Isolation of specific messenger RNA by adsorption of polysomes to matrix-bound antibody.

A procedure is presented for the purification of specific mRNAs, which exploits the ability of antibodies prepared against a native protein to bind to the nascent polypeptide on the polysome. Rather than precipitating these soluble antibody-polysome complexes with anti-antibody, which can lead to nonspecific trapping of polysomes, we have linked the anti-antibody to an insoluble matrix. Thus, the antibody-polysome complex binds to the anti-antibody support and nonspecific polysomes can easily be removed by several washes. We have found para-aminobenzyl cellulose (PAB cellulose), to be a suitable matrix for this purpose. This support can bind large quantities of anti-antibody and it displayed no detectable nonspecific affinity for polysomes or RNA. Using this procedure, we have obtained an apparently homogeneous preparation of ovalbumin mRNA.     

一种嗜热厌氧纤维素降解细菌的分离纯化方法

根据纤维素降解细菌对不溶性纤维素底部的粘附作用,利用Ｈｕｎｇａｔｅ厌氧操作技术直接以不溶性纤维素粉为基质进行滚管,分离和纯化获得嗜热厌氧纤维素降解细菌。     

Importance of the carbohydrate-binding module of Clostridium stercorarium Xyn10B to xylan hydrolysis

The Clostridium stercorarium xylanase Xyn10B is a modular enzyme comprising two thermostabilizing domains, a family 10 catalytic domain of glycosyl hydrolases, a family 9 carbohydrate-binding module (CBM), and two S-layer homologous (SLH) domains [Biosci. Biotechnol. Biochem., 63, 1596-1604 (1999)]. To investigate the role of this CBM, we constructed two derivatives of Xyn10B and compared their hydrolytic activity toward xylan and some preparations of plant cell walls; Xyn10BdeltaCBM consists of a catalytic domain only, and Xyn10B-CBM comprises a catalytic domain and a CBM. Xyn10B-CBM bound to various insoluble polysaccharides including Avicel, acid-swollen cellulose, ball-milled chitin, Sephadex G-25, and amylose-resin. A cellulose binding assay in the presence of soluble saccharides suggested that the CBM of Xyn10B had an affinity for even monosaccharides such as glucose, galactose, xylose, mannose and ribose. Removal of the CBM from the enzyme negated its cellulose- and xylan-binding abilities and severely reduced its enzyme activity toward insoluble xylan and plant cell walls but not soluble xylan. These findings clearly indicated that the CBM of Xyn10B is important in the hydrolysis of insoluble xylan. This is the first report of a family 9 CBM with an affinity for insoluble xylan in addition to crystalline cellulose and the ability to increase hydrolytic activity toward insoluble xylan.     

An adhesion-defective mutant of Ruminococcus albus SY3 is impaired in its capability to degrade cellulose

An adhesion-defective mutant of Ruminococcus albus SY3 was isolated by a subtractive enrichment procedure, which involved repetitive adsorption of cellobiose-grown cells to cellulose. The growth characteristics of the mutant were compared with those of the wild type. Like the wild-type cells, the mutant was capable of growing on soluble substrates, i.e. cellobiose and xylan. However, in contrast to the wild type strain, the mutant was impaired in its capacity to utilize insoluble substrates, e.g. crystalline cellulose, acid-swollen cellulose or alfalfa cell walls. Scanning electron microscopy revealed protuberance-like surface structures on the wild-type strain which were absent on the mutant. The levels of endoglucanase and xylanase enzymatic activities released into the extracellular culture fluid were higher in the wild type compared to the mutant. However, Avicelase activity was not detected in the extracellular culture fluid of either strains when grown on cellobiose.     

Determination of the molecular states of the processive endocellulase Thermobifida fusca Cel9A during crystalline cellulose depolymerization

Detailed understanding of cell wall degrading enzymes is important for their modeling and industrial applications, including in the production of biofuels. Here we used Cel9A, a processive endocellulase from Thermobifida fusca, to demonstrate that cellulases that contain a catalytic domain (CD) attached to a cellulose binding module (CBM) by a flexible linker exist in three distinct molecular states. By measuring the ability of a soluble competitor to reduce Cel9A activity on an insoluble substrate, we show that the most common state of Cel9A is bound via its CBM, but with its CD unoccupied by the insoluble substrate. These findings are relevant for kinetic modeling and microscopy studies of modular glycoside hydrolases.     

