a protein that lacked the sequence between the 2nd and 24th residues at the NH(2) terminus were amplified by polymerase chain reactions and cloned into an expression vector. The full-length and truncated proteins were expressed in Escherichia coli, producing active enzymes of 0.25 and 305 milliunits, respectively, per milliliter of the medium in the lysate of E. coli. The truncated enzyme was much more soluble without detergent than the full-length enzyme. The enzyme catalyzed the transfer of N-acetylneuraminic acid from CMP-N-acetylneuraminic acid to disaccharides, such as lactose and N-acetyllactosamine, with low apparent K(m) and to monosaccharides, such as alpha-methyl-galactopyranoside and beta-methyl-galactopyranoside, with much lower apparent K(m). Thus, this sialyltransferase is unique and should be very useful for achieving high productivity in E. coli with a wide substrate range.     

Extracellular enzyme system utilized by the fungus Sporotrichum pulverulentum (Chrysosporium lignorum) for the breakdown of cellulose. 3. Purification and physico-chemical characterization of an exo-1,4-beta-glucanase.

An exo-1,4-beta-glucanase from culture solution of the rot fungus Sporotrichum pulverulentum (formerly called Chrysosporium lignorum) grown on powder cellulose as the sole carbon source has been extensively purified and characterized with respect to some physico-chemical properties. The purification has been carried out in a five-step procedure comprising chromatography on DEAE-Sephadex, gel filtration on polyacrylamide P-150, activation on a Dowex 2-X8 anion exchanger, chromatography on Concanavalin A-Sepharose and chromatography on SP-Sephadex. The purified enzyme was found to be pure and homogeneous by analytical polyacrylamide electrophoresis, by electrophoresis on dodecylsulphate gels and by analytical polyacrylamide electrophoresis, by electrophoresis on dodecylsulphate gels and by analytical isoelectric focusing. A single symmetrical peak was obtained with the free zone electrophoresis method. The purification factor is about 15 and the yield of exo-1,4-beta-glucanase activity 7%. After purification, the enzyme showed no viscosity-decreasing activity towards carboxymethyl-cellulose solutions. The exo-1,4-beta-glucanase was isoelectric at pH 4.3 (4 degrees C). A molecular weight of 48600 was calculated on the basis of a knowledge of the partial specific volume, ultracentrifugation data and the amino acid composition. The enzyme contained no carbohydrate.     

Thermus aquaticus ATCC 33923 amylomaltase gene cloning and expression and enzyme characterization: production of cycloamylose

The amylomaltase gene of the thermophilic bacterium Thermus aquaticus ATCC 33923 was cloned and sequenced. The open reading frame of this gene consisted of 1,503 nucleotides and encoded a polypeptide that was 500 amino acids long and had a calculated molecular mass of 57,221 Da. The deduced amino acid sequence of the amylomaltase exhibited a high level of homology with the amino acid sequence of potato disproportionating enzyme (D-enzyme) (41%) but a low level of homology with the amino acid sequence of the Escherichia coli amylomaltase (19%). The amylomaltase gene was overexpressed in E. coli, and the enzyme was purified. This enzyme exhibited maximum activity at 75 degrees C in a 10-min reaction with maltotriose and was stable at temperatures up to 85 degrees C. When the enzyme acted on amylose, it catalyzed an intramolecular transglycosylation (cyclization) reaction which produced cyclic alpha-1,4-glucan (cycloamylose), like potato D-enzyme. The yield of cycloamylose produced from synthetic amylose with an average molecular mass of 110 kDa was 84%. However, the minimum degree of polymerization (DP) of the cycloamylose produced by T. aquaticus enzyme was 22, whereas the minimum DP of the cycloamylose produced by potato D-enzyme was 17. The T. aquaticus enzyme also catalyzed intermolecular transglycosylation of maltooligosaccharides. A detailed analysis of the activity of T. aquaticus ATCC 33923 amylomaltase with maltooligosaccharides indicated that the catalytic properties of this enzyme differ from those of E. coli amylomaltase and the plant D-enzyme.     

Cloning, overexpression and nucleotide sequence of a thermostable DNA ligase-encoding gene.

