Miniaturized enzyme production and development of micro-assays for cellulolytic and xylanolytic enzymes

Miniaturized fungal cultivation and enzyme assays were developed. Cultivation for enzyme production was performed in 50 mL conical tubes. In addition, the miniaturized enzyme assays reduced the amount of enzymes and reagents necessary. These procedures can be adopted in screening fungi to determine if they produce cellulolytic and xylanolytic enzymes.     

Simultaneous production and induction of cellulolytic and xylanolytic enzymes in aStreptomyces sp.

Extracellular cellulolytic and xylanolytic enzymes ofStreptomyces sp. EC22 were produced during submerged fermentation. The cell-free culture supernatant of the streptomycete grown on microcrystalline cellulose contained enzymes able to depolymerize both crystalline and soluble celluloses and xylans. Higher cellulase and xylanase activities were found in the cell-free culture supernatant of the strain when grown on microcrystalline cellulose than when grown on xylan. Total cellulase and endoglucanase [carboxymethyl-cellulase (CMCase)] activities reached maxima after 72 h and xylanase activity was maximal after 60h. Temperature and pH optima were 55°C and 5.0 for CMCase activity and 60°C and 5.5 for total crystalline cellulase and xylanase activities. At 80°C, approximate half-lives of the enzymes were 37, 81 and 51 min for CMCase, crystalline cellulose depolymerization and xylanase, respectively.     

Influence of vanillin on the production of cellulolytic and xylanolytic enzymes from a wood-rotting fungus,Coriolus versicolor

To examine the influence of a phenolic compound on the production of cellulolytic and xylanolytic enzymes of a woodrotting fungusCoriolus versicolor, a two-dimensional map of enzyme activity was constructed with various concentrations of cellobiose and vanillin. The productions of CMCase, xylanase, β-glucosidase, and β-xylosidase increased with higher cellobiose concentration and were markedly enhanced by addition of vanillin. Higher ratio of vanillin/cellobiose activated the production of these enzymes. Only acetyl esterase, which is not actively produced at the ligninolytic stage ofC. versicolor, was inhibited by the monolignol vanillin. As the presence of vanillin is considered to approximate conditions of wood decay more closely than its absence, the present result demonstrates that addition of vanillin, a phenolic compound, enhanced the production of cellulolytic and xylanolytic enzymes for wood cell wall degradation.     

Production of cellulolytic and xylanolytic enzymes by an Arthrographis species

A fungal isolate, Arthrographis sp. strain F4, when grown in shake-flask culture, produced cellulolytic and xylanolytic enzymes optimally at 30°C with an initial pH of 5.0 to 6.0. Coarsely-ground filter paper was the most suitable carbon substrate for production of the enzymes. Inorganic nitrogen sources gave higher activities of the enzymes than organic nitrogen sources: NH₄NO₃ and yeast extract was the most effective combination. Significant stimulation (P<0.05) of enzyme production was achieved with 0.1% (v/v) Tween 80.B.C. Okeke was and S.K.C. Obi is with the Department of Microbiology, University of Nigeria, Nsukka, Nigeria. B.C. Okeke is now with the Department of Bioscience and Biotechnology, Royal College Building, University of Strathclyde, Glasgow G1 1XW, UK     

Fermentation of cellulose and production of cellulolytic and xylanolytic enzymes by anaerobic fungi from ruminant and non-ruminant herbivores

Four anaerobic fungi were grown on filter paper cellulose and monitored over a 7–8 days period for substrate utilisation, fermentation products, and secretion of cellulolytic and xylanolytic enzymes. Two of the fungi (N1 and N2) were Neocallimastix species isolated from a ruminant (sheep) and the other two fungi were Piromyces species (E2 and R1) isolated from an Indian Elephant and an Indian Rhinoceros, respectively. The tested anaerobic fungi degraded the filter paper cellulose almost completely and estimated cellulose digestion rates were 0.25, 0.13, 0.21 and 0.18 g · 1^-1 · h^-1 for strains E2, N1, N2, R1, respectively. All strains secreted cellulolytic and xylanolytic enzymes, including endoglucanase, exoglucanase, -glucosidase and xylanase. Strain E2 secreted the highest levels of enzymes in a relatively short time. The product formation on avicel by enzymes secreted by the four fungi was studied. Both in the presence and absence of glucurono-1,5--lactone, a specific inhibitor of -glucosidase, mainly glucose was formed but no cellobiose. Therefore the exoglucanase secreted by the four fungi is probably a glucohydrolase.     

