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.     

High activity xylanase from thermotolerantStreptomyces T7: Cultural conditions and enzyme properties

Summary A thermotolerantStreptomyces T₇ produced 70–72 U/ml of extracellular xylanase activity when grown at 50°C in submerged culture, in à medium containing 5% wheat bran as a carbon source. Among the various sugars tested, maltose showed the highest activity of 8 U/ml. Pure xylan was less effective as an inducer as compared to wheat bran. Ammonium sulphate at a concentration of 0.7% was found to be optimum for maximum yield of the enzyme. The optimum period and pH for maximum production were 72th and 7.0, respectively. The culture filtrate was devoid of amylase, cellulase and B-xylosidase activity. The xylanase was exceptionally stable and did not show any loss in activity after storage at 50°C at pH 5.0 for 6 days.     

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.     

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.     

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.     

Fungus fruit body lytic enzyme from a myxomycete,Badhamia utricularis

Chitinase,β-1,3-glucanase, cellulase, xylanase and protease activity were detected in a crude enzyme preparation obtained from a slime mold (Badhamia utricularis) which was grown on autoclaved mycelia ofPholiota nameko in a petri dish. The optimal pH of the enzyme preparation for lytic activity against fruit bodies ofLentinus edodes was 4.0, and those ofβ-1,3-glucanase and cellulase were the same. On the other hand, chitinase and protease showed optimal activity at pH 5.0 and 8.0, respectively. The lytic activity was stable below 40°C but completely inactivated at 70°C, and was most stable at pH 5.0. The studies of the optimal pH, thermal stability, and pH stability, and isoelectric focusing analysis of the enzyme preparation suggest that chitinase,β-1,3-glucanase and cellulase activities may be responsible for lysis of fruit bodies of some mushrooms. The crude enzyme preparation from the slime mold lysed fruit bodies of several mushrooms more efficiently than did commercial lytic enzymes preparations (Driselase and Usukizyme).     

Quantification of supplemental enzymes in animal feedingstuffs by radial enzyme diffusion

Methods are described which facilitate quantification of supplemental cellulase, protease and -amylase when added to animal feedingstuffs at normal industrial inclusion levels. The methods entail extraction of the enzymes from the feedingstuffs by agitation in buffer followed by quantification of extract activity using radial diffusion techniques. A linear relationship between the diameter of the zone of hydrolyzed substrate and the log of the enzyme activity applied is observed over a broad activity range. Assay of a feedingstuff supplemented with 1 kg t^–1 cellulase, protease and -amylase yielded net supplemental activity recoveries of 104±11.7%, 91.3±6.74% and 126±29.5%, respectively. A similar assay method did not prove sufficiently sensitive to facilitate detection of xylanase at typical in-feed inclusion levels. The levels of endogenous cellulase, protease and -amylase activity detected in the unsupplemented feedingstuffs were equivalent to 6.4±0.47%, 6.6±0.82% and 29.0±14.1%, respectively, of a 1 kg t^–1 supplement. The methods are technically straightforward and will facilitate determination of enzyme stabilities during processes such as high-temperature pelleting of feedingstuffs, as well as allowing more rigorous quality control related to enzyme-supplemented animal feedingstuffs.     

Studies on the enzymatic activities related to varied pattern of diets in the air-breathing cat fish,clarias batrachus (Linn.)

1. Specific activity of amylase, cellulase, protease and lipase in the intestines of the air-breathing catfish, Clarias batrachus (Linn.) has been studied.
2. Excepting amylase and protease, the activity of lipase and cellulase showed practically no changes with change in the nutritional status of the diets.
3. pH optima of all enzymes were between 6.9 and 7.6
4. There is reason to believe from cellulase and high amylase activity in the intestine of the species that its culture operation could be done more economically by giving them a supplementary diet from indigeneously available raw material particularly from plant origin.

     

Xylanase active at high pH from an alkalotolerant Cephalosporium species

Summary An alkalotolerant Cephalosporium (NCL 87.11.9) strain capable of rapid growth and xylanase secretion over a wide pH range (pH 4–10) has been isolated from soil samples. When grown in shake flasks on a wheat bran, yeast extract medium for 96 h it produced 15 to 18 IU/ml. The novel feature of this study is that it is the first report of an extracellular fungal xylanase which is active and stable at high alkaline pH (8–9.5). The culture filtrate did not show any significant cellulase activity. Gel filtration studies indicated two peaks of xylanase activities corresponding to molecular weights of 70,000 and 30,000 in the proportion of 10:90.     

