Regulation of β-xylosidase formation by xylose in Trichoderma reesei

The soft-rot fungus Trichoderma reesei forms -xylosidase (EC 3.2.1.37) activity during cultivation on xylan and xylose, but not on glucose. When mycelia precultivated on glycerol were washed and transferred to fresh medium without a carbon and nitrogen source, -xylosidase formation was induced by xylan, xylobiose and xylose. A supply of 4 mm xylose and a pH of 2.5 provided optimal conditions for induction. -Xylosidase accounted for the major portion of total extracellular protein under these conditions, and could be purified to physical homogeneity by a single anion exchange chromatography step. A recombinant strain of T. reesei that carries multiple copies of the homologous xylanase II-encoding gene has a six-fold increased xylanase activity, but forms comparable -xylosidase activities. This shows that the rate of xylan hydrolysis has no effect on the induction of -xylosidase. Methyl--d-xyloside inhibited -xylosidase competitively and was a weak -xylosidase inducer. The induction by xylobiose and xylan was strongly enhanced by the simultaneous addition of methyl--d-xylosidese and xylan or xylobiose. The results suggest that a slow supply of xylose is a trigger for -xylosidase induction.     

Xylanase and β-xylosidase production byAspergillus niger NCIM 1207

Summary Aspergillus niger NCIM 1207 producing significantly high levels of -glucosidase was found to secrete hemicellulolytic enzymes (xylanase and -xylosidase) in the culture medium. High yields of -xylosidase were obtained when it was grown on either xylan (3%) or wheat bran (4%). Cellulose was a poor inducer of -xylosidase. The pH and temperature optima for-xylosidase were 4.5 and 65°C respectively.NCL Communication No. 3751     

Comparison of different strains ofCellulomonas for production of cellulolytic and xylanolytic enzymes from biomass produced on saline lands

A comparison of the rate of carboxymethyl cellulase (CMCase), avicelase, xylanase, -glucosidase and -xylosidase production and rates of growth by 4 different strains ofCellulomonas revealed a wide range of behaviour; with some strains producing more CMCase, avicelase, xylanase, -glucosidase and -xylosidase from complex lignocellulosic (LC) biomass (from saline land) and CMC while some others producing small amounts of these enzymes. One strain,C. biazotea, was better with respect to enzyme production potential and growth behaviour than most of the other strains and has been chosen as a starting strain for genetic improvement for producing enzymes of the cellulase complex.     

Xylan-hydrolysing enzymes of the yeast Pichia stipitis

Summary Two xylanolytic enzymes, xylanase and -xylosidase from the yeast Pichia stipitis were purified to homogeneity and characterized. Both enzymes are secreted into the culture medium upon growth on xylan. The xylanase is a glycoprotein with an approximate molecular mass of 43 kDa. The N-linked carbohydrate content was estimated to be 26% by endoglycosidase H digestion. The -xylosidase protein has a molecular mass of 37 kDa as determined by sodium dodecyl sulphate gel electrophoresis. Synthesis of xylanase was found to be inducible by xylan and repressible by xylose and glucose. By contrast, -xylosidase is synthesized constitutively to a considerable degree. The purified -xylosidase is able to hydrolyse aryl--D-glucosides with an even higher rate than -xylosides. Thus, this enzyme may not be a specific component of the xylan-degrading system of P. stipitis. Offprint requests to: M. Ciriary     

Induction of xylanase and β-xylosidase in Cellulomonas flavigena growing on different carbon sources

When Cellulomonas flavigena CDBB-531 was grown on glucose, xylose, glycerol, solka floc, sugarcane bagasse or xylan, xylanase activity was found only in the fermentation broth, while -xylosidase activity was always associated with the cells. Both enzymes were inducible, sugar-cane bagasse was the best inducer, solka floc and avicel were moderately good, while xylan was poor. A synergistic effect on xylanase and -xylosidase synthesis was observed when cellulose and hemicellulose were used together as carbon sources. When this strain was grown on glucose, cellobiose, arabinose or xylose, only low levels of both enzymes were detected. These results indicate that xylanase and -xylosidase were carbon-source-repressed by readily metabolizable substrates. The effect of glycerol on enzymes that were already induced was studied. The addition of glycerol caused a significant decrease in the levels of xylanases, while -xylosidase activity remained unchanged.     

