9-24-2010

Last night/ this morning I completed my list of properties and their respective sources the compound of interest was Benzene.
[In addition to the original value you need to convert to common units - that also means solubility at 15C can't be compared with room temperature and you need numbers. For links that don't go to the number provide a cropped screenshot also. ChemSpider links should be to permalinks only. JCB]
[The ChemSpider link is not a primary source JCB]
[specify when values are predicted JCB]

Property
Source
Value
Melting Point
http://en.wikipedia.org/wiki/Benzene
5.5 °C

http://www.alfa.com/content/msds/english/L14012.pdf
5.51 °C

http://www.epa.gov/oms/regs/toxics/airtox1b.pdf
5.5 C

http://www.cdc.gov/niosh/npg/npgd0049.html
42°F/5.5 °C

http://www.hbcpnetbase.com/ (see Benzene Properties bmp file below)
5.49°C
Boiling Point
http://en.wikipedia.org/wiki/Benzene
80.1 °C

http://www.alfa.com/content/msds/english/L14012.pdf
80.1 °C

http://www.epa.gov/oms/regs/toxics/airtox1b.pdf
80.1°C

http://www.cdc.gov/niosh/npg/npgd0049.html
176 °F/80°C

http://www.hbcpnetbase.com/(see Benzene Properties bmp file below)
80.09 °C
Flashpoint
http://en.wikipedia.org/wiki/Benzene
-11°C

http://www.sciencelab.com/xMSDS-Benzene-9927339
-11.1°C

http://www.alfa.com/content/msds/english/L14012.pdf
-11°C

http://www.cdc.gov/niosh/npg/npgd0049.html
12°F/-11°C

http://www.epa.gov/oms/regs/toxics/airtox1b.pdf
-11.1°C
Vapor Pressure
http://en.wikipedia.org/wiki/Benzene_(data_page)
100 mm Hg @ 26.1°C

hSpecialty Gases MSDS
75 mmHg ( 20 °C)

http://www.epa.gov/oms/regs/toxics/airtox1b.pdf
98.8 mmHg @ 25°C

http://www.cdc.gov/niosh/npg/npgd0049.html
75 mmHg

http://www.sciencelab.com/xMSDS-Benzene-9927339
75 mmHg@ 20°C
Density
http://en.wikipedia.org/wiki/Benzene
0.8765(20) g/mL

http://www.alfa.com/content/msds/english/L14012.pdf
0.8794 g/mL @ 25 °C

http://www.epa.gov/oms/regs/toxics/airtox1b.pdf
0.879 g/mL

http://www.engineersedge.com/fluid_flow/fluid_data.htm
0.879 g/mL

http://www.hbcpnetbase.com/
0.8765 g/mL





11.1.10

The following is my research article summary
The nomenclature follows that of the article the third number indicates the number of the paragraph in that part of the article the first two numbers indicate the chapter (author's nomenclature) except for the first chapter which has just one part and two paragraphs.

Jung, H., D. B. Wilson, et al. (2002). "Binding of Thermobifida fusca CDCel5A, CDCel6B and CDCel48A to easily hydrolysable and recalcitrant cellulose fractions on BMCC." Enzyme and Microbial Technology 31(7): 941-948.
[Full Marks JCB]
external image clear.gifdoi:10.1016/S0141-0229(02)00181-3

1.1Green chemistry has spurred interest in polysaccharide degrading enzymes “PSDEs” as a source of energy and chemical generations. A crucial step in realizing the full potential of PSDEs is understanding their enzyme substrate interaction. One way to do this is to investigate the interaction of the enzymes’ (in this case cellulase) catalytic domain (CD) and carbohydrate binding modules (CBM) with insoluble cellulose.

1.2 Previous work charactereized the binding mechanism of three related CBMs. The current work will focus on the binding and catalytic mechanisms of the CDs complimentary to the prior studied CBMs.

2.1.1This paragraphs details how the CD’s were produced. The desired genes were expressed in bacteria and then the desired cells were cultured.

2.1.2 Another paragraph detailing how the CD’s were produced.

2.2.1 This paragraph details how the CD protein was purified from the crude cells. It was centrifuged then run on a column.

2.2.2 The fractions of highest purity were then collected. Then the protein was purified by column again and a final purification was performed by extrusion. Lastly protein concentrations were measured spectrophotometrically.