Measurement of saccharifying cellulase

This article sets forth a simple cellulase assay procedure. Cellulose is variable in nature, insoluble and resistant to enzymatic attack. As a result there have been a bevy of bewildering cellulase assays published that yielded irrational results. Certain protocols focused on the rapidity of the assay while ignoring that only the most readily susceptible cellulose regions were being hydrolyzed. Other assays simplified the system by using modified soluble substrates and yielded results that bore no relationship to the real world hydrolysis of insoluble cellulose. In this study Mandels, Andreotti and Roche utilized a common substrate, Whatman filter paper. Hydrolysis of a 50 mg sample of the paper was followed to roughly 4% degradation, which circumvented the problems of attack of only the most susceptible zones. This common hydrolysis target range also resulted in some balance with regard to the interaction of the several cellulase components. The method was subsequently widely adopted.     

Screening of cellulases for biofuel production: online monitoring of the enzymatic hydrolysis of insoluble cellulose using high-throughput scattered light detection

A new prospective cellulase assay simultaneously combining high-throughput, online analysis and insoluble cellulosic substrates is described. The hydrolysis of three different insoluble cellulosic substrates, catalysed by a commercial cellulase preparation from Trichoderma reesei (Celluclast), was monitored using the BioLector - allowing online monitoring of scattered light intensities in a continuously shaken microtiter plate. Cellulase activities could be quantitatively assayed using the BioLector. At low cellulase/cellulose ratios, the Michaelis-Menten parameters of the cellulase mixture were mainly affected by the crystallinity index of the cellulose. Here, the apparent maximum cellulase activities inversely correlated with the crystallinity index of the cellulose. At high cellulase/cellulose ratios the particle size of the cellulose, defining the external surface area accessible to the cellulases, was the key determining factor for cellulase activity. The developed technique was also successfully applied to evaluate the pH optimum of cellulases. Moreover, the non-hydrolytic deagglomeration of cellulose particles was investigated, for the first time, using high-throughput scattered light detection. In conclusion, this cellulase assay ideally links high-throughput, online analysis and realistic insoluble cellulosic substrates in one simple system. It will considerably simplify and accelerate fundamental research on cellulase screening.     

Considering water availability and the effect of solute concentration on high solids saccharification of lignocellulosic biomass

Milliliter scale (ligno)cellulose saccharifications suggest general solute concentration and its impact on water availability plays a significant role in detrimental effects associated with high solids lignocellulose conversions. A microtumbler developed to enable free‐fall mixing at dry solids loadings up to 35% (w/w) repeatedly produced known detrimental conversion trends on cellulose, xylan and pretreated lignocellulose with commercial enzymes. Despite this, high concentrations of insoluble nonhydrolysable dextrans did not depress saccharification extents in 5% (w/w) cellulose slurries suggesting mass transfer limitations may not significantly limit hydrolysis extents at high solids loadings. Interestingly, cellulose saccharification by purified cellulases showed increased conversions with increasing dry solids loadings. This prompted investigations into impacts the concentration of soluble species, such as sugar alcohols, low molecular weight enzyme preparation components, and monomer hydrolysis products, have on the hydrolysis environment. Such substances significantly depress conversion rates and were shown to correlatively lower water activity (A_w) in the hydrolysis environment while high insoluble solids concentrations did not. Furthermore, low‐field NMR on concentrated slurries of insoluble complex carbohydrates, including the nonhydrolysable dextrans, showed all solids constrained water significantly more than high concentrations of soluble species (inhibitory) suggesting water constraint may not be as problematic an issue at high solids loadings compared to the availability of water in the system. Additionally, the introduction of soluble species lessened overall water constraint in high solids systems and appears to shift the distribution of water away from insoluble surfaces. This is potentially a critical issue for industrial processes operating at high dry solids levels. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Isolation and properties of a major cellobiohydrolase from the cellulosome of Clostridium thermocellum.