Molecular cloning of the genes for xylan degradation of Bacillus pumilus and their expression in Escherichia coli

Summary The 7.7 Mdal PstI fragment of Bacillus pumilus IPO containing genes for xylan degradation, xylanase, and -xylosidase was inserted at the PstI site of pBR322 and cloned in E. coli C600. The hybrid plasmid thus formed was named pOXN29. The amount of xylanase and -xylosidase expressed in E. coli harboring pOXN29 was about 6% and 20% of the activity produced by the donor, B. pumilus. The reverse orientation of the inserted fragment resulted respectively in 5 times and 50 times increases in xylanase and -xylosidase productivities. Both enzymes expressed in E. coli transformants were shown to be indistinguishable from those of B. pumilus by immunological and chemical criteria. Digestion of pOXN29 with BglII produced two fragments; one was 6.7 Mdal in size and contained the whole pBR322 and the -xylosidase gene, and the other was 3.7 Mdal and coded for xylanase. Analysis of enzymes expressed in the transformant cells indicated that neither enzyme was secreted into the culture medium, periplasm nor membrane bound, although xylanase but not -xylosidase, was secreted into the medium in a B. pumilus culture.     

Induction of cellulose- and xylan-degrading enzyme complex in the yeast Trichosporon cutaneum

The specificity of induction of cellulose- and xylan-degrading enzymes was investigated on the yeast strain Trichosporon cutaneum CCY 30-5-4 using series of compounds structurally related to cellulose and xylan, including monosaccharides, glycosides, glucooligosaccharides and xylooligosaccharides. Determination of activities of secreted cellulase and -xylanase, intracellular, cell wall bound and extracellular -glucosidase and -xylosidase revealed that: (1) The synthesis of xylan-degrading enzymes is induced in the cell only by xylosaccharides, 1,3--xylobiose, 1,2--xylobiose, 1,4--xylosyl-L-arabinose, 1,4--xylobiose and thioxylobiose being the best inducers. The xylan-degrading enzymes show different pattern of development in time and discrete cellular localization, i.e. intracellular -xylosidase precedes extracellular -xylanase. (2) A true cellulase is not inducible by glucosaccharides and cellulose. Negligible constitutive cellulase activity was detected which was about two orders lower than an induced cellulase in the typical cellulolytic fungus Trichoderma reesei QM 9414. (3) The best inducer of intracellular -glucosidase splitting cellobiose was thiocellobiose in a wide range of concentration (0.1–10 mM), whereas xylosaccharides at high concentrations induced -xylosidase of xylobiose type and a non-specific aryl -D-glucosidase.The results were confirmed by growing cells on cellulose and xylan. T. cutaneum was found to be a xylan-voracious yeast, unable to grow on cellulose.     

Degradation of different [(glucurono)arabino]xylans by a combination of purified xylan-degrading enzymes

The degradation of [({4-O-methyl-}glucurono)arabino]xylans from rice bran, oat spelts, wheat flour, larchwood, and birchwood with two types of endo-(1,4)--xylanase (I and III), (1,4)--xylosidase, (1,4)--d-arabinoxylan arabinofuranohydrolase (AXH), and an acetyl xylan esterase (AE), single and in combinations was investigated. The endo-(1,4)--xylanases showed the highest initial release of reducing end-groups on oat spelt xylan, followed successively by larchwood xylan, wheat flour xylan, birchwood xylan and rice bran xylan. The extent of degradation governed by degree and pattern of substitution was highest for oat spelts, followed by wheat flour and larchwood xylan. The extent of hydrolysis for the commercially available birchwood xylan was low, due to the partly insoluble fraction. Rice bran arabinoxylan could only partly be degraded by the combined action of endo-(1,4)--xylanase and AXH. The combination of endo-(1,4)--xylanase I or III, with (1,4)--xylosidase and AXH, or AE, resulted in the highest degree of hydrolysis after 24 h of incubation. Correspondence to: A. G. J. Voragen     

Production of xylanolytic enzymes by Streptomyces flavogriseus

Summary Cultures of Streptomyces flavogriseus produced considerable amounts of xylanase when grown on xylan containing media. Comparatively lower yields of this enzyme were obtained when hay or avicel served as main carbon source, -xylosidase was synthesized intracellularly and appeared less dependent on the fermentation substrate. The strain produced simultaneously various enzymes of the cellulase complex and the xylose induced glucose isomerase.     

Thermostable xylanolytic enzymes from Rhodothermus marinus grown on xylan

The thermophilic eubacterium Rhodothermus marinus was cultivated in a fermentor and studied with respect to activities of induced xylanolytic enzymes. Growth in the fermentor on xylan occurred with a maximum specific growth rate of 0.43 h^–1 for a batch culture. The final cell concentration was 4 g cell dry weight (CDW)/l for cells grown on xylan compared to 2 g CDW/l for cells grown without xylan in the cultivation medium. At least two xylanolytic enzymes, endo-1,4--xylanase and xylan 1,4--xylosidase, were secreted into the culture medium when cells were cultivated on xylan. Of the three cellulolytic enzymes tested for activity, -glucosidase activity was in the range of the xylanolytic enzyme activities whereas cellulose-1,4--cellobiosidase and cellulase activities were hardly detectable. The expression of endo-1,4--xylanase activities during cultivation indicates the existance of more than one xylanase in R. marinus. This is also observed in fractions from gel filtration. The xylanolytic enzymes are heat-stable. At 90°C and at pH 7.0 the half-life of the endo-1,4--xylanase was about 14 h and that of xylan 1,4--xylosidase was 45 min. Correspondence to: L. Dahlberg     