2.3.1 Here the authors detail a binding and catalysis study wherein the concentrations of bound CD protein and free saccharide concentrations were studied over time. The study was performed under two conditions: Enzyme concentration varied; Temperature constant and temperature constant; enzyme concentration varied.

2.3.2 Continued discussion about the experimentals : after the experiment the reactions were centrifuged and samples were extracted for analysis

2.4.1 Details a similar temperature experiment wherein the CD samples were reacted as in 2.3.2 at 5 oC and then transferred to a 50oC environment where binding and hydrolysis measurements were taken over time.

2.5.1 Describes an experiment where the substrate was pretreated with cellulase, then quenched and exposed to the sample CD’s. Binding and hydrolysis of the pretreated cellulose was measured as a function of time.

2.6.1 Bound enzyme concentration was estimated based on the initial total nzyme concentration and the free enzyme concentration.

2.6.2 The reduced sugar concentration was determined by reacting it with p-hydroxybenzoic acid hydrazide which induced a chemical change in the analyte allowing its concentration to be measured spectrophotometrically.
2.7.1 The authors describe the parameters of an inhibition study where they added cellobiose (a small water soluble substrate) to the reaction mixture (CD+large insoluble substrate.)

3.1.1 Describes a figure relating concentration of bound enzyme and time. The authors describe how maximum binding occurred just after adding the enzyme, and suggest this is due to the destruction of binding sites by hydrolysis over time. The authors also propose an equation for determination of the concentration of bound enzyme as a function of time.

3.1.2 Discusses the binding constants obtained from the above equation. The author states that the model (two term single exponential) fit the data well. Furthermore binding activity increased proportional to the CD concentration until saturation levels were reached. Data collected shows that binding to the easily hydrosylable portions of substrate was variable except for one of the CDs that was tested suggesting that this particular species binds the easily hydrolysable domains first.

3.1.3 Data showed that more of the overall CDs were bound to the less hydrolysable domains. This trend was particularly pronounced for the exocellulases species. A last point the authors make is that the desorption constant of the exocellulases decreased as concentration of CD increased while that of the endogluconases did not.

3.1.4 Discuses the structure of endo and exogluconase structure. Endogluconases have a cleft type active site while exogluconases have a tunnel shaped active site characterized by hydrogen bonding and hydrophobic stacking (the authors do not discuss the endogluconase binding characteristics.)

3.2.1For two of the CD species increases in temperate were proportional to rate of binding, however increased temperature inhibited the binding of CD­Cel6B

3.3.1 The authors discussed the enzymes ability to convert the substrate to reduced sugars. After 10 hours the percent conversion was less than 1% and most of this is complete within the first two hours. The authors suggest that this is correlated to the time it takes for steady state binding to be achieved.

3.3.2 The statistics for conversion at seven hours with elevated temperature are low as well

3.4.1 Results of the pretreated experiment show no desorption confirming that the desorption is due to lack of binding sites after hydrolysis. Percent conversion is also lower

3.4.2 Results of the pretreatment experiment show a dichotomy exists between the endo and exogluconases. The Endogluconases had hydrolyzed less than the exogluconases suggesting that the Endogluconases prefer the easily accessible substrate.

3.5.1 The authors note that the addition of soluble substrate to the reaction system acted to inhibit binding. The inhibitory effect was most notable on the easily hydrolyzed portions of the substrate. Inhibition ceased after ~1 hour of reaction time implying that the inhibition is due to substrate structural change.

4.1.1 The authors conclude that maximal binding is achieved immediately after the CDs are added and slowly decreases over time due to catalysis of substrate.

4.1.2 Here the authors conclude that the results showing that endogluconases preferentially bind easily hydrolyzed substrates indicate a possible explanation why intact cellulases preferentially bind easily hydrolysable substrates. The results showing that the exocellulases bind equally to the easily hydrolyzed and normal substrate suggest that exogluconase activity is strictly concerned with attacking the chain ends regardless of their environment or ease of hydrolysis.

4.1.3 Here the authors reiterate that the conversion rate maximized soon after the binding reached steady state. Additionally the exogluconase hydrolysis leveled off due to lack of binding and the exocellulases conversion increased gradually with the reaction time.

4.1.4 Reports that as reaction temperature increased the rate of binding increased for some CDs and decreased for others. At this time the authors give no reason for this discrepancy (possible thermodynamics of binding).

