Douglas E. Eveleigh

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.Douglas E Eveleigh

Characteristics of fungal cellulases

Abstract Fungi colonize diverse ecologic niches, yet their cellulases are based on a central theme — exo- and endo-splitting glucanases acting co-operatively and in some instances, synergistically. The characterization of cellulases has proved to be complex. This is due in part to the ill-defined nature of the natural substrates, plus the multiple nature of cellulase components. The rationales for this multiplicity continues to be explored. Cloning studies have shown far less diversity at the genomic level. Additionally, the endo- and exo-splitting glucanases have been shown to be comprised af a tripartite structure with both enzyme forms containing a cellulose binding site, hinge region and catalytic domain. Oxidative enzymes (cellobios quinone oxido-reductase, cellobiose oxidase and lactonase) though minor in quantitative terms, may have important roles in the regulation/initiation of cellulase synthesis per se.

Intracellular Symbionts and Other Bacteria Associated with Deer Ticks (Ixodes scapularis) from Nantucket and Wellfleet, Cape Cod, Massachusetts

ABSTRACT The diversity of bacteria associated with the deer tick (Ixodes scapularis) was assessed using PCR amplification, cloning, and sequencing of 16S rRNA genes originating from seven ticks collected from Nantucket Island and Wellfleet, Cape Cod, Mass. The majority of sequences obtained originated from gram-negative proteobacteria. Four intracellular bacteria were detected including strains of Ehrlichia, Rickettsia, and Wolbachia and an organism related to intracellular insect symbionts from the Cytophaga-Flavobacterium-Bacteroides group. Several strains of members of the Sphingomonadaceae were also detected in all but one tick. The results provide a view of the diversity of bacteria associated with I. scapularis ticks in the field.

Cloning and characterization of the glucooligosaccharide catabolic pathway beta-glucan glucohydrolase and cellobiose phosphorylase in the marine hyperthermophile Thermotoga neapolitana

Characterization in Thermotoga neapolitana of a catabolic gene cluster encoding two glycosyl hydrolases, 1,4-beta-D-glucan glucohydrolase (GghA) and cellobiose phosphorylase (CbpA), and the apparent absence of a cellobiohydrolase (Cbh) suggest a nonconventional pathway for glucan utilization in Thermotogales. GghA purified from T. neapolitana is a 52.5-kDa family 1 glycosyl hydrolase with optimal activity at pH 6.5 and 95 degrees C. GghA releases glucose from soluble glucooligomers, with a preference for longer oligomers: k(cat)/K(m) values are 155.2, 76.0, and 9.9 mM(-1) s(-1) for cellotetraose, cellotriose, and cellobiose, respectively. GghA has broad substrate specificity, with specific activities of 236 U/mg towards cellobiose and 251 U/mg towards lactose. With p-nitrophenyl-beta-glucoside as the substrate, GghA exhibits biphasic kinetic behavior, involving both substrate- and end product-directed activation. Its capacity for transglycosylation is a factor in this activation. Cloning of gghA revealed a contiguous upstream gene (cbpA) encoding a 93.5-kDa cellobiose phosphorylase. Recombinant CbpA has optimal activity at pH 5.0 and 85 degrees C. It has specific activity of 11.8 U/mg and a K(m) of 1.42 mM for cellobiose, but shows no activity towards other disaccharides or cellotriose. With its single substrate specificity and low K(m) for cellobiose (compared to GghAs K(m) of 28.6 mM), CbpA may be the primary enzyme for attacking cellobiose in Thermotoga spp. By phosphorolysis of cellobiose, CbpA releases one activated glucosyl molecule while conserving one ATP molecule per disaccharide. CbpA is the first hyperthermophilic cellobiose phosphorylase to be characterized.

Cellulase secretion from a hyper-cellulolytic mutant of Trichoderma reesei Rut-C30

Two strains of Trichoderma reesei, wild type QM6a and mutant Rut-C30, were grown in meida containing an inducer, insoluble crystalline cellulose (Avicel PH101), as carbon source for 11 days. The cell growth, expressed as myceliar protein content, of Rut-C30 was 4–5 times higher than QM6a. The lack of ultrastructural disorganization, and absence of intracellular enzyme release into the growth medium, indicated that none of these two strains had undergone any significant autolysis during the entire growth phase. Cellulase activities, mainly endoglucanase, cellobiase and filter paper degrading activity (disc) were enhanced in Rut-C30 cells. A major change was observed in the endoglucanase activity which was 30 times higher in Rut-C30 than QM6a, whereas, both β-glucosidase and disc activities were 3 times enhanced in Rut-C30 compared to QM6a. In addition to synthesis, cellulase secretion was also enhanced in Rut-C30. Both the organisms contained same amounts of intracellular marker enzyme activities (e.g., inosine diphosphatase, thiamine pyrophosphatase, alkaline phosphatase). Finally, the enahncement of secretory activity of Rut-C30 was correlated with the proliferation of rough endoplasmic reticulum (RER) and increased phospholipid content. It appears that Rut-C30 is not only a hypercellulolytic but also a hypersecretor mutant.