Comparison of the xylanolytic and cellulolytic activities of Cellulomonas

Summary A highly cellulolytic Cellulomonas mutant, CS1-17, has been shown to be improved over the original parent strain, CS1-1, with respect to xylanase and -xylosidase activities. From induction studies during growth on xylan, crystalline cellulose and carboxymethylcellulose it can be deduced that, although both activities have been similarly affected by the mutation, xylanolytic activity is distinct from cellulolytic activity; however, the possibility of some cross-specificity has not been eliminated.     

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.     

A semi-continuous culture system for production of cellulolytic and xylanolytic enzymes by the anaerobic fungus Piromyces sp. strain E2

Summary A system was developed for the semi-continuous cultivation of an anaerobic fungus, Piromyces sp. strain E2 (isolated from an Indian elephant), on Avicel (microcrystalline cellulose). The fungus was grown in a semi-continuous culture system: solids and fungal biomass was retained by means of a simple filter construction whereas the culture fluid was removed continuously. The production of fermentation products (acetate, ethanol, formate, lactate, hydrogen or methane), cellulolytic and xylanolytic enzymes, and protein by the fungus in monoculture or co-culture with Methanobacterium formicicum during growth on Avicel was monitored up to 45 days. These productions stabilized after an adaptation period of 24 and 30 days in the semi-continuous co-culture and monoculture, respectively. After this period the average (±SD) avicelase, -glucosidase, endoglucanase, and xylanase production in the semi-continuous monoculture were 27±6, 140±16, 1057±120 and 5012±583 IU.l^–1.dya^–1, respectively. Co-culture with the methanogen caused a shift in fermentation products to more acetate, and less ethanol and lactate. Furthermore, the production of all cellulolytic enzymes increased (40%) and xylanolytic enzyme production decreased (35%).Correspondence to: H. J. M. Op den Camp     

Novel process for the production of cellulolytic enzymes

A novel process for the production of extracellular carboxymethylcellulase (CMCase) and xylanase by fermentation under nonaseptic or nonsterile conditions is described. The fermentation process is carried out under very acidic conditions of pH 2.0 by using a acidophilic cellulolytic fungus. Microbial contamination is avoided or minimized to an insignificant level under this acid pH condition. The culture medium for this production consists of a carbon source from cellulosics or lignocellulosics, such as Na-CMC, xylan, Avicel cellulose, cellulose powder, alpha-cellulose, sawdust, etc., or a mixture of the forementioned together with simple ingredients such as (NH(4))(2)SO(4), K(2)HPO(4), MgSO(4) and NaNO(3). The fermentation is carried out at room temperature (28-30 degrees C), under aerobic conditions, and without controlling the pH. The CMCase and xylanase produced are stable under very simple storage conditions, such as in the fresh culture medium not containing the substrate for a period of 3 days, at any temperature from 0 to 30 degrees C. These extracellular enzymes have an optimum pH around 3, with the best range of pH from 2.0 to 3.6, for any temperature between 15 and 60 degrees C. The optimum temperatures are 55 degrees C for CMCase activity and 25-50 degrees C for xylanase activity, at any pH between 2.0 and 5.2. The apparent Michaelis constants Km are 2.6 and 1.5 mg/mL for CMCase and xylanase of the culture filtrate, respectively.     