In the anaerobic, thermophilic, cellulolytic bacterium Clostridium thermocellum, efficient solubilization of the insoluble cellulose substrate is accomplished largely through the action of a cellulose-binding multienzyme complex, the cellulosome. A major cellobiohydrolase activity from the cellulosome has been traced to its Mr 75,000 S8 subunit, and an active fragment of this subunit was prepared by a novel procedure involving limited proteolytic cleavage. The truncated Mr 68,000 fragment, termed S8-tr, was purified by gel filtration and high-performance ion-exchange chromatography. The purified protein adsorbed weakly to amorphous cellulose, and its enzymatic action yielded cellobiose as the major end product from both amorphous and crystalline cellulose preparations. The high ratio of exo- to endo-beta-glucanase activities was supported by viscosimetric measurements. The use of model substrates showed that the smallest cellodextrin to be degraded was cellotetraose, but cellopentaose was degraded at a much greater rate. Cellobiose dramatically inhibited the cellulolytic activities. In the absence of calcium or other bivalent metal ions, both the truncated cellobiohydrolase activity of S8-tr and the true cellulase activity of the parent cellulosome were relatively unstable at temperatures above 50 degrees C. Cysteine further enhanced the stabilizing effect of calcium. This is the first report of a defined cellobiohydrolase in C. thermocellum. Its association with the cellulosome and the correspondence of several of their major distinctive properties suggest that this cellobiohydrolase plays a key role in the solubilization of cellulose by the intact cellulosomal complex.     

Hydrolysis of cellulose by a mixture of Trichoderma reesei cellobiohydrolase and Aspergillus niger endoglucanase

Two endoglucanase-containing fractions were separated from Aspergillus niger cellulase by gel filtration and fast protein liquid chromatofocusing (FPLC). They possessed no ability to bind to or hydrolyze insoluble microcrystalline cellulose (Avicel) but were active toward soluble carboxymethylcellulose. No synergism was observed between Trichoderma reesei cellobiohydrolase I and either endoglucanase from A. niger. These findings may indicate that the role of the endoglucanase component of cellulase in insoluble microcrystalline cellulose hydrolysis is dependent upon its ability to be adsorbed upon the substrate.     

Prevention of fungal colonization and digestion of cellulose by the addition of methylcellulose

When the attachment of cellulolytic rumen fungi to cellulose is blocked by the addition of methylcellulose, cellulose digestion is entirely inhibited. Even after these fungi have colonized and penetrated the cellulosic fibers of filter paper, the addition of methylcellulose effectively halts cellulose digestion. This effect of methylcellulose is accompanied by the complete inhibition of fungal attachment to cellulose fibers; the addition of methylcellulose does not affect the growth of these organisms on soluble substrates. We conclude that fungal cellulose digestion, like bacterial cellulose digestion, requires the spatial juxtaposition of the cellulolytic organism and its insoluble substrate. The simultaneous inhibition of both attachment and digestion by the same inhibitor suggests that these two processes are functionally linked in the fungi.     

Fusion of cellulose binding domain from Trichoderma reesei CBHI to Cryptococcus sp. S-2 cellulase enhances its binding affinity and its cellulolytic activity to insoluble cellulosic substrates

Cryptococcus sp. S-2 carboxymethyl cellulase (CSCMCase) is active in the acidic pH and lacks a binding domain. The absence of the binding domain makes the enzyme inefficient against insoluble cellulosic substrates. To enhance its binding affinity and its cellulolytic activity to insoluble cellulosic substrates, cellulose binding domain (CBD) of cellobiohydrolase I (CBHI) from Trichoderma reesei belonging to carbohydrate binding module (CBM) family 1 was fused at the C-terminus of CSCMCase. The constructed fusion enzymes (CSCMCase-CBD and CSCMCase-2CBD) were expressed in a newly recombinant expression system of Cryptococcus sp. S-2, purified to homogeneity, and then subject to detailed characterization. The recombinant fusion enzymes displayed optimal pH similar to those of the native enzyme. Compared with rCSCMCase, the recombinant fusion enzymes had acquired an increased binding affinity to insoluble cellulose and the cellulolytic activity toward insoluble cellulosic substrates (SIGMACELL^® and Avicel) was higher than that of native enzyme, confirming the presence of CBDs improve the binding and the cellulolytic activity of CSCMCase on insoluble substrates. This attribute should make CSCMCase an attractive applicant for various application.     

