Thermocellum Cellulase Research Articles

The degradation pattern of cellulose by extracellular cellulases of aerobic and anaerobic microorganisms

Cellulose (150 mg) was incubated with equal amounts of carboxymethylcellulase activity (5·0 IU) from cell-free culture filtrates of Clostridium themocellum, Neocallimastix frontalis, Trichoderma koningii and Penicillium pinophilum. No change in the crystallinity index was detected during enzymatic hydrolysis from 1 to 7 days. This finding suggests the simultaneous solubilization of both crystalline and amorphous regions. Major differences between different enzymes were detected with respect to the length of the cellulose chains. Although a considerable weight loss of the cellulosic substrate occurred no change in the chain length distribution of cellulose could be detected with the C. thermocellum cellulases, whereas a considerable shift to smaller units was seen when cellulose was incubated with cellulases of all other microorganisms. The main soluble degradation product of C. thermocellum cellulase was cellobiose, whereas all other enzyme systems liberated glucose as the main soluble sugar.

Addition of cloned β-glucosidase enhances the degradation of crystalline cellulose by the Clostridium thermocellum cellulase complex

A thermostable β-glucosidase from Clostridium thermocellum which is expressed in Escherichia coli was used to determine the substrate specificity of the enzyme. A restriction map of the β-glucosidase gene cloned in plasmid pALD7 was determined. Addition of the E.coli cell extract (containing the β-glucosidase) to the cellulase complex from C.thermocellum increased the conversion of crystalline cellulose (Avicel) to glucose. The increase was specifically due to hydrolysis of the accumulated cellobiose. A cellulose degradation process using β-glucosidase to assist the potent cellulase complex of C.thermocellum, as shown here can open the way for industrial saccharification of cellulose to glucose.

Breakdown of crystalline cellulose by synergistic action between cellulase components fromClostridium thermocellumandTrichoderma koningii

Certain isolated components of fungal cellulases, which cannot effect the breakdown of highly ordered cellulose individually, interact together synergistically to do so when recombined. Suprisingly, not all fungal cellulase components exhibit this property, and no such synergism has been observed so far between fungal and bacterial cellulases. The cellulase complex of Clostridium thermocellum cannot effect the extensive breakdown of highly ordered cellulose unless Ca2+ and dithiothreitol (DTT) are present. However, we now report that isolated cellobiohydrolase from Trichoderma koningii can combine with C. thermocellum cellulase to effect the breakdown of cellulose in the absence of Ca2+ and DTT. enhanced activity is observed if Ca2+ and DTT are present. This finding may have important applications in industry: it certainly has important implications for those interested in the basic mechanism of cellulase action in C. thermocellum.

High-level expression of Clostridium thermocellum cellulase genes in Escherichia coli

The Clostridium thermocellum cellulase genes celA and celC encoding endoglucanase A and C were subcloned in a temperature-regulated Escherichia coli expression vector containing the leftward promoterpl of bacteriophage lambda. The level of gene expression was controlled by thermal inactivation of the heat-sensitive lambda cI857 repressor. Under optimal conditions the recombinant endoglucanases A and C were expressed to a level of 10–15% of total cellular protein. Endoglucanase A was partially exported into the periplasmic space, whereas endoglucanase C was found sequestered within the cytoplasm. Overexpression of the celA gene resulted in decreased cell viability concomitant with the accumulation of endoglucanase A in the membrane fraction. In contrast, high-level synthesis of the celC gene product was well tolerated by the host cell. Overproduced endoglucanase C accumulated as a soluble enzyme without detectable formation of inactive inclusion bodies.

Hydrolysis of dilute acid pretreated mixed hardwood and purified microcrystalline cellulose by cell‐free broth from Clostridium thermocellum

The cellulase activity in cell-free broths from the thermophilic, ethanol-producing anaerobic bacterium Clostridium thermocellum is examined on both dilute-acid-pretreated mixed hardwood (90% maple, 10% birch) and Avicel. Experiments were conducted in vitro in order to distinguish properties of the cellulase from properties of the organism and to evaluate the effectiveness of C. thermocellum cellulase in the hydrolysis of a naturally occurring, lignin-containing substrate. The results obtained establish that essentially quantitative hydrolysis of cellulose from pretreated mixed hardwood is possible using this enzyme system. Pretreatment with 1% H(2)SO(4) and a 9-s residence time at 220, 210, 200, and 180 degrees C allowed yields after enzymatic hydrolysis (percentage of glucan solubilized/ glucan potentially solubilized) of 97.8, 86.1, 82.0, and 34.6%, respectively. Enzymatic hydrolysis of mixed hardwood with no pretreatment resulted in a yield of 10.1%. Hydrolysis yields of >95% were obtained from approximately 0.6 g/L mixed hardwood pretreated at 220 degrees C in 7 h at broth strengths of 60 and 80% (v/v) and in approximately 48 h with 33% broth. Hydrolysis of pretreated mixed hardwood is compared to hydrolysis of Avicel, a pure microcrystalline cellulose studied previously. The initial rate of Avicel hydrolysis saturates with respect to enzyme, whereas the initial rate of hydrolysis of pretreated wood is proportional to the amount of enzyme present. Initial hydrolysis rates for pretreated wood and Avicel at 0.6 g/L are greater for wood at low broth dilutions (1.25: 1 to 5 :1) by up to 2.7-fold and greater for Avicel at high broth dilutions (5 : 1 to 50 : 1) by up to 4.3-fold. Maximum rates of hydrolysis are achieved at <2 g substrate/L for both pretreated wood and Avicel. The substrate concentration at one-half the maximum observed rate for C. thermocellum broths is smaller for pretreated mixed hardwood than for Avicel and decreases with increasing broth dilution for both substrates. An initial activity per volume broth of approximately 11 mumol soluble glucose equivalent produced/L broth/min is observed for mixed hardwood pretreated at 220 degrees C and for Avicel at high broth dilutions; the initial activity per volume broth for Avicel is lower at low broth dilutions. The results indicate that pretreated wood is hydrolyzed at rates comparable to Avicel under many conditions and at rates significantly faster than Avicel under several conditions.

The application of a mathematical model to an appraisal of the cellulose-azure method for determining cellulase activity

The use of a dye bound substrate, cellulose-azure, for determining cellulase activity was investigated and found to be of comparable sensitivity to determinations involving measurment of sugar release from cellulose power. A mathematical model of cellulose breakdown was developed and used in conjunction with the cellulose-azure method to estimate several important characteristics of cellulolysis. The methodology was compared with traditional techniques using Clostridium thermocellum cellulase. Further applications of the model are discussed.