Production of multiple xylanolytic and cellulolytic enzymes by thermophilic fungus Myceliophthora sp. IMI 387099

This study reports the production of xylanolytic and cellulolytic enzymes by a thermophilic fungal isolate Myceliophthora sp. using a cheap medium containing rice straw and chemically defined basal medium under solid-state culture. A combination of one factor at a time approach followed by response surface methodology using Box-Behnken design of experiments resulted in 2.5, 1.25, 1.28 and 4.23 fold increase in xylanase, endoglucanase, beta-glucosidase and FPase activity, respectively. The zymograms developed against IEF gels showed that multiple isoforms of xylanase (5), endoglucanase (4) and beta-glucosidase (2) were produced under optimized culture conditions. Moreover, thiol containing serine proteases produced during the growth of the culture had no role in the post-translational modification of these xylanases.     

Purification and characterization of cellulolytic enzymes produced by Aspergillus nidulans

Three exo-glucanases, two endo-glucanases and two beta-glucosidases were separated and purified from the culture medium of Aspergillus nidulans. The optimal assay conditions for all forms of cellulase components ranged from pH 5.0 to 6.0 and 50 degrees C and 65 degrees C for exo-glucanases and endo-glucanases but 35 degrees C and 65 degrees C for beta-glucosidases. A close relation of enzyme stability to their optimal pH range was observed. All the cellulase components were stable for 10 min at 40-50 degrees C. Exo-II and Exo-III (Km, 38.46 and 37.71 mg/ml) had greater affinity for the substrate than Exo-I (Km, 50.00 mg/ml). The Km values of Endo-I and Endo-II (5.0 and 4.0 mg/ml) and their maximum reaction velocities (Vmax, 12.0 and 10.0 IU/mg protein) were comparable. beta-Glucosidases exhibited Km values of 0.24 and 0.12 mmol and Vmax values of 8.00 and 0.67 IU/mg protein. The molecular weights recorded for various enzyme forms were: Exo-I, 29,000; Exo-II, 72,500; Exo-III, 138,000; Endo-I, 25,000; Endo-II, 32,500; beta-Gluco-I, 14,000 and beta-Gluco-II, 26,000. Exo- and endo-glucanases were found to require some metal ions as co-factors for their catalytic activities whereas beta-glucosidases did not. Hg2+ inhibited the activity of all the cellulase components. The saccharification studies demonstrated a high degree of synergism among all the three cellulase components for hydrolysis of dewaxed cotton.     

Purification and characterization of cellulolytic enzymes produced by Aspergillus nidulans

Three exo-glucanases, two endo-glucanases and two β-glucosidases were separated and purified from the culture medium of Aspergillus nidulans. The optimal assay conditions for all forms of cellulase components ranged from pH 5.0 to 6.0 and 50°C and 65°C for exo-glucanases and endo-glucanases but 35°C and 65°C for β-glucosidases. A close relation of enzyme stability to their optimal pH range was observed. All the cellulase components were stable for 10 min at 40–50°C. Exo-II and Exo-III ( K _m, 38.46 and 37.71 mg/ml) had greater affinity for the substrate than Exo-I ( K _m, 50.00 mg/ml). The K _m values of Endo-I and Endo-II (5.0 and 4.0 mg/ml) and their maximum reaction velocities ( V _max, 12.0 and 10.0 IU/mg protein) were comparable. β-Glucosidases exhibited K _m values of 0.24 and 0.12 mmol and V _max values of 8.00 and 0.67 IU/mg protein. The molecular weights recorded for various enzyme forms were: Exo-I, 29000; Exo-II, 72500; Exo-III, 138000; Endo-I, 25000; Endo-II, 32500; β-Gluco-I, 14000 and β-Gluco-II, 26000. Exo- and endo-glucanases were found to require some metal ions as co-factors for their catalytic activities whereas β-glucosidases did not. Hg²⁺ inhibited the activity of all the cellulase components. The saccharification studies demonstrated a high degree of synergism among all the three cellulase components for hydrolysis of dewaxed cotton.     