4.1.3 Concludes that the addition of soluble substrate decreased the binding to the easily hydrolyzed portions of the insoluble substrate and did not show a decrease in binding to the less easily hydrolyzed portions of the insoluble substrate. These results suggest that the inhibition is due to structural changes in the substrate as opposed to product related inhibition


~smg67

11.30.2010





An Introduction to the Current Literature on Cellulose, Cellulase and the Hydrolytic Enzymatic Reaction of Cellulase on Cellulose

Abstract

Cellulolytic organisms have a key role on earth by recycling cellulose, the most plentiful macromolecule on the planet. Cellulose has a simple polymer microstructure, but it forms insoluble, crystalline microfibrils, macrostructure which is resistant to enzymatic hydrolysis. All of the known organisms that degrade cellulose efficiently produce a group of synergistic enzymes with different specialties, which act together. Cellulase research at the molecular level has elucidated some of the structures and mechanisms that contribute to their activity. Despite the considerable variety, sequence comparisons show that the catalytic cores of cellulases belong to a limited number of families. Within each family, the current data show that the variety of enzymes share a common folding pattern, the same catalytic amino acid residues, and the same reaction mechanisms, (either single substitution with inversion of configuration or double substitution with retention of the β-configuration at the anomeric carbon). In addition to the catalytic domains, many cellulolytic enzymes (cellulases, xylanases etc.) contain domains not directly concerned with catalysis, but rather which act in substrate binding, multi-enzyme subunit complex formation, or attachment to the cell surface. It is assumed, these domains assist in the catalysis of crystalline cellulose by not allowing the enzymes to be washed from the surface of the substrate, by focusing catalysis on certain areas in which the substrate is synergistically weakened by multiple cutting event. In most cellulolytic organisms, cellulase synthesis is inhibited in the presence of easily hydrolyzed, soluble carbon sources and promoted in the presence of cellulose. Research shows that the promotion of cellulases is effected by soluble products created from cellulose by cellulolytic enzymes synthesized continuously at a low concentration. Several applications of cellulases and hemicellulases exist for textile, food, and paper pulp processing. These applications rely on the modification of cellulose and hemicellulose by partial hydrolysis. Total hydrolysis of cellulose into glucose is not yet economically feasible. However, the desire to reduce greenhouse gas emissions provides an added incentive for the development of processes generating fuels from cellulose, a major renewable carbon source.



The eminent prospect of producing fuel from cellulosic biomass, the largest carbon source on earth, has resulted in large investments in the biofuel industry in recent past (Schubert, 2006; Sheridan, 2008; Waltz, 2007). Cellulosic ethanol is produced via four major consecutive steps: pretreatment, hydrolysis, fermentation, and separation. Currently improvements are necessary in the enzymatic hydrolysis of cellulosic biomass in order to economically realize cellulosic ethanol. Compared to corn ethanol and petroleum derived gasoline cellulosic ethanol is rather uneconomical (Galbe and Zacchi, 2002; Lynd et al., 2008; Sun and Cheng, 2002). The current challenges include poor rate of hydrolysis, cost of cellulases, and lack of information about the cellulase cellulose “system” and its governing properties. The benefits of cellulosic ethanol over current hydrolysis methods, such as acid hydrolysis, include less expense (cooling water, gas, and electricity), decreased disposal cost and no corrosion issues (Sun and Cheng, 2002). Cost reduction is the main driving factor behind the current surge of interest in cellulosic fuels and subsequent cellulose-cellulase interest (Lynd et al., 2008). Better technologies can be created by improving upon the current cellulases as well as by improving the cellulosic substrates (Himmel et al., 2007). To do so, it is necessary to understand the enzyme-substrate interactions and identify the contribution of various properties of the substrate, enzyme and environment to the hydrolysis process. This paper will discuss in depth the chemical structure and biological morphology of several types of celluloses and cellulases with an emphasis on research presented up to the present.