Characterization of a family 5 glycoside hydrolase isolated from the outer membrane of cellulolytic Cytophaga hutchinsonii

Cytophaga hutchinsonii is an abundant aerobic cellulolytic bacterium that rapidly digests crystalline cellulose in a contact-dependent manner. The different roles of various predicted glycoside hydrolases and the detailed mechanism used by C. hutchinsonii in cellulose digestion are, however, not known. In this study, an endoglucanase belonging to glycoside hydrolase family 5 (GH5) named as ChCel5A was isolated from the outer membrane of C. hutchinsonii. The catalytic domain of ChCel5A exhibited typical endoglucanase activity and was capable of hydrolyzing insoluble cellulose with cellobiose and cellotriose as the predominant digestion products. Site-directed mutagenesis identified two aromatic amino acids in ChCle5A, W61 and W308, that dramatically decreased its hydrolytic activity toward filter paper while causing only a slight decrease in carboxymethylcellulase (CMCase) activity. Disruption of chu_1107 encoding ChCel5A caused no drastic effect on the growth parameters on cellulose for the resulting mutant strain with negligible reduction in the specific CMCase activities for intact cells. The demonstration of targeted gene inactivation capability for C. hutchinsonii has provided an opportunity to improve understanding of the details of the mechanism underlying its efficient utilization of cellulose.     

Different effects of carbohydrate binding modules on the viscoelasticity of nanocellulose gels

Many cellulose degrading and modifying enzymes have distinct parts called carbohydrate binding modules (CBMs). The CBMs have been shown to increase the concentration of enzymes on the insoluble substrate and thereby enhance catalytic activity. It has been suggested that CBMs also have a role in disrupting or dispersing the insoluble cellulose substrate, but dispute remains and explicit evidence of such a mechanism is lacking. We produced the isolated CBMs from two major cellulases (Cel6A and Cel7A) from Trichoderma reesei as recombinant proteins in Escherichia coli. We then studied the viscoelastic properties of native unmodified cellulose nanofibrils (CNF) in combination with the highly purified CBMs to detect possible functional effects of the CBMs on the CNF. The two CBMs showed clearly different effects on the viscoelastic properties of CNF. The difference in effects is noteworthy, yet it was not possible to conclude for example disruptive effects. We discuss here the alternative explanations for viscoelastic effects on CNF caused by CBMs, including the effect of ionic cosolutes.     

Fusion of family VI cellulose binding domains to Bacillus halodurans xylanase increases its catalytic activity and substrate-binding capacity to insoluble xylan

A tandem repeat of the family VI cellulose binding domain (CBD) from Clostridium stercorarium xylanase (XylA) was fused at the carboxyl-terminus of Bacillus halodurans xylanase (XylA). B. halodurans XylA is an enzyme which is active in the alkaline region of pH and lacks a CBD. The constructed chimera was expressed in Escherichia coli, purified to homogeneity, and then subjected to detailed characterization. The chimeric enzyme displayed pH activity and stability profiles similar to those of the parental enzyme. The optimal temperature of the chimera was observed at 60 °C and the enzyme was stable up to 50 °C. Binding studies with insoluble polysaccharides indicated that the chimera had acquired an increased affinity for oat spelt xylan and acid-swollen cellulose. The bound chimeric enzyme was desorbed from insoluble substrates with sugars and soluble polysaccharides, indicating that the CBDs also possess an affinity for soluble sugars. Overall, the chimera displayed a higher level of hydrolytic activity toward insoluble oat spelt xylan than its parental enzyme and a similar level of activity toward soluble xylan.     