Extracellular xylanolytic enzymes ofPaecilomyces varioti

Summary Paecilomyces varioti produced an extracellular xylanase and B-xylosidase when cultured in a medium containing xylan and corn steep liquor. Xylose (2%, w/v) totally inhibited production of both enzymes. The enzymes were purified and both had a pH optimum of 4.0. The xylanase had a molecular weight of 20,000, an isoelectric point of 5.2 and was inactive on all substrates tested except xylan. The -xylosidase, a glycoprotein, had a molecular weight of 67,000, an isoelectric point of 4.0 and had highest activity on p-nitrophenyl--D-xyloside. The xylanase had a Km of 49.5 mg/ml for xylan and the -xylosidase had a Km of 5.4 mM for p-nitrophenyl--D-xyloside.     

Production and localization of cellulases and β-glucosidase from the thermophilic fungusThielavia terrestris

Summary The enzyme production and localization ofThielavia terrestris strains C464 and NRRL 8126 were compared to determine their optimum temperature and pH for cellulase activity. High levels of intracellular -glucosidase activity were detected in the former strain. The intracellular -glucosidase of both strains were more thermostable than the extracellular enzyme; the half life ofT.terrestris (C464) endoglucanase activity at 60°C was greater than 96 hrs.     

Fates and expression ofBacillus subtilis endo-β-1, 4-glucanase gene andAlcaligenes faecalis β-glucosidase gene introduced inEscherichia coli andBacillus cells

Summary Two kinds of cellulase genes coding for endo--1, 4-glucanase and -glucosidase, isolated fromBacillus subtilis andAlcaligenes faecalis respectively, were separately or combinedly put on a newly constructedEscherichia coli-Bacillus shuttle vector plasmid. When the recombinant plasmids having cellulase gene(s) were introduced intoE. coli orBacillus cells, drastic differences in fates and expression of the two genes were observed.     

Growth and enzyme production by Polyporus hirsutus in presence of ammonium sulphate

Summary Growth of Polyporus hirsutus on rice straw rapidly increases its susceptibility to cellulase and xylanase. Addition of ammonium sulphate to the straw (0.1 g/g) enhances cellulase and xylanase production but does not affect laccase production by the fungus although it appears to inhibit its growth.     

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.     

Media carbon induction of extracellular cellulase activities inNeocallimastix frontalis isolate EB188

Extracellular cellulase induction in the ruminal fungusNeocallimastix frontalis isolate EB188 was followed. Glucose media-established cultures produced cellulase when switched to a variety of cellulose-containing media. High levels of cellulase and xylanase activities were present in cultures switched to sigma cell 100, solka floc, avicel, sisal fiber, and wheat straw, but not those switched to glucose, carboxymethylcellulose, or wood chips. Several assay substrates were used to show differential cellulase induction as well as-glucosidase activity. Cellulases hydrolyzed short oligosaccharides and released glucose from insoluble cellulose. Cellobiase activity was also indicated. Cellulase activity tolerated brief exposure to high temperature, was insensitive to certain metal ions, and possessed pH optima between 5.0 and 6.5.     

Ethylene biosynthesis-inducing protein from cellulysin is an endoxylanase

The proteinaceous ethylene biosynthesis-inducing factor (EIF) that was purified from Cellulysin was also shown to contain a xylanase activity. In all nondenaturing protein separation methods employed (Sephacryl S-200 chromatography, and preparative isoelectric focusing and agarose electrophoresis), xylanase activity copurified with the ethylene biosynthesis-inducing activity. Treatment with heat (60°C) or proteases in 8 molar urea inhibited both ethylene-inducing and xylanase activities. Antibodies raised against purified EIF, which contains three polypeptides of 18, 14, and 10 kilodaltons, immunoprecipitated both ethylene biosynthesis-inducing and xylanase activities. The purified EIF contained no detectable cellulase, polygalacturonase, or protease activity. Other hydrolytic activities as estimated by using p-nitrophenyl derivatives of several sugars as substrates also were not detected. Different commercially available hydrolytic enzyme preparations were tested for both ethylene biosynthesis-inducing and xylanase activities. All enzymes tested contained xylanase activity, but only a few induced ethylene biosynthesis. Western blots of proteins separated by SDS-PAGE, using antibodies prepared against the non-denatured purified EIF, revealed two major bands of about 18 and 14 kilodaltons in EIF. These antibodies seem to be specific for these proteins from Trichoderma viride, because there was little cross-reactivity with the other proteins in Cellulysin and other commercial enzyme preparations. Based on these data, we suggest that EIF contains a specific xylanase activity which is involved in inducing ethylene biosynthesis.     