I. Cellulose

Cellulose forms the chief element of green plants and wood. It is the most abundantly available natural material and nature produces between 1010 and 1011 tons of cellulose each year. Cellulose is a linear polysaccharide of repeating d-glucopyranose units linked by β-(1,4) linkages. The degree of polymerization of cellulose depends largely on the plant species but is approximately 10,000 for wood and 15,000 for cotton. Natural cellulose is a semicrystalline polymer with crystalline sections produced by polymer alignment and held together by strong hydrogen bonding (in-plane) and Van der Waals interactions (between planes). There are four different allomorphs (similar forms) for cellulose: cellulose I (Iα and Iβ), cellulose II, cellulose III (IIII and IIIII) and cellulose IV (IVI and IVII), and they can easily be transformed from one form to another by chemical treatment (O’Sullivan, 1997). While cellulose III is thermodynamically the most stable form, it is cellulose I which is most commonly found in nature. The Iα allomorph is predominant in primitive organisms, while Iβ is the predominant allomorph in higher plants. Iβ can also be obtained from Iα by water treatment at 260–280 ◦C for 15–30 min (Debzi et al., 1991). Often found in cell-walls accompanying cellulose is hemicellulose, which is a term first coined by Schulze (Schulze 1891). This group of polysaccharides is obtained by extracting materials containing cell-walls with alkali. Hemicelluloses have a random amorphous structure and are comprised of several polysaccharides (Muralikrishna and Rao, 2007). Starch is a naturally occurring blend of two polymers: amylase and amylopectin. Both are α-d-glucopyranose polymers. For standard starches, the amylose to amylopectin ratio varies from 1:4 to 1:2 (Tester and Karkalas, 2004). “Amylose-rich” and “waxy” starch varieties are enriched in amylose and amylopectin, respectively. Amylose is a linear homopolysaccharide formed with d-glucopyranose units linked together with α-(1,4) linkages. The average degree of polymerization (DP) of amylose is between 300 and 25,000 and the exact DP depends on the origin of the starch. Amylopectin is a branched homopolysaccharide formed with d-glucopyranose units. The rate of branching is about 4–5% (every 20 units). In each chain, units are linked by α-(1,4) linkages; the side-chains are linked to the main chain thanks to α-(1,6) linkages. The number of glucose units is between 105 and 109, but, again, depends on the botanical origin of starch. Starch a is a semicrystalline polymer found in three different allomorphs: the A allomorph is found in cereals, the B allomorph is found in tubers, while the C allomorph is found in legumes, roots, fruits and is actually a combination of both A and B allomorphs (Veregin, Fyfe, and Marchessault, 1986; Buleon, Gerard, and Chanzy, 1998). The crystalline sections are formed by alignment of the sidechains of amylopectin alongside the backbone. The crystallinity is thus affected by the amylopectin content and both the extent and length of branching.

Lignin is the second most abundant, naturally occurring macromolecule following cellulose. It is found as cell-wall component in all vascular plants and in the woody stems of arborescent angiosperms (hardwoods) and gymnosperms (softwoods) and thus coexists with cellulose (Hergert and Sarkanen, 1971). As lignin is largely hydrophobic and cellulose is hydrophilic, compatibility is obtained through hemicelluloses, which contains both hydrophilic and hydrophobic sections. The lignin content in woody stems varies between 15 and 40% where it acts as water sealant in the stems and plays an important part in controlling water transport through the cell-wall. It also protects plants against biological attack by hampering enzyme penetration. Finally, lignin also acts as permanent glue, binding cells together in the woody stems, and thus giving the stems their well-known rigidity and impact resistance. Payen discovered lignin in 1838 during the treatment of wood with nitric acid and alkaline solutions (Payen, 1838). These treatments yielded an insoluble fraction designated cellulose, and a soluble fraction which he called incrustant. This soluble material was later named “lignin” by Schulze (Schulze, 1891). Lignin is generally defined as polymeric natural products derived from an enzyme catalyzed “dehydrogenative polymerization” of three precursors: trans-coniferyl, trans-sinapyl and trans-ρ-coumaryl alcohol (). Lignin is normally used to address the lignin extracted from wood, while proto-lignin is used for lignin still associated with cells. The enzyme-initiated polymerization results in bonds of outstanding stability: biphenyl carbon-carbon linkages between aromatic carbons, alkyl-aryl carbon-carbon linkages between an aliphatic and aromatic carbon, and hydrolysis-resistant ether linkages. The only linkage which is relatively weak and hydrolysable is the α-aryl ether bond. The stable linkages make lignin very resistant against degradation and harsh pulping conditions are therefore needed if one wants to hydrolyse the other, more stable, ether bonds. The plant type determines the final structure of lignin. However, due to the similar base structure of the lignin precursors, every lignin molecule consists of sequence of phenyl–propane units. Lignins are divided into two major groups: guaiacyl and guaiacyl–syringyl lignins (Gibbs, 1958). The guaiacyl lignins include most gymnosperm lignins, while all angiosperm and herbaceous lignins belong to the guaiacyl–syringyl lignin class (Wardrop, 1971). This division is not absolute as different lignins can coexist, even within the same plant. As can be deduced from the class names, guaiacyl lignins only contain p-hydroxyphenyl propane (no–OCH3 on the aromatic ring, from trans-p-coumaryl inclusion) and guaiacyl propane units (one–OCH3 group on the aromatic ring from trans-coniferyl), while the guaiacyl-syringyl lignins also contain syringyl propane units (two –OCH3 on the aromatic ring from trans-sinapyl) (Nimtz, 1973).