The enzymic properties of a modified ox heart myosin adenosine triphosphatase on covalent binding to an insoluble cellulose matrix

The preparation of ox heart myosin and its partial digestion with cellulose-bound papain is described. A procedure is outlined by which heavy meromyosin subfragment 1 can be covalently bound to a cellulose ion-exchange matrix. Attachment of heavy meromyosin subfragment 1 to the insoluble matrix results in a change in the ion specificity towards ATP hydrolysis. Unlike the soluble enzyme the bound form is activated by both Ca(2+) and Mg(2+). Maximal activation by Ca(2+) occurred at a lower concentration for the bound enzyme. Mg(2+) activates at a concentration which causes near-maximal inhibition of the Ca(2+)-activated adenosine triphosphatase (ATPase) of the non-bound enzyme. The Mg(2+)-activated ATPase of the bound enzyme was in turn inhibited by the presence of Ca(2+). The activation by Mg(2+) resembles the characteristic enzymic action of the actin-subfragment 1 complex.     

Specific Polysome Immunoadsorption to Purify an Ammonium-Inducible Glutamate Dehydrogenase mRNA from Chlorella sorokiniana and Synthesis of Full Length Double-Stranded cDNA from the Purified mRNA

A specific polysome immunoadsorption procedure, employing soluble rabbit anti-NADP-GDH IgG and sheep anti-rabbit IgG covalently-linked to an insoluble cellulose matrix, was used to immunoselect polysomes translating mRNA for a chloroplastic ammonium-inducible NADP-GDH in fully induced cells of Chlorella sorokiniana. The immunoselected polysomes were dissociated, and the NADP-GDH mRNA was recovered by oligo (dT)cellulose chromatography. The translatable NADP-GDH mRNA was estimated to be 0.07 and 90% of the total polysomal poly(A)⁺RNA before and after immunoselection of the polysomes, respectively. The immunoadsorption procedure resulted in an 83% recovery and 1,291-fold purification of translatable NADP-GDH mRNA. In vitro translation of the immunoselected poly(A)⁺RNA yielded a single radioactive protein (on sodium dodecyl sufate polyacrylamide gels) with a molecular weight of 58,500, i.e. size of the putative precursor-protein of the NADP-GDH subunit in the holoenzyme in fully induced cells. The purified NADP-GDH mRNA was used for synthesis of a high proportion of nearly full-length single-stranded cDNA and double-stranded cDNA molecules.     

Purification and chemical studies on rat urinary kallikrein.

A highly purified kallikrein was obtained from rat urine by chromatography on DE-32 cellulose, affinity chromatography on Bio-gel P-200-Aprotinin and gel filtration over Sephadex G-100 coarse and superfine. A molecular weight of 32,000 by sodium dodecyl sulfate polyacrylamide disc gel electrophoresis was estimated. The aminoacid composition and the esterase activity of the purified material were determined. Biological characterization of the purified kallikrein was tested by liberation of a kinin from rat plasma kininogen, by direct action on the isolated rat uterus and by the lowering of rat arterial pressure after intravenous injection of the enzyme. The preparation of insoluble derivative of Aprotinin is described herein. The polymer used as insoluble support (Bio-gel P-200) was before changed to its corresponding azide, which reacts with Aprotinin; the product maintained the binding property of the Aprotinin with urinary kallikrein.     

Endoglucanase activity and relative expression of glycoside hydrolase genes of Fibrobacter succinogenes S85 grown on different substrates

The endoglucanase activity of cells and extracellular culture fluid of Fibrobacter succinogenes S85 grown on glucose, cellobiose, soluble polysaccharides (beta-glucan, lichenan) and intact plant polysaccharides, was compared. The specific activity of cells grown on cellulose or forages was 6- to 20-fold higher than that of cells grown on soluble substrates, suggesting an induction of endoglucanases by the insoluble substrates. The ratios of cells to extracellular culture fluid endoglucanase activities measured in cultures grown on sugars or insoluble polysaccharides suggested that the endoglucanases induced by the insoluble polysaccharides remained attached to the cells. The mRNA of all the F. succinogenes glycoside hydrolase genes sequenced so far were then quantified in cells grown on glucose, cellobiose or cellulose. The results show that all these genes were transcribed in growing cells, and that they are all overexpressed in cultures grown on cellulose. Endoglucanase-encoding endB and endA(FS) genes, and xylanase-encoding xynC gene appeared the most expressed genes in growing cells. EGB and ENDA are thus likely to play a major role in cellulose degradation in F. succinogenes.