scholarly journals Processive action of cellobiohydrolase Cel7A from Trichoderma reesei is revealed as ‘burst’ kinetics on fluorescent polymeric model substrates

2005 ◽  
Vol 385 (2) ◽  
pp. 527-535 ◽  
Author(s):  
Kalle KIPPER ◽  
Priit VÄLJAMÄE ◽  
Gunnar JOHANSSON
Keyword(s):  
Bacterial Cellulose ◽  
Trichoderma Reesei ◽  
Anthranilic Acid ◽  
Degree Of Polymerization ◽  
Cellulose Substrates ◽  
Quantitative Monitoring ◽  
End Groups ◽  
Alternative Means ◽  
Burst Kinetics ◽  
Hydrolysis Of

Reaction conditions for the reducing-end-specific derivatization of cellulose substrates with the fluorogenic compound, anthranilic acid, have been established. Hydrolysis of fluorescence-labelled celluloses by cellobiohydrolase Cel7A from Trichoderma reesei was consistent with the active-site titration kinetics (burst kinetics), which allowed the quantification of the processivity of the enzyme. The processivity values of 88±10, 42±10 and 34±2.0 cellobiose units were found for Cel7A acting on labelled bacterial cellulose, bacterial microcrystalline cellulose and endoglucanase-pretreated bacterial cellulose respectively. The anthranilic acid derivatization also provides an alternative means for estimating the average degree of polymerization of cellulose and, furthermore, allows the quantitative monitoring of the production of reducing end groups on solid cellulose on hydrolysis by cellulases. Hydrolysis of bacterial cellulose by cellulases from T. reesei revealed that, by contrast with endoglucanase Cel5A, neither cellobiohydrolases Cel7A nor Cel6A produced detectable amounts of new reducing end groups on residual cellulose.

scholarly journals Fungal cellulase systems. Comparison of the specificities of the cellobiohydrolases isolated from Penicillium pinophilum and Trichoderma reesei

1989 ◽  
Vol 261 (3) ◽  
pp. 819-825 ◽  
Author(s):  
M Claeyssens ◽  
H Van Tilbeurgh ◽  
P Tomme ◽  
T M Wood ◽  
S I McRae
Keyword(s):  
Trichoderma Reesei ◽  
Specific Binding ◽  
Degree Of Polymerization ◽  
Penicillium Pinophilum ◽  
Random Manner ◽  
Chromophoric Group ◽  
Apparent Kinetic Parameters ◽  
Fungal Cellulase ◽  
Hydrolysis Of ◽  
Reaction Patterns

Reaction patterns for the hydrolysis of chromophoric glycosides from cello-oligosaccharides and lactose by the cellobiohydrolases (CBH I and CBH II) purified from Trichoderma reesei and Penicillium pinophilum were determined. They coincide with those found for the parent unsubstituted sugars. CBH I enzyme from both organisms attacks these substrates in a random manner. Turnover numbers are, however, low and do not increase appreciably as a function of the degree of polymerization of the substrates. The active-site topology of the CBH I from T. reesei was further probed by equilibrium binding experiments with cellobiose, cellotriose, lactose and some of their derivatives. These point to a single interaction site (ABC), spatially restricted as deduced from the apparent independency of the thermodynamic parameters. It appears that the putative subsite A can accommodate a galactopyranosyl or glucopyranosyl group, and subsite B a glucopyranosyl group, whereas in subsite C either a glucopyranosyl or a chromophoric group can be bound, scission occurring between subsites B and C. The apparent kinetic parameters (turnover numbers) for the hydrolysis of cello-oligosaccharides (and their derivatives) by the CBH II type enzyme increase as a function of chain length, indicative of an extended binding site (A-F). Its architecture allows for specific binding of beta-(1-4)-glucopyranosyl groups in subsites A, B and C. Binding of a chromophore in subsite C produces a non-hydrolysable complex. The thermodynamic interaction parameters of some ligands common to both type of enzyme were compared: these substantiate the conclusions reached above.

Properties and Applications of Modified Bacterial Cellulose-Based Materials

2020 ◽  
Vol 16 ◽  
Author(s):  
Munair Badshah ◽  
Hanif Ullah ◽  
Fazli Wahid ◽  
Taous Khan
Keyword(s):  
Surface Modification ◽  
Bacterial Cellulose ◽  
Degree Of Polymerization ◽  
Biomedical Science ◽  
Hydroxyl Groups ◽  
Market Demand ◽  
Ideal Candidate ◽  
Fibrous Network ◽  
Potential Applications ◽  
Surface Modification Techniques