Xylanase production under solid-state fermentation and its characterization by an isolated strain of <Emphasis Type="Italic">Aspergillus foetidus</Emphasis> in India

Summary A locally isolated strain of Aspergillus foetidus MTCC 4898 was studied for xylanase (EC 3.2.1.8) production using lignocellulosic substrates under solid state fermentation. Corncobs were found as the best substrates for high yield of xylanases with poor cellulase production. The influence of various parameters such as temperature, pH, moistening agents, moisture level, nitrogen sources and pretreatment of substrates were evaluated with respect to xylanase yield, specific activity and cellulase production. Influence of nitrogen sources on protease secretion was also examined. Maximum xylanase production (3065 U/g) was obtained on untreated corncobs moistened with modified Mandels and Strenberg medium, pH 5.0 at 1 5 moisture levels at 30 °C in 4 days of cultivation. Submerged fermentation under the same conditions gave higher yield (3300 U/g) in 5 days of cultivation, but productivity was less. Ammonium sulphate fractionation yielded 3.56-fold purified xylanase with 76% recovery. Optimum pH and temperature for xylanase activity were found to be 5.3 and 50 °C respectively. Kinetic parameters like K_m and V_max were found to be 3.58 mg/ml and 570 μmol/mg/min. Activity of the enzyme was found to be enhanced by cystiene hydrochloride, CoCl₂, xylose and Tween 80, while significantly inhibited by Hg⁺⁺, Cu⁺⁺ and glucose. The enzyme was found to be stable at 40 °C. The half life at 50 °C was 57.53 min. However thermostability was enhanced by glycerol, trehalose and Ca⁺⁺. The crude enzyme was stable during lyophilization and could be stored at less than 0 °C.     

Inductive formation of celluases by L-sorbose in Trichoderma reesei

Summary L-Sorbose, which is known as an inhibitor of -1,3-glucan synthesis in fungi, induces the production of cellulases in strains belonging to Trichoderma reesei. Especially, mutant strains PC-3–7 and X-31, which were obtained by several steps of mutation from QM 9414, have the most effective cellulase inducibility by L-sorbose comparing with other mutants of Trichoderma reesei. They synthesized cellulases effectively in liquid culture, whenever the alkaline treated sugarcane bagasse was used as a main carbon source for lowering the cost of cellulase production.     

Production and characterization of xylanases of a Bacillus strain isolated from soil

Xylanase production was performed by growing a Bacillus isolate on agricultural by-products, wheat straw, wheat bran, corn cobs and cotton bagasse. A maximum xylanase activity of 180 U/ml was obtained together with a cellulase activity of 0.03 U/ml on 4 (w/v) corn cobs. Electrophoretic analysis showed the presence of three endo--1, 4-xylanases having molecular weights of about 22, 23 and 40 kDa. Xylanolytic activity was stable up to 50 °C in the pH range of 4.5–10 and the highest activity was observed at 70 °C and pH 6.5.     

Different temperature profiles of enzyme secretion by two common strains ofTrichoderma reesei

Summary We have investigated the effects of high and low temperature on the synthesis and secretion of cellulases and other enzymes by two common and readily available strains ofTrichoderma reesei. While some effects were similar in both strains QM9414 and RUT-C30 (a reduction in cellulase production but stimulation of xylanase production at high temperature, and alterations in expression of the cellulase complex at low temperature), some specific differences between the strains were determined, most significantly an enhanced specific secretion rate (secretion/growth) at low growth temperature for QM9414.