Localization and release mechanism of cellulases in Trichoderma reesei QM 9414

Summary A significant increase in the extracellular yield of -glucosidase was observed when Trichoderma reesei QM 9414 was cultivated on a cellulose medium containing chitin. Measurement of enzyme activities in the various fractions of the mycelium revealed that endoglucanase was truly extracellular while -glucosidase was cell wall bound. Treatment of Trichoderma mycelium with cell wall degrading enzymes (produced from Trichoderma) led to a release of -glucosidase from the mycelium. Apparently chitin, in the presence of cellulose, induces the synthesis of chitinase and other cell wall lytic enzymes which promote release of the intramural -glucosidase into the medium.     

Cellulase production byTalaromyces emersonii CBS 814.70 co-immobilized with cellulose in calcium alginate

Summary The thermophilic fungusTalaromyces emersonii CBS 814.70 was co-immobilized with cellulose in calcium alginate beads. Cellulose production by this system was compared with a conventional process. The immobilized system continued to produce enzyme in a batch-fed reactor system while the non-immobilized had ceased production; levels of enzyme produced by the immobilized system were almost twice the maximum produced by the non-immobilized system.     

Extracellular invertase from Aspergillus athecius: Isolation and immobilization

Summary A fungal strain isolated from soil and identified asAspergillus athecius, when grown on moistened wheat bran produced large amounts of extracellular invertase. Most of the invertase from the moldy bran was easily extracted by low ionic strength buffer (0.005 M, pH 5.7). The crude invertase immobilized on DEAE cellulose showed not only increased activity (45%) but also greater thermal and storage stability than the free enzyme. The free and the bound enzymes showed a temperature optimum of 50–55°C and a pH optimum of 5.7 and 4.8 respectively. The K_m app. of the bound enzyme was lower than that of the free enzyme.     

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.     

A one step process for the conversion of cellulose to ethanol using anaerobic microorganisms in mono- and co-culture

Summary The reducing sugars, glucose, and ethanol produced during growth of the anaerobes Clostridium thermocellum and Acetivibrio cellulolyticus on cellulose were assayed. Zymomonas mobilis was grown under similar conditions and could ferment glucose to ethanol. The ethanol production by the cellulolytic bacteria alone and in co-culture with Zymomonas is described. Approximately 27% of a 1% cellulose substrate could be converted to ethanol by this co-culture.     

Enhancement of ethanol production in the presence of metabolic inhibitors inClostridium thermocellum strain SS8

Summary Clostridium thermocellum strain SS8 produced 0.25g of ethanol and 0.24g of acetic acid per g cellulose consumed. Enhancement in ethanol production upto 0.39g/g substrate was observed in the presence of 0.15mM concentration of sodium azide and 7% polyethyleneglycol along with significant repression in acetic acid formation.     

Biomass protein formation by filamentous fungi on woody substrates

Summary Growth and biomass protein formation by filamentous fungi grown on pretreated tropical woods of Mesta (Hibiscus cannabinus Linn.) and Subabul [Leucaena leucocephala (Lam.) de Witt] as well as their isolated hemicellulose and cellulose fractions have been studied. Penicillium janthinellum and Penicillium funiculosum produced a biomass having 20 to 30% crude protein when grown on either hemicellulose, while growth on pretreated (autoclaved in 1% NaOH) wood or isolated cellulose fractions was comparatively poor and crude protein content only 5 to 8% in the biomass.NCL Communication no.3550     

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 cellulase from <Emphasis Type="Italic">E. coli</Emphasis> EgRK2 recombinant based oil palm empty fruit bunch

Oil Palm Empty Fruit Bunch (OPEFB) is an abundant biomass resource in Indonesia, which contains 41.3 ~ 46.5% (w/w) of cellulose. This research examined the production of cellulase by the E. coli EgRK2 recombinant strain using an OPEFB substrate. The production of the enzyme was initially examined to identify optimum growth conditions, by observing the growth and activity of E. coli EgRK2 compared to its wild type. Our results showed that the optimum production time, pH and temperature of the recombinant growth and cellulase activity were achieved at 24 h, and at 7 and 40°C, respectively. Using these optimum conditions, the enzyme was produced, and experiments were carried out to examine the enzyme characteristics, produced from both strains, on hydrolysis of cellulose from OPEFB. Our results showed that the activity of the enzyme produced by the recombinant almost doubled compared to that of the wild type, although the optimum pH for both strains was pH 6. Higher activity was achieved by the recombinant compared to the wild type strain, and values were 1.905 and 1.366 U/mL, respectively. The optimum temperature for hydrolysis by cellulase occurred at 50°C for Bacillus sp. RK2, and 60°C for Bacillus sp. EgRK2. The Michaelis-Menten constant (Km) and maximum velocity (Vmax) for OPEFB degradation by E. coli EgRK2 were 0.26% and 1.750 μmol/mL/sec, which were significantly better values than those of the wild type. Control experiments for the degradation test using CMC also showed a better Vmax value for E. coli EgRK2 compared to the wild type, which is 2.543 and 1.605 μmol/mL/sec, respectively.     

Relation between filter paper activity and saccharifying ability of a Trichoderma cellulase system

Summary Tests made to study the relation between filter paper activity and actual saccharifying ability of Trichoderma cellulases show that 30 IU/g of cellulose were sufficient to achieve over 80% hydrolysis of a 25 g/L cellulose suspension in 24 h. With the same enzyme/substrate ratio, but double the concentration of substrate, about 60% hydrolysis was achieved. End- product inhibition is one factor which seriously limits the degree of hydrolysis and therefore the concentration of sugars achievable by enzymatic hydrolysis at high levels of substrate concentration or enzyme/substrate ratio.     