Background: Bacterial cellulose (BC) is purest form of cellulose as it is free from pactin, lignin, hemicellulose and other active constituents associated with cellulose derived from plant sources. High biocompatibility and easy molding into desired shape make BC an ideal candidate for applications in biomedical field such as tissue engineering, wound healing and bone regeneration. In addition to this, BC has been widely studied for applications in the delivery of proteins and drugs in various forms via different routes. However, BC lacks therapeutic properties and resistance to free movement of small molecules i.e., gases and solvents. Therefore, modification of BC is required to meet the research ad market demand. Methods: We have searched the updated data relevant to as-synthesized and modified BC, properties and applications in various fields using Web of science, Science direct, Google and PubMed. Results: As-synthesized BC possesses properties such as high crystallinity, well organized fibrous network, higher degree of polymerization, and ability of being produced in swollen form. The large surface area with abundance of free accessible hydroxyl groups makes BC an ideal candidate for carrying out surface functionalization to enhance its features. The various reported surface modification techniques including, but not limited to, are amination, methylation and acetylation. Conclusion: In this review, we have highlighted various approaches made for BC surface modification. We have also reported enhancement in the properties of modified BC and potential applications in different fields ranging from biomedical science to drug delivery and paper-making to various electronic devices.

scholarly journals Purification and properties of three (1→3)-β-d-glucanase isoenzymes from young leaves of barley (Hordeum vulgare)

1993 ◽  
Vol 289 (2) ◽  
pp. 453-461 ◽  
Author(s):  
M Hrmova ◽  
G B Fincher
Keyword(s):  
Hordeum Vulgare ◽  
Elevated Temperatures ◽  
Gel Filtration ◽  
Degree Of Polymerization ◽  
Exchange Chromatography ◽  
Purification And Properties ◽  
Young Leaves ◽  
Monomeric Proteins ◽  
Ph Range ◽  
Hydrolysis Of

Three (1->3)-beta-D-glucan glucanohydrolase (EC 3.2.1.39) isoenzymes GI, GII and GIII were purified from young leaves of barley (Hordeum vulgare) using (NH4)2SO4 fractional precipitation, ion-exchange chromatography, chromatofocusing and gel-filtration chromatography. The three (1->3)-beta-D-glucanases are monomeric proteins of apparent M(r)32,000 with pI values in the range 8.8-10.3. N-terminal amino-acid-sequence analyses confirmed that the three isoenzymes represent the products of separate genes. Isoenzymes GI and GII are less stable at elevated temperatures and are active over a narrower pH range than is isoenzyme GIII, which is a glycoprotein containing 20-30 mol of hexose equivalents/mol of enzyme. The preferred substrate for the enzymes is laminarin from the brown alga Laminaria digitata, an essentially linear (1->3)-beta-D-glucan with a low degree of glucosyl substitution at 0-6 and a degree of polymerization of approx. 25. The three enzymes are classified as endohydrolases, because they yield (1->3)-beta-D-oligoglucosides with degrees of polymerization of 3-8 in the initial stages of hydrolysis of laminarin. Kinetic analyses indicate apparent Km values in the range 172-208 microM, kcat. constants of 36-155 s-1 and pH optima of 4.8. Substrate specificity studies show that the three isoenzymes hydrolyse substituted (1->3)-beta-D-glucans with degrees of polymerization of 25-31 and various high-M(r), substituted and side-branched fungal (1->3;1->6)-beta-D-glucans. However, the isoenzymes differ in their rates of hydrolysis of a (1->3;1->6)-beta-D-glucan from baker's yeast and their specific activities against laminarin vary significantly. The enzymes do not hydrolyse (1->3;1->4)-beta-D-glucans, (1->6)-beta-D-glucan, CM-cellulose, insoluble (1->3)-beta-D-glucans or aryl beta-D-glycosides.

scholarly journals On-site integrated production of cellulases and β-glucosidases by Trichoderma reesei Rut C30 using steam-pretreated sugarcane bagasse

2018 ◽  
Author(s):  
Marcella Fernandes de Souza ◽  
Elba Pinto da Silva Bon ◽  
Ayla Sant’ Ana da Silvab
Keyword(s):  
Carbon Source ◽  
Lignocellulosic Biomass ◽  
Trichoderma Reesei ◽  
Sugarcane Bagasse ◽  
Enzyme Preparation ◽  
Integrated Approach ◽  
Aspergillus Awamori ◽  
Pretreated Sugarcane Bagasse ◽  
Hydrolysis Of ◽  
Rut C30

AbstractThe high cost of commercial cellulases still hampers the economic competitiveness of the production of fuels and chemicals from lignocellulosic biomasses. This cost may be decreased by the on-site production of cellulases with the integrated use of the lignocellulosic biomass as carbon source. This integrated approach was evaluated in the present study whereby steam-pretreated sugarcane bagasse (SPSB) was used as carbon source for the production of cellulases by Trichoderma reesei Rut C30 and the produced enzymes were subsequently used for SPSB hydrolysis. An enzyme preparation with a high cellulase activity, of 1.93 FPU/mL, was obtained, and a significant β-glucosidase activity was achieved in buffered media, indicating the importance of pH control during enzyme production. The hydrolysis of SPSB with the laboratory-made mixture resulted in a glucose yield of 80%, which was equivalent to those observed for control experiments using commercial enzymes. Even though the supplementation of this mixture with external β-glucosidase from Aspergillus awamori was found to increase the initial hydrolysis rates, it had no impact on the final hydrolysis yield. It was shown that SPSB is a promising carbon source for the production of cellulases and β-glucosidases by T. reesei Rut C30 and that the enzyme preparation obtained is effective for the hydrolysis of SPSB, supporting the on-site integrated approach to decrease the cost of the enzymatic hydrolysis of lignocellulosic biomass.