兼性厌氧纤维素降解菌的筛选和产酶研究

从我国天津地区堆肥中分离筛选出一株兼性厌氧的纤维素分解细菌D1,初步鉴定为纤维单孢菌(Cellulomonas sp.)。产酶最适碳源为葡萄糖,氮源为复合蛋白胨,酶的最适作用温度和pH值是50℃和6．4,在70℃以下和pH在5．2～8．4里稳定。     

Morphology and biomass production of prairie cordgrass on marginal lands

Prairie cordgrass (Spartina pectinata Link.) is indigenous throughout most of the continental United States and Canada to 60°N latitude and is well suited to marginal land too wet for maize (Zea mays L.) and switchgrass (Panicum virgatum L.). Evaluations of prairie cordgrass in Europe and North America indicated it has high potential for biomass production, relative to switchgrass, in short‐season areas. Our objective was to describe morphology and biomass production and partitioning in mature stands of ‘Red River’ prairie cordgrass and determine biomass production of natural populations on marginal land. This study was conducted from 2000 to 2008 in eastern South Dakota. Mean biomass production of mature stands of Red River was 12.7 Mg ha^?1. Leaves composed >88% of the biomass, and 60% of the tillers had no internodes. Belowground biomass to a depth of approximately 25 cm, not including roots, was 21 Mg ha^?1. Tiller density ranged from 683 tillers m^?2 for a 10‐year‐old stand to 1140 tillers m^?2 for a 4‐year‐old stand. The proaxis was composed of about eight phytomers, with rhizomes originating at proximal nodes and erect tillers at distal nodes. Vegetative propagation was achieved by both phalanx and guerilla growth. Differences among natural populations for biomass were expressed on gravelly marginal land. However, production, averaged across populations, was low (1.37 Mg ha^?1) and comparable to ‘Cave‐In‐Rock’ switchgrass (1.67 Mg ha^?1) over a 4‐year period. The large carbon storage capacity of prairie cordgrass in proaxes and rhizomes makes it useful for carbon sequestration purposes. Prairie cordgrass should be compared with switchgrass and other C₄ perennial grasses along environmental gradients to determine optimum landscape positions for each and to maximize bioenergy production and minimize inputs.     

Fermentation of cassava peels for the production of cellulolytic enzymes

Cassava peels were used as a substrate for the production of cellulolytic enzymes. Under solid substrate fermentation conditions and a Rhizopus sp., thermostable cellulolytic enzymes were produced. Optimal production temperature and pH were 45°C and 5.6 respectively. Kinetic studies of the enzymes showed that the cellulase C₁ activity was optimal at pH 5.0 and 50°C, whereas that of cellulase C_x was optimal at pH 7.0 and 60°C. The enzymes degraded ca 44% of sorghum grains in 6 h, thus suggesting a possible use in saccharification processes. The results also showed the possibility of re-cycling cassava peels as a cheap substrate for the enzyme industry. and accepted 6 June 1989     

The production of cellulolytic enzymes by fungal cultures

Summary Twelve fungal cultures belonging to the genera ofAspergillus, Tricboderma, Chaetomium, Stachybotrys, andHypocrea were screened for the production of cellulolytic activity. All twelve were found to degrade xylan, avicel, and carboxymethylcellulose, More cellulolytic activity was obtained with shaken cultures than with still cultures and the addition of citrate-phosphate buffer to the media greatly depressed the levels of cellulolytic activity. Varying the composition of the mineral salts in the medium had no effect on the cellulolytic activity.The growth ofAspergillus wentii under controlled conditions in a bioreactor showed that the cellulolytic activity was not affected by the aeration rate or the type of stirrer. The rate of stirring, however, did effect the cellulolytic activity, as at lower stirring speeds considerable wall growth occurred which resulted in low levels of cellulolytic activity.Culture supernatant fromAspergillus wentii was found to hydrolyze from 30–32% of Solka-Floc and from 2–10% of corn cobs, wheat straw, and newsprint. The extensive hydrolysis of the Solka-Floc indicates that with suitably treated cellulosic wastes and appropriate enzymes, appreciable amounts of sugars could be obtained.     

