Fermentation of Cellodextrins by Different Yeast Strains

The fermentation of cellodextrins by eight yeast species capable of fermenting cellobiose was monitored. Only two of these species, Torulopsis molischiana and T. wickerhamii, were able to ferment β-glucosides with a degree of polymerization between one and six. These two species showed exocellular β-glucosidase activity. Four other species were able to ferment cellotriose, and the last two species only fermented cellobiose. These latter six species produced a β-glucosidase capable of attacking cellodextrins, but this enzyme was endocellular.     

Characterization of a multi-function processive endoglucanase CHU_2103 from Cytophaga hutchinsonii

Cytophaga hutchinsonii is a Gram-negative gliding bacterium which can efficiently degrade crystalline cellulose by an unknown strategy. Genomic analysis suggests the C. hutchinsonii genome lacks homologs to an obvious exoglucanase that previously seemed essential for cellulose degradation. One of the putative endoglucanases, CHU_2103, was successfully expressed in Escherichia coli JM109 and identified as a processive endoglucanase with transglycosylation activity. It could hydrolyze carboxymethyl cellulose (CMC) into cellodextrins and rapidly decrease the viscosity of CMC. When regenerated amorphous cellulose (RAC) was degraded by CHU_2103, the ratio of the soluble to insoluble reducing sugars was 3.72 after 3 h with cellobiose and cellotriose as the main products, indicating that CHU_2103 was a processive endoglucanase. CHU_2103 could degrade cellodextrins of degree of polymerization ≥3. It hydrolyzed p-nitrophenyl β-d-cellodextrins by cutting glucose or cellobiose from the non-reducing end. Meanwhile, some larger-molecular-weight cellodextrins could be detected, indicating it also had transglycosylation activity. Without carbohydrate-binding module (CBM), CHU_2103 could bind to crystalline cellulose and acted processively on it. Site-directed mutation of CHU_2103 demonstrated that the conserved aromatic amino acid W197 in the catalytic domain was essential not only for its processive activity, but also its cellulose binding ability.     

Expression and characterization of Pichia etchellsii β-glucosidase in Escherichia coli

The β-glucosidase enzyme is important as the terminal enzyme involved in hydrolysis of cellobiose and short-chain cellodextrins generated during enzymatic cellulose degradation. Under controlled reaction conditions the enzyme also displays cello-oligosaccharide synthesizing ability (based on either the thermodynamic or kinetic approach). We present here the purification of the enzyme β-glucosidase (BGL) of Pichia etchellsii from recombinant pBG55 Escherichia coli clone. The kinetic parameters, substrate specificity and oligosaccharide synthesizing ability of the purified enzyme are also reported. The purified 200-kDa protein (tetramer of 50 kDa) was identified as a broad-substrate-specificity enzyme exhibiting increased temperature and glucose tolerance compared to the native yeast enzyme. Temperature directed substrate specificity for aryl β,1–4 linkage, and β(1–2), β(1–4), β(1–6) and β(2-1) linkages in various natural disaccharides was observed. Glycosylation of the enzyme was found to be unimportant for enzyme activity. With both cellobiose and glucose, oligosaccharide synthesis was detected. The implications of this information with regard to cellulose hydrolysis and oligosaccharide synthesis are discussed.     

Cellodextrin and laminaribiose ABC transporters in Clostridium thermocellum

Clostridium thermocellum is an anaerobic thermophilic bacterium that grows efficiently on cellulosic biomass. This bacterium produces and secretes a highly active multienzyme complex, the cellulosome, that mediates the cell attachment to and hydrolysis of the crystalline cellulosic substrate. C. thermocellum can efficiently utilize only β-1,3 and β-1,4 glucans and prefers long cellodextrins. Since the bacterium can also produce ethanol, it is considered an attractive candidate for a consolidated fermentation process in which cellulose hydrolysis and ethanol fermentation occur in a single process. In this study, we have identified and characterized five sugar ABC transporter systems in C. thermocellum. The putative transporters were identified by sequence homology of the putative solute-binding lipoprotein to known sugar-binding proteins. Each of these systems is transcribed from a gene cluster, which includes an extracellular solute-binding protein, one or two integral membrane proteins, and, in most cases, an ATP-binding protein. The genes of the five solute-binding proteins were cloned, fused to His tags, overexpressed, and purified, and their abilities to interact with different sugars was examined by isothermal titration calorimetry. Three of the sugar-binding lipoproteins (CbpB to -D) interacted with different lengths of cellodextrins (G₂ to G₅), with disassociation constants in the micromolar range. One protein, CbpA, binds only cellotriose (G₃), while another protein, Lbp (laminaribiose-binding protein) interacts with laminaribiose. The sugar specificity of the different binding lipoproteins is consistent with the observed substrate preference of C. thermocellum, in which cellodextrins (G₃ to G₅) are assimilated faster than cellobiose.     

Cloning,expression, and characterization of a cellobiose dehydrogenase from Thielavia terrestris induced under cellulose growth conditions

The enzyme cellobiose dehydrogenase (CDH) is of considerable interest, not only for its biotechnological applications, but also its potential biological role in lignocellulosic biomass breakdown. The enzyme catalyzes the oxidation of cellobiose and other cellodextrins, utilizing a variety of one- and two-electron acceptors, although the electron acceptor employed in nature is still unknown. In this study we show that a CDH is present in the secretome of the thermophilic ascomycete Thielavia terrestris when grown with cellulose, along with a mixture of cellulases and hemicellulases capable of breaking down lignocellulosic biomass. We report the cloning of this T. terrestris CDH gene (cbdA), its recombinant expression in Aspergillus oryzae, and purification and characterization of the T. terrestris CDH protein (TtCDH). The TtCDH shows spectral properties and enzyme activity similar to other characterized CDH enzymes. Substrate specificity was determined for a number of carbohydrate electron donors in the presence of the two-electron acceptor 2,6-dichlorophenol-indophenol. The TtCDH also shows dramatic synergy with Thermoascus aurantiacus glycoside hydrolase family 61A protein in the presence of a β-glucosidase for the cleavage of cellulose.     