STRUCTURE OF A GALACTURONAN FROM SUNFLOWER PECTIC ACID

1966 ◽  
Vol 44 (11) ◽  
pp. 1275-1282 ◽  
Author(s):  
V. Zitko ◽  
C. T. Bishop
Keyword(s):  
Galacturonic Acid ◽  
Periodate Oxidation ◽  
Degree Of Polymerization ◽  
Critical Assessment ◽  
Molar Ratio ◽  
Aqueous Methanol ◽  
Pectic Acid ◽  
Potassium Borohydride ◽  
Rate Of Hydrolysis ◽  
Hydrolysis Of

Fractions of sunflower pectic acid containing 89.8%, 94.2%, and 91.4% of D-galacturonic acid were carboxyl reduced as their methyl or ethylene glycol esters by potassium borohydride. Critical assessment of the effects of three different solvents (water, 80% aqueous dimethyl sulfoxide, and 80% aqueous methanol) on the efficiency of reduction showed that the latter solvent was best. The reductions caused a decrease in the degree of polymerization from 270 to 21. Measurement of the rates of hydrolysis of partially reduced pectic acids containing 90%, 41.6%, 19.9%, 11.0%, and 0.65% of D-galacturonic acid showed that the rate of hydrolysis was directly related to the proportion of galacturonosidic linkages present. Methylation and hydrolysis of the carboxyl-reduced pectic acid fractions yielded 2,3,4,6-tetra-O-methyl-D-galactose and 2,3,6-tri-O-methyl-D-galactose in an approximate molar ratio of 1:20. Results of the periodate oxidation of the carboxyl-reduced pectic acid supported the conclusion inferred from the methylation results that the pectic acid was a linear polymer of 1 → 4 linked α-D-galacturonic acid units.

scholarly journals Production of Recombinant Trichoderma reesei Cellobiohydrolase II in a New Expression System Based on Wickerhamomyces anomalus

2017 ◽  
Vol 2017 ◽  
pp. 1-8 ◽  
Author(s):  
Dennis J. Díaz-Rincón ◽  
Ivonne Duque ◽  
Erika Osorio ◽  
Alexander Rodríguez-López ◽  
Angela Espejo-Mojica ◽  
...  
Keyword(s):  
Trichoderma Reesei ◽  
Recombinant Expression ◽  
Expression System ◽  
Enzymatic Extract ◽  
Native Strain ◽  
Wickerhamomyces Anomalus ◽  
Optimum Ph ◽  
Hydrolysis Of Cellulose ◽  
Cellobiohydrolase Ii ◽  
Hydrolysis Of

Cellulase is a family of at least three groups of enzymes that participate in the sequential hydrolysis of cellulose. Recombinant expression of cellulases might allow reducing their production times and increasing the low proteins concentrations obtained with filamentous fungi. In this study, we describe the production of Trichoderma reesei cellobiohydrolase II (CBHII) in a native strain of Wickerhamomyces anomalus. Recombinant CBHII was expressed in W. anomalus 54-A reaching enzyme activity values of up to 14.5 U L−1. The enzyme extract showed optimum pH and temperature of 5.0–6.0 and 40°C, respectively. Enzyme kinetic parameters (KM of 2.73 mM and Vmax of 23.1 µM min−1) were between the ranges of values reported for other CBHII enzymes. Finally, the results showed that an enzymatic extract of W. anomalus 54-A carrying the recombinant T. reesei CBHII allows production of reducing sugars similar to that of a crude extract from cellulolytic fungi. These results show the first report on the use of W. anomalus as a host to produce recombinant proteins. In addition, recombinant T. reesei CBHII enzyme could potentially be used in the degradation of lignocellulosic residues to produce bioethanol, based on its pH and temperature activity profile.

Acetylation of Bacterial Cellulose: Preparation of Cellulose Acetate Having a High Degree of Polymerization

1998 ◽  
Vol 62 (7) ◽  
pp. 1451-1454 ◽  
Author(s):  
Mari TABUCHI ◽  
Kunihiko WATANABE ◽  
Yasushi MORINAGA ◽  
Fumihiro YOSHINAGA
Keyword(s):  
Cellulose Acetate ◽  
Bacterial Cellulose ◽  
Degree Of Polymerization ◽  
High Degree
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