II. Cellulase

Cellulases, glycosyl hydrolase enzymes, hydrolyze oligosaccharides and polysaccharides (Henrissat, 1991; Henrissat et al., 1989, 1998). Cellulases are modular in nature; each module consists of a successive portion of the polypeptide chain and forms a distinct unit. While there are modular variations, all cellulases possess a catalytic domain, which is a glycosyl hydrolase according to its amino acid sequence .Within a given family, the positions of the catalytic residues are conserved. The three-dimensional structures of cellulases and related enzymes from 15 different families, which coincide with 5 different protein folds, are currently known (Davies and Henrissat, 1995; Henrissat and Davies, 1997; White and Rose, 1997).

The cellulosomal (the cellulosome is a complex consisting of multiple cellulases and related enzyme subunits) enzyme subunits are not different from related free cellulases; both free and cellulosomal enzymes contain common types of catalytic domains from one family of glycosyl hydrolase. The important difference between the two types of enzymes is that all cellulosomal enzymes currently known contain a dockerin domain, which harness it to the cellulosome, whereas noncellulosomal enzymes lack the dockerin domain (Bayer et al., 1994; Be´guin and Lemaire, 1996). In bacteria, the dockerin domain measures approximately seventy amino acids long, of which twenty two residues repeat (Tokatlidis et al., 1991).

In place of a dockerin domain, noncellulosomal cellulases usually contain at least one copy of a cellulose-binding module (CBM). The CBM serves to direct a given catalytic domain to the substrate (Tomme et al., 1995). Several cellulosomal enzymes also contain CBMs, however the presence of a CBM seems not to be a definitive character of cellulosomal cellulases, and its role is not always that of a targeting mediator. For this task, the cellulosomal enzymes collectively rely on a special CBM, carried by a scaffolding subunit.

C. thermocellum has become the model organism for cellulase research due to the numerous studies on it to date. In addition its cellulase system displays an exceptional wealth, diversity, and intricacy of enzymatic components, rendering C. thermocellum the foremost cellulose-degrading organism currently known. The cellulosomal enzymes from C. thermocellum vary in size from forty to one hundred eighty kiloDaltons. The smaller cellulases (70 kDa) are characterized by a single catalytic domain attached to a dockerin domain. As the cellulosomal enzymatic subunits become larger, they generally take on additional modules. One exemption is the comparatively large CelS, which appears to contain only a single catalytic domain together with its dockerin (Kruus et al., 1993). This enzyme contains a large, catalytic domain. The native CelS catalytic domain, proteolytically clipped from its neighboring dockerin domain, was isolated from the cellulosome in viable form, and its properties were investigated (Lamed et al., 1991). CelS was described as the chief cellulosomal cellobiohydrolase, which shows a processive type of activity whereby a cellulose chain is cleaved sequentially. The native enzyme was shown to be inhibited by cellobiose (product of enzymatic hydrolysis), a typical property of the intact cellulosome


Carbohydrate Binding Modules

An additional important cellulase has a “Family-9” catalytic domain. The C. thermocellum cellulosome includes at least four such enzymes ( CelD, CelF, CbhA, and CelJ). Crystal structures, showing subtypes of these glycosyl hydrolases are currently known. Comparison of the crystal structure conveys important implications about the modular nature of cellulases. The structure of the Family-9 catalytic domain is very similar, displaying an (α/α)6-barrel fold and equivalent catalytic machinery. They seem to differ in their neighboring modules which co-crystallized with the catalytic domains. The catalytic domain of the cellulosomal CelD from C. thermocellum was the first cellulase to have been crystallized, also the structure showed an N-terminal neighboring seven-stranded immunoglobulin like (Ig) domain of unknown function (Juy et al., 1992). Another crystal structure for a different type of Family-9 catalytic domain has been reported on from a thermophilic aerobe (Thermomonospora fusca) (Sakon et al., 1997). This cellulase (equivalent to CelF from the C. thermocellum cellulosome) lacks the Ig domain; however its Family-9 catalytic domain is linked to a C-terminal, Family-III CBM. This CBM is unusual.