Xylan-hydrolysing enzymes from thermophilic and mesophilic fungi

Screening of 40 mesophilic and 13 thermophilic fungi indicated that enzyme activities capable of degrading oat spelt xylan extensively were produced by only a few of the mesophilic species investigated. The relatively low degree of hydrolysis effected by the enzymes from thermophilic organisms could be explained, in part, by their lack of -xylosidase. Several strains of Aspergillus awamori and Aspergillus phoenicis were notable in producing high xylanase and -xylosidase and low protease activities. Of the fungl tested, 13 produced activities capable of removing O-acetyl, arabinosyl, 4-O-methylglucuronyl, feruloyl and coumaroyl substituents from the backbone of xylan polysaccharides as well as endo-1,4--d-xylanase and -1,4-xylosidase. When the growth medium contained oat spelt xylan as carbon source, higher levels of xylanase, -xylosidase and acetyl xylan esterase were found than in cultures containing meadow fescue grass but the latter were richer in ferulic acid and coumaric acid esterases and 4-O-methylglucuronidase. No single organism or carbon source used was capabie of producing high levels of all the debranching enzymes as well as high levels of enzymes capable of cleaving the glycosidic linkages of the xylan backbone. The best ballnce of enzymes was obtained in cultures of A. awamori IMI 142717 and NRRL 2276 and A. phoenicis IMI 214827. Either of these would be suitable for strain improvement studies.The authors are with The Rowett Research Institute. Bucksburn, Aberdeen, AB2 9SB, UK.T.M. Wood is the corresponding author.     

Cloning theClostridium thermocellum thermostable laminarinase gene inEscherichia coli: The properties of the enzyme thus produced

Summary We have cloned a 1.9-kb-long fragment ofClostridium thermocellum DNA which encodes laminarinase (EC 3.2.1.39). The enzyme hydrolyzes the -1,3-glucoside bonds in -1,3-and in mixed -1,3-1,4-polyglucans. The enzyme's optimum pH value is around 8.5, temperature optimum –70°C. PAGE-determined mol. weight –32 kDa.Abbreviations used CMC carboxymethyl cellulose - pNPC p-nitrophenyl D cellobioside - pNPLac p-nitrophenyl- D-lactoside - pNPG p-nitrophenyl D glucopyranoside - pNPGal p-nitrophenyl- D galactopyranoside - pNPXyl p-nitrophenyl- - D xylopyranoside - Ap ampicillin - SDS-PAGE SDS polyacrylamide gel electrophoresis     

Detection and assay of xylanolytic enzymes in a Cellulomonas isolate

Summary The accuracy of xylanase assay was tested and improved. The assay was used to monitor xylanase production by a Cellulomonas isolate and to demonstrate that this activity is distinct from the organism's -xylosidase activity.     

Production and characterization of cellulolytic and xylanolytic enzymes from the ligninolytic white-rot fungus Phanerochaete chrysosporium grown on sugarcane bagasse

The ligninolytic white-rot fungus Phanerochaete chrysosporium BKM-F-1767 produced extracellular cellulolytic enzymes (carboxymethylcellulase, CMCase and -glucosidase) and xylanolytic enzymes (xylanase and -xylosidase) in liquid medium containing 1.0% sugarcane bagasse with or without 1.0% glucose. The changes in pH and soluble protein content were monitored in the culture filtrates. The results obtained showed that the pH decreased after 3 days and then increased. The soluble protein content increased and reached the maximum value after 12 days. The results showed that the activities of enzymes were higher in the case of sugarcane bagasse without glucose. The characterization study indicated that the optimum pH values were 4.6, 4.2, 5.0 and 5.0 for CMCase, -glucosidase, xylanase and -xylosidase, respectively and the optimum temperatures were 60, 70, 65 and 60 °C for the investigated enzymes, respectively. The results showed also that after prolonged heating (5 h) at 60 °C, CMCase, -glucosidase, xylanase and -xylosidase retained 81.2, 86.8, 51.5 and 27.4% activity, respectively.     