Influence of dietary fiber on xylanolytic and cellulolytic bacteria of adult pigs

Xylanolytic and cellulolytic bacteria were enumerated over an 86-day period from fecal samples of 10 8-month-old gilts that were fed either a control or a 40% alfalfa meal (high-fiber) diet. Fecal samples were collected from all pigs on days 0, 3, 5, 12, 25, 37, 58, and 86. Overall, the numbers of xylanolytic bacteria producing greater than 5-mm-diameter zones of clearing on 0.24% xylan roll tube medium after 24 to 36 h of incubation were 1.6 X 10(8) and 4.2 X 10(8)/g (dry weight) of feces for the control pigs and those fed the high-fiber diet, respectively. After 1 week of incubation, a large number of smaller zones of clearing (1 to 2 mm) appeared. Besides Bacteroides succinogenes and Ruminococcus flavefaciens, which produced faint zones of clearing in xylan roll tubes, three strains which closely resembled B. ruminicola hydrolyzed and used xylan for growth. The overall numbers of cellulolytic bacteria producing zones of clearing in 0.5% agar roll tube medium were 0.36 X 10(8) and 4.1 X 10(8)/g for the control pigs and those fed the high-fiber diet, respectively. B. succinogenes was the predominant cellulolytic isolate from both groups of pigs, and R. flavefaciens was found in a ratio of approximately 1 to 15 with B. succinogenes. Degradation of xylan and cellulose, measured by in vitro dry matter disappearance after inoculation with fecal samples, was significantly greater for pigs fed the high-fiber diet than that for the controls. These data suggest that the number of fibrolytic microorganisms and their activity in the large intestine of the adult pig can be increased by feeding pigs high-alfalfa-fiber diets and that these organisms are similar to those found in the rumen.     

Methods for discovery and characterization of cellulolytic enzymes from insects

Abstract Cellulosic ethanol has been identified as a crucial biofuel resource due to its sustainability and abundance of cellulose feedstocks. However, current methods to obtain glucose from lignocellulosic biomass are ineffective due to recalcitrance of plant biomass. Insects have evolved endogenous and symbiotic enzymes to efficiently use lignocellulosic material as a source of metabolic glucose. Even though traditional biochemical methods have been used to identify and characterize these enzymes, the advancement of genomic and proteomic research tools are expected to allow new insights into insect digestion of cellulose. This information is highly relevant to the design of improved industrial processes of biofuel production and to identify potential new targets for development of insecticides. This review describes the diverse methodologies used to detect, quantify, purify, clone and express cellulolytic enzymes from insects, as well as their advantages and limitations.     

The effect of different cellulosic growth substrates and pH on the production of cellulolytic enzymes by Trichoderma reesei

The effect of different cellulosic growth substrates on the production of cellulolytic enzymes by Trichoderma reesei was investigated. It was observed that growth on Avicel, Solka Floc and wheat straw produced different pH/time profiles in cultures. Over a range of controlled pH it was demonstrated that the production of cellulolytic and xylanolytic activity by T. reesei is dependent on culture pH and the type of growth substrate. The effect of pH on enzyme production varies with the nature of the growth substrate. Furthermore, it was shown that the optimum culture pH and growth substrate for the production of enzyme preparations for the extensive saccharification of cellulosic materials depends on the type of material to be saccharified.     

Selection of thermotolerant yeast strains for biomass production from sudanese molasses

Summary Yeast isolates were obtained from different stages in the sugar refining process in an attempt to isolate thermotolerant strains which would grow on a molasses urea medium. Several strains which gave biomass yields of 30–41% at 40° were isolated and identified. Four of these strains were shown to be more resistant to a 15 minute incubation at 55° than three mesophilic strains.