Most of the cellulose-binding residues are not conserved, and the CBMs of this subtype have thus been classified as a subfamily (IIIc). Family-IIIc CBMs have been proposed to act with the catalytic domain by binding loosely to the incoming cellulose chain, which is then “fed” into the active-site cleft for hydrolysis. This type of CBM is not involved in the initial binding to crystalline cellulose, but acts more so in the catalytic function by promoting processivity (Bayer et al., 1998b; Gal et al., 1997; Irwin et al., 1998).

The information from the Family-9 enzymes suggests that the catalytic domains can be adapted by partner modules, which can alter the overall properties of an enzyme. The persistent appearance in nature of a given type of module nearby to a specific type of catalytic domain suggests a significant theme. Another theme among cellulolytic enzymes is the connection of an N-terminal Family-IV CBM with an Ig-associated Family-9 cellulase. These themes suggests a more general role for certain types of CBM and other modules. Their connection with certain types of catalytic domain could signify a ‘‘helper’’ role, whereby they bind a specific conformation of the cellulose chain and thread the chain into the active-site of its nearby catalytic domain.

Multiple Substrate Enzymes


Many of the larger cellulosomal enzyme subunits in C. thermocellum possess more than one catalytic domain. In this context, some microbial xylanases (enzymes closely related to cellulase) contain an additional enzymatic module that exhibits esterase. The two catalytic domains may act in concert on the hemicellulose of the plant cell wall; hydrolyzing the xylan chain while separating it from the lignin component. Similarly, other examples of paired catalytic domains, may promote concerted action on cellulosic substrates.

Scaffoldin

The enzymatic subunits of the cellulosome are organized by a specialized scaffoldin subunit. The scaffoldin subunits are very large, modular polypeptides. Each contains: a single Family-IIIa CBM, one or more conserved hydrophilic modules (called X modules) of unknown function, and, most importantly, multiple copies of cohesin domains. There are two known types of scaffoldins. The C. thermocellum scaffoldin carries an internal CBM and a C-terminal type-II dockerin domain, which anchors it to the cell surface. The other species have N-terminal CBMs and lack a type-II dockerin. The dockerin domains of the Catalytic domains bind with the cohesin of the scaffoldin protein to give the cellulosome ordered structure. The structural organization of the cellulosomes in nature might show much greater diversity.

Although the overall structure of the cellulosome is not completely known, the general principles that appear to govern the inclusion of its enzymatic subunits into the cellulosomal complex seem to be resolved. The dockerin domains of the enzymatic subunits engage in a very stable type of binding interaction with the cohesin domains of the scaffoldin subunit. The binding is calcium dependent (Choi and Ljungdahl, 1996; Lytle et al., 1996; Yaron et al., 1995). There seems to be no specificity in the binding among the cohesins and the dockerins within a given cellulosome. The various cohesins seem to recognize nearly all of the dockerins equally. It would seems that the enzymatic subunits would be incorporated into the cellulosome in a random manner, which would generate heterogeneity in cellulosome composition. Evidence of this has been reported for, C. papyrosolvens (Pohlschroder et al., 1995). However the question of random or selective incorporation of the cellulosomal subunits into the complex is still an open issue.

III Hydrolysis

The hydrolysis reaction catalyzed by glycosidases (including cellulases and xylanases) proceeds via an acid-base mechanism involving two specific residues. The first residue acts as an acid catalyst and protonates the oxygen of the osidic bond. The other residue acts as a nucleophile, which either interacts with the oxocarbonium intermediate or promotes the formation of an (OH-) ion from a water molecule. The reaction proceeds via retention or inversion of configuration. Reactions leading to retention of configuration require a two-step mechanism, including a double inversion of configuration at the anomeric carbon, and the formation of an oxocarbonium intermediate (Sinnot, 1990). The model of this type of reaction is the mechanism of lysozyme (Kelly et al., 1979). Reactions leading to inversion of configuration occur via a nucleophilic substitution (Sinnot, 1990) ). Enzymes of family 5, 7, 10 and 11 proceed with retention of configuration. Enzymes of family 6 and 9 proceed with inversion of configuration. Proton donating residues have been identified in T. reesei CBH II (Asp-221) C. thermocellum endoglucanase CelD (Glu-555) (Juy et al., 1992), B. pumilus xylanase XynA (Glu-182) (Ko et al.,1992), Bacillus polymyxa and Bacillus subtills endoglucanases (Baird et al.,1990), Erwinia chrysanthemi endoglucanase CelZ (Py et al,1991), Clostridium cellulolyticum endoglucanase CelCCA and C. thermocellum endoglucanase CelC (Glu-140). Nucleophilic residues have been identified on the basis of structural analysis and mutagenesis data in C. thermocellum CelD (Asp-201) (Juy et al., 1992) and B. pumilus xylanase XynA (Glu-93) (Ko et al.,1992).