Preparation of octyl β-d-xylobioside and xyloside by xylanase-catalyzed direct transglycosylation reaction of xylan and octanol

1-Octyl -d-xylobioside and xyloside were prepared by direct transglycosylation reaction of xylan and 1-octanol using purified xylanase from Aureobasidium pullulans. 2-Ethylhexyl -d- xylobioside and xyloside were also prepared in the same way. The maximum yields of 2-ethylhexyl -d-xylobioside and 2-ethylhexyl -d-xyloside were 110 and 54 mg/g xylan, respectively. The proposed mechanism for production of octyl xylobioside and xyloside involved the reaction of xylan and octanol by xylanase to produce octyl xylobioside and xylotrioside, the latter of which was simultaneously hydrolyzed by xylanase into octyl xyloside and xylobiose.     

Influence of growth conditions on the production of xylanolytic enzymes by Trametes trogii

Various nitrogen and carbon sources were examined as inducers of the production of endoxylanase and -xylosidase by Trametes trogii. T. trogii grown on xylan plus crystalline cellulose provided supernatants with the highest enzymatic activities. Organic nitrogen sources (especially asparagine and casamino acids) were the best for enzyme production. The increase in xylan concentration stimulated endoxylanase production whereas significant differences were not attained in -xylosidase production with more than 5g xylan/l in the culture medium. pH 4.0 was optimal for endoxylanase production, while -xylosidase production was maximum at pH 5.5. Temperatures in the range of 23–28°C stimulated enzyme production. The endoxylanase activity in the crude culture filtrate was greatest at 50°C and pH around 5.0. The optimum pH and temperature for -xylosidase activity were 5.5 and 50°C respectively.     

Isolation of aClostridium thermocellum gene encoding a thermostable β-1, 3-glucanase (laminarinase)

Summary AClostridium thermocellum gene directing the synthesis of a thermostable -glucanase was localized on a 1.9-kb DNA fragment by subcloning intoEscherichia coli plasmid vectors. The enzyme was highly efficient in degrading glucans with alternating -1, 3- and -1,4-linkages such as lichenan and barley glucan. It was also active towards the -1, 3-glucan laminarin, but lacked activity on cellulosic substrates and -glucans. The enzyme was therefore classified as -1, 3-glucanase (laminarinase) and the corresponding gene was designatedlicA. With barley -glucan as substrate the enzyme had a pH optimum around pH 6.5 and a temperature optimum at 65°C. It was stable for several hours at 60°C in the absence of substrate.     

Production and characterization of xylanase from Thielaviopsis basicola

Summary The black rot fungus Thielaviopsis basicola has the ability to grow on cellulosic biomass, producing xylanase. Of the four cellulosic substrates tested, rice straw was found to be the best for production of xylanase. A xylanase activity of 34 U/ml was obtained with rice straw which was more than three times that obtained with larchwood xylan. The -xylosidase activities obtained with these two substrates were 0.05 U/ml and 0.016 U/ml respectively. Both enzymes are active at pH 5 but the temperature optima of xylanase and -xylosidase activities are 60°C and 40°C respectively. The xylanase activity is stable over a pH range of 4–8 but the stability towards temperature falls sharply above 50°C.     

Cellulase and beta-glucosidase production by mexied culture of trichoderma reesei rut C30 and aspergillus phoenicis

Summary When lignocellulosic materials are hydrolysed using cellulase fromTrichoderma reesei, cellobiose accumulates due to a deficiency in -glucosidase activity of the enzyme complex. Cellobiose decreases the rate and extent of hydrolysis through feedback inhibition of exo -1,4-glucanase. Cellulase produced through mixed culture ofTrichoderma andAspergillus showed increased -glucosidase activity and greatly improved hydrolytic potential.     

Very stable enzymes from extremely thermophilic archaebacteria and eubacteria

Summary Thirty-six thermophilic archaebacteria and nine extremely thermophilic eubacteria have been screened on solid media for extracellular amylase, protease, hemicellulase (xylanase), cellulase, pectinase and lipase activities. Extracellular enzymes were detected in 14 archaebacteria belonging to three different orders. Twelve of these were able to degrade starch and casein and the two Thermofilum strains were able to degrade starch, xylan and carboxymethylcellulose. Three of the eubacteria could degrade only starch. The other six (including four Thermotoga strains) all had activity against starch, xylan and carboxymethylcellulose, and one also had activity against casein. Some of the amylolytic archaebacteria released -glucosidase, -glucosidase, amylase and transglucosylase activities into liquid media containing starch or maltose. Thermotoga strain FjSS3B.1 released amylase, xylanase, cellulase and -glucosidase activities into the medium when grown in the presence of substrates. When the partially purified enzymes from Thermotoga and some of the archaebacteria were compared with known thermostable enzymes the majority were found to be the most thermostable of their type. The -glucosidase, xylanase and cellulase from Thermotoga and two -glucosidases, a -glucosidase, an amylase and a pullulanase from archaebacteria all have half-lives of at least 15 min at 105°C.