Enhanced cellulase production using Solka Floc in a fed-batch fermentation

Summary Use of a fed-batch mode of cultivation of T. reesei has permitted high concentrations of substrate to be consumed. This has resulted in the production of high titre cellulase preparations around 30 FPU/ml at high volumetric productivities (177 IU/L.hr).Perhaps the most obvious area for major improvement in the process of cellulose utilization is the production of cellulase enzyme for hydrolysis of wood and agricultural residues. It has been estimated that some 50% of the cost of producing glucose from cellulosic material is attributable to enzyme production alone (Perez, et al., 1980). Improvements in the area would therefore have a dramatic impact, and are of paramount importance if economical hydrolysis processes are to be realized. The first major thrust in the area has been the development of improved mutant strains of T. reesei, free from catabolite repression and capable of constitutive cellulase production (Montenecourt and Eveleigh, 1977; Gallo, 1982).While this effort continues to develop further high yielding mutants, improvement must also come from developments in fermentation techniques. A major advance is the use of fed-batch cultivation, which provides a means of avoiding the agitation and aeration difficulties, as well as repression effects encountered with high substrate concentration batch fermentation. This report briefly compares batch and fed-batch operation over a range of substrate concentrations.     

Hyper production of β-glucosidase by an Aspergillus sp.

Summary An Aspergillus sp. was isolated which secreted high levels of -glucosidase in growth medium. The maximum activity(10 IU/ml of -glucosidase and 22.6 IU/ml of cellobiase) was obtained in cellulose medium supplemented with wheat bran. The pH and temperature optima for this enzyme were 4.5 and 65°C respectively.NCL Communication No. 3616     

Cellulase and xylanase production by Trichoderma reesei Rut C-30

Summary When grown on cellulose or xylan, Trichoderma reesei (strain Rut C-30) produced extra-cellular enzymes which could hydrolyze both cellulose and xylan to their respective monosaccharides. At low O₂ saturation, -glucosidase activity is greatly reduced for cellulose-grown but not xylan-grown cells.     

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.     

Affinity chromatography of extracellular cellulase from chaetomium cellulolyticum

Summary An endo--glucanase of C.cellulolyticum was purified by a procedure involving concanavalin A (Con A)-Sepharose chromatography and polyacrylamide gel electrophoresis (PAGE). The enzyme produced G1 and oligosaccharides from CMC. Chromatography on Procion Red HE3B-Agarose proved to be useful in the separation of cellobiase from cellobiose dehydrogenase.Abbreviations CMC carboxymethyl cellulose - CM-cellulase carboxymethyl cellulase - FP-cellulase filter paper degrading cellulase - G1 glucose - G2 cellobiose - G3 cellotriose - G4 cellotetraose - G5 cellopentaose - G6 cellohexaose - G7 celloheptaose - -MG methyl--D-glucoside - pNPG p-nitrophenyl--glucopyranoside - pNP p-nitrophenol - CBDH cellobiose dehydrogenase     

Formation of a raw starch-hydrolyzing alpha-amylase by Clostridium 2021: Effect of carbon sources

Summary Clostridium 2021 was found to produce -amylase effective at hydrolyzing raw starch. Of the carbohydrates examined, starch at 3 % concentration was found to be the best carbon source for enzyme production. The products of -amylase action on starch were: maltose. glucose and higher dextrins.     

β-Amylolytic activities ofEmericella nidulans (eidam) vuill

Summary Screening of fungal isolates led to selection of a strain ofEmericella nidulans 45 producing exocellular -amylase in a starch medium. Studies of dialysed enzyme and the formulation of the medium for the enzyme production are presented.     

DNA-[N4-Cytosine]-Methyltransferase from Bacillus amyloliquefaciens: Mechanism of Action Derived from Steady-State Kinetics

Kinetic analysis of methyl group transfer from S-adenosyl-L-methionine (SAM) to the 5"-GGATCC recognition site catalyzed by the DNA-[N4-cytosine]-methyltransferase from Bacillus amyloliquefaciens [EC 2.1.1.113] has shown that the dependence of the rate of methylation of the 20-meric substrate duplex on SAM and DNA concentration are normally hyperbolic, and the maximal rate is attained upon enzyme saturation with both substrates. No substrate inhibition is observed even at concentrations many times higher than the K _M values (0.107 M for DNA and 1.45 M for SAM), which means that no nonreactive enzyme–substrate complexes are formed during the reaction. The overall pattern of product inhibition corresponds to an ordered steady-state mechanism following the sequence SAMDNAmetDNA SAH (S-adenosyl-L-homocysteine). However, more detailed numerical analysis of the aggregate experimental data admits an alternative order of substrate binding, DNA SAM }, though this route is an order of magnitude slower.     

Mutants ofStreptomyces hygroscopicus deregulated in amylase and α-glucosidase formation

Summary Two mutants, M36 and M39, of turimycin-producingS. hygroscopicus JA 6599/PR1 obtained by directed selection in a chemostat displayed altered pattern of amylase and -glucosidase production as revelaed by both constitutive enzyme formation and higher enzyme levels.     

Cellulase and hemicellulase production by Cellulomonas flavigena NIAB 441

Summary Cellulomonas flavigena (strain NIAB 441) produced cellulase and hemicellulase activities when grown on Leptochloa fusca L. Kunth (Kallar grass), found to be the best inducer for enzyme production. The enzyme possessed the potential to saccharify bagasse, Kallar grass straw, wheat straw, carboxymethyl cellulose (CMC) and xylan to reducing sugars.