IV Summary/Conclusion


Cellulose is an organic polysaccharide (C6H10O5)n made up of a linear chain of several hundred to thousands of β(1→4) linked D-glucose units and is the structural component of the cell wall of green plants. Cellulose is the most abundant organic compound on Earth. It is insoluble in water and most organic solvents. It can be broken down by naturally occurring enzymes and chemically into its glucose units by acid treatment at high temperature. Cellulose molecules adopt an extended and stiff rod-like conformation, aided by the equatorial conformation of the glucose residues. The multiple hydroxyl groups on the glucose from one chain form hydrogen bonds with oxygen molecules on the same or on a neighbor chain, holding the chains firmly together side-by-side and forming microfibrils with high tensile strength. Cellulose is also very crystalline. Several different crystalline structures of cellulose are known, corresponding to the location of hydrogen bonds between and within strands. Many properties of cellulose depend on its chain length or degree of polymerization, the number of glucose units that make up one polymer molecule. Plant-derived cellulose is usually found in a mixture with hemicellulose, lignin, pectin and other substances, while microbial cellulose is quite pure, has a much higher water content, and consists of long chains.

Cellulosomes are produced by anaerobic microbes and deconstruct plant cell wall polysaccharides. Molecular integration of cellulases and hemicellulases into cellulosomal multienzyme complexes results from the high-affinity interaction established between type I dockerin domains of the modular enzymes and type I cohesin modules of a noncatalytic scaffoldin. Cellulosomes bind the plant cell wall primarily through a scaffoldin-borne carbohydrate binding module (CBM). In addition, cellulosomes may be anchored to the microbial cell surface through the interaction of a type II dockerin located in the scaffoldin and type II cohesin domains located at the cell envelope. Integration of the microbial biocatalysts into cellulosomes potentiates catalysis through the maximization of enzyme synergism afforded by enzyme proximity and efficient substrate targeting. Cellulosomal dockerins display a dual cohesin-binding interface that may introduce enhanced flexibility in the quaternary organization of the multienzyme complex.

The hydrolysis reaction proceeds via an acid-base mechanism involving two specific residues. The reaction proceeds via retention or inversion of configuration. Reactions leading to retention of configuration require a two-step mechanism, including a double inversion of configuration at the anomeric carbon, and the formation of an oxocarbonium intermediate. Reactions leading to inversion of configuration occur via a nucleophilic substitution.


V Bibliography

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Bayer, E. A.; Morag, E.; Lamed, R., The cellulosome -- A treasure-trove for biotechnology. Trends in Biotechnology 1994, 12 (9), 379-386.
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Bayer, E. A.; Shimon, L. J. W.; Shoham, Y.; Lamed, R., Cellulosomes - Structure and ultrastructure. J. Struct. Biol. 1998, 124 (2-3), 221-234.
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Beguin, P.; Lemaire, M., The cellulosome: An exocellular, multiprotein complex specialized in cellulose degradation. Crit. Rev. Biochem. Mol. Biol. 1996, 31 (3), 201-236.

http://informahealthcare.com/doi/pdf/10.3109/10409239609106584

Belaich, A.; Fierobe, H. P.; Baty, D.; Busetta, B.; Bagnara-Tardif, C.; Gaudin, C.; Belaich, J. P., The catalytic domain of endoglucanase A from Clostridium cellulolyticum: effects of arginine 79 and histidine 122 mutations on catalysis. J. Bacteriol. 1992, 174 (14), 4677-4682.

http://jb.asm.org/cgi/reprint/174/14/4677

Choi, S. K.; Ljungdahl, L. G., Structural Role of Calcium for the Organization of the Cellulosome of Clostridium thermocellum†. Biochemistry 1996, 35 (15), 4906-4910.
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H.H. Nimtz, Tappi J. 56 (1973), p. 124.