Thermostable DNA ligase has been harnessed for the detection of single-base genetic diseases using the ligase chain reaction [Barany, Proc. Natl. Acad. Sci. USA 88 (1991) 189-193]. The Thermus thermophilus (Tth) DNA ligase-encoding gene (ligT) was cloned in Escherichia coli by genetic complementation of a ligts 7 defect in an E. coli host. Nucleotide sequence analysis of the gene revealed a single chain of 676 amino acid residues with 47% identity to the E. coli ligase. Under phoA promoter control, Tth ligase was overproduced to greater than 10% of E. coli cellular proteins. Adenylated and deadenylated forms of the purified enzyme were distinguished by apparent molecular weights of 81 kDa and 78 kDa, respectively, after separation via sodium dodecyl sulfate-polyacrylamide-gel electrophoresis.     

High-level production and purification of Escherichia coli N-acetylneuraminic acid aldolase (EC 4.1.3.3).

The Escherichia coli gene which encodes N-acetylneuraminic acid aldolase was isolated by the polymerase chain reaction, cloned into the inducible expression vector pTTQ18, and overexpressed in E. coli. The high yield of aldolase was achieved through both optimum growth of cells and efficient expression of the aldolase gene (20-30% soluble cellular protein). The recombinant enzyme was purified to homogeneity with an activity of 1.2-2.2 U/mg, which compared favorably with that of commercial preparations of E. coli aldolase (1.1 U/mg) and Clostridium perfringens aldolase (0.4 U/mg). The cloning strategy, fermentation conditions, purification protocol, and activity assay are described.     

Exo-mode of action of cellobiohydrolase Cel48C from Paenibacillus sp. BP-23. A unique type of cellulase among Bacillales.

Sequence analysis of a Paenibacillus sp. BP-23 recombinant clone coding for a previously described endoglucanase revealed the presence of an additional truncated ORF with homology to family 48 glycosyl hydrolases. The corresponding 3509-bp DNA fragment was isolated after gene walking and cloned in Escherichia coli Xl1-Blue for expression and purification. The encoded enzyme, a cellulase of 1091 amino acids with a deduced molecular mass of 118 kDa and a pI of 4.85, displayed a multidomain organization bearing a canonical family 48 catalytic domain, a bacterial type 3a cellulose-binding module, and a putative fibronectin-III domain. The cloned cellulase, unique among Bacillales and designated Cel48C, was purified through affinity chromatography using its ability to bind Avicel. Maximum activity was achieved at 45 degrees C and pH 6.0 on acid-swollen cellulose, bacterial microcrystalline cellulose, Avicel and cellodextrins, whereas no activity was found on carboxy methyl cellulose, cellobiose, cellotriose, pNP-glycosides or 4-methylumbeliferyl alpha-d-glucoside. Cellobiose was the major product of cellulose hydrolysis, identifying Cel48C as a processive cellobiohydrolase. Although no chromogenic activity was detected from pNP-glycosides, TLC analysis revealed the release of p-nitrophenyl-glycosides and cellodextrins from these substrates, suggesting that Cel48C acts from the reducing ends of the sugar chain. Presence of such a cellobiohydrolase in Paenibacillus sp. BP-23 would contribute to widen up its range of action on natural cellulosic substrates.     

Cloning and expression of a Bacillus subtilis Endo-1,3-1,4-beta-D-glucanase gene in Escherichia coli K12

EcoRI fragments of DNA from Bacillus subtilis NCIB 8565, a high producer of an endo-1,3-1,4-beta-D-glucanase, were 'shot-gun' cloned in the plasmid vector pBR325. A 3.5 kb insert, carrying single restriction sites for AvaI, BglII, ClaI, PvuI and PvuII, was shown to direct the synthesis of beta-glucanase in Escherichia coli K12. Enzyme activity was demonstrated in extracellular fractions of E. coli harbouring the beta-glucanase gene; however, the largest proportion (greater than 50%) of total enzyme activity was periplasmic in location. beta-Glucanase activity and cellular location were independent of the orientation of the 3.5 kb fragment in pBR325.     

Extracellular enzyme system utilized by the fungus Sporotrichum pulverulentum (Chrysosporium lignorum) for the breakdown of cullulose. Functional characterization of five endo-1,4-beta-glucanases and one exo-1,4-beta-glucanase.

A strong synergistic response was observed between the five endo-1,4-beta-glucanases and the exo-1,4-beta-glucanase obtained from culture solutions of the rot fungus Sporotrichum pulverulentum (formerly called Chrysosporium lignorum), when these enzymes were allowed to degrade de-waxed cotton and Avicel. No synergism was observed if Walseth cellulose, an acid-swollen cullulose, was used. If de-waxed cotton was pretreated with endo-1,4-beta-glucanases, the exo-1,4-beta-glucanase enzyme released much more degradation products than from an untreated cotton...