Increasing Yields of Extracellular Enzymes

Publisher Summary This chapter discusses the current rationales and methodologies employed to increase the extracellular enzyme yields. The four aspects considered are strain selection, environmental control, genetic regulatory controls, genetic recombination, and gene amplification techniques. Two practical approaches should be emphasized with regard to strain selection: effectiveness of the screening assay, and selection of economic high-yielding strains. The limits of the screening assay should be defined and correlated with the chances of success in screening. The selected high yielding strains should possess the rapid growth rate of the parent strain in order to ensure high productivity. The screening for hyperproducing mutants allow for large-scale general selection of strains (106 colonies per screen), of which a high proportion (70%) are mutants. The potential of more rapid strain development programs is now possible based on mutants that are resistant to environmental stress (temperature), catabolic repression, and feedback inhibition. Many environmental parameters influence the yield of an extracellular enzyme, such as, pH, temperature, aeration, and ionic strength.

Zymomonas — an alcoholic enigma

The bacterium Zymomonas mobilis can produce alcohol more than twice as fast as yeast. The possible large-scale production of ethanol by this organism has been restricted in part because its substrate range is limited to glucose, fructose and sucrose. Recent developments in the genetics and biotechnology of this ethanologen are reviewed.

Isolation and characterization of a cellulolytic actinomycete Microbispora bispora

SummaryProtocols for the isolation of cellulolytic actinomycetes are described, and their use illustrated in the selection of thermophilic bacteria from soil. One isolate, Microbispora bispora, was selected for further study. It grew readily at 55°C, produced an extracellular cellulase in good yield (endoglucanase, 5.9 U/ml) that had a broad pH range (pH 5.5–7.2) and was thermally stable. Its aryl-β-glucosidase was cell-associated and was relatively resistant to end-product inhibition.

CHITOSANASES: OCCURRENCE, PRODUCTION AND IMMOBILIZATION

Microbial soil populations were found to contain chitosan-degrading bacteria comprising 1-7% of the heterotrophic bacteria. The basis for this considerable component of the total microbial population is discussed. The production of chitosanase from one such lytic soil bacterium, Bacillus circulans, was optimized. The enzyme was efficiently purified through affinity chromatography and its general characteristics determined. The chitosanase was immobilized by a variety of methods, the best being via covalent binding to carbodiimidazole-activated agarose beads. The role of chito sanas es within the context of microbial mineralization of chitin and chitosan is discussed.

Sphaeroplast formation and regeneration in Trichoderma reesei

SummarySphaeroplasts from several genetically marked strains of Trichoderma reesei were readily prepared through enzymatic hydrolysis of mycelial suspensions utilizing Driselase, a commercially available lytic enzyme preparation. Sphaeroplasts were released from the apical tips of hyphae after 90 min exposure to the enzyme and, with longer treatments, from other regions of the hyphae. The efficiency of sphaeroplast formation was dependent upon a number of factors. Young mycelium was far more susceptible to lysis than older hyphae. Additionally, the yields of sphaeroplasts were directly proportional to the mycelial concentration within the range tested (0.7–7.0 mg dry weight of mycelium). Ammonium sulfate (0.6 M) was the most effective osmotic support in relation to sphaeroplast stability during their release from the mycelium and subsequent purification. The most prominent enzymatic activities of Driselase were endo-β-(1→4)-glucanase, β-glucosidase and β-(1→3)-glucanase. No chitinase activity was detected. The combined application of Driselase and chitinase (Streptomyces griseus) to mycelial suspensions resulted in increased sphaeroplast yields from young (18 h) as well as older (24 h) mycelia. The parameters defined here allow the rapid (<6h), inexpensive production of 107 sphaeroplasts/ml. Regeneration of T. reesei sphaeroplasts proceeded by the production of chains of sphaeroplast-like cells followed by true hyphal formation. The frequency of regeneration to mycelial form was dependent upon the length of exposure of mycelium to the lytic enzyme. Less than 1% of the sphaeroplasts prepared from 24 h hydrolytic treatment of mycelia regenerate to form colonies while virtually 100% of the viable sphaeroplasts prepared from 6 h treatment form colonies in regeneration medium.