Biochemical and Electron Microscopic Studies of the Streptomyces reticuli Cellulase (Avicelase) in Its Mycelium-Associated and Extracellular Forms

Streptomyces reticuli is able to grow efficiently with crystalline cellulose (Avicel) as the sole carbon source. Cultivation in the presence of the nonionic detergent Tween 80 at a concentration of 0.1% led to a 10-fold increase in extracellular cellulolytic activity. Under these conditions, one single 82-kDa cellulase (Avicelase) capable of degrading crystalline and soluble cellulose as well as cellodextrins and p-nitrophenylcellobioside was purified to apparent homogeneity by a procedure which consisted of two consecutive anion-exchange chromatographies followed by chromatofocusing. Aggregation, which was a major problem during protein purification, could be avoided by including Triton X-100 at a concentration of 0.1% in every chromatographic step. The Avicelase was identified in extracellular and mycelium-associated forms, the latter of which could be released efficiently by nonionic detergents. In addition, a 42-kDa truncated form retaining cellulolytic activity was identified which had been generated from the 82-kDa enzyme by a protease. Antibodies raised against the mycelium-associated Avicelase reacted with the 42-kDa derivative and the extracellular form. The mycelial association of the enzyme was confirmed by immunofluorescence and immunoelectron microscopies.     

Comparison of the kinetics and factors affecting the stabilities of chitin-immobilized naringinases from two fungal sources

Naringinases from both Penicillium sp. and Aspergillus niger were compared for their enzyme kinetics and the effects of sugars on the enzyme activities. Lineweaver-Burk plots showed that glucose, fructose and rhamnose were all competitive inhibitors for α-rhamnosidase of naringinase from Penicillium sp. and non-competitive inhibitors for the same enzyme from A. niger, When naringinase from Penicillium sp. was immobilized on chitin and used successively for the hydrolysis of p-nitrophenyl-α-rhamnoside or naringin in a simulated fruit juice system or grapefruit juice, it was observed that the enzyme column was very stable. Such results are in contrast to what has bee observed for naringinase fron A. niger. Therefore, it is quite possible that the sugars in the fruit juice which play a role as competitive or non-competitive inhibitors on naringinase may account for the stability of the enzyme column during successive debittering of grapefruits juice.     

The action on cellulose and its derivatives of a purified 1,4-beta-glucanase from Trichoderma koningii.

The specific properties have been examined of the 1,4-beta-glucanase component of Trichoderma koningii that participates in an early and effective stage of random breakdown of native cellulose to short fibres. The enzyme was purified and freed from associated components of the cellulase complex (particularly beta-glucosidase) that interfere with, and complicate interpretation of, the action of such enzymes. Purification increased the specific activity 25-fold over culture filtrates; the enzyme hydrolysed CM-cellulose faster than the purified beta-glucosidase from the same organism hydrolysed any of its substrates (cellobiose or cellodextrins). The specificity of the glucanase was directed towards soluble derivatives of cellulose, CM-cellulose and cellodextrins, and not to insoluble cellulose or alpha-linked polymers. The approximate Km was 2.5 mg of CM-cellulose . ml-1 at 37 degrees C at the optimum pH, 5.5, where enzymic activity was maximal with 6--7 mg of CM-cellulose . ml-1 and inhibited by higher concentrations. The temperature optimum was 60 degrees C. The glucanase attacked larger cellodextrins (cellohexaose to cellotetraose, in that order) much more readily than smaller dextrins (cellobiose and cellotriose) and released a mixture of products, glucose up to cellopentaose, which was quantitatively determined after chromatography on charcoal. Similar examination of hydrolysates of the reduced cellodextrins showed clearly the high specificity of the enzyme for the central bond of its natural substrates (the cellodextrins), whatever their chain length, and indicated the nature of the enzyme as an endoglucanase. Outer bonds shared a weaker, but similar, susceptibility to enzymic cleavage. Transferase activity was absent and no larger dextrins than the initial substrate were formed.     

Cellulose synthesis by Acetobacter xylinum III. Matrix,primer and lipid requirements and heat stability of the cellulose-forming enzymes

The addition of soluble cellodextrins of increasing size to a cell envelope preparation of Acetobacter xylinum stimulated cellulose synthesis from UDPG. This stimulation was attributed to both acceptor and activator effects. Enzymes required for cellulose synthesis were found to be heat-unstable and those required for synthesis of glycosylated lipid components from UDPG, heat-stable. Both heat-inactivated envelope fragments and supernatant fluid from whole cells were necessary for cellulose synthesis from UDPG. Cellulose was not formed from UDPG in the presence of either supernatant fluid alone or heat-inactivated envelopes alone.The combined results of this and previous studies suggest that either the cell envelope is necessary for synthesis of a more immediate precursor to cellulose than UDPG, or that the synthesis from UDPG requires a matrix. The former suggestion and its possible link with lipid intermediate involvement was strengthened by the observation of inefficient glycoxylated lipid formation by a celluloseless mutant strain of A. xylinum. The possible locations of various enzyme activities required for the synthesis of the cellulose precursor are indicated and a possible microfibril nucleation process is discussed.     

Purification and properties of cellobiose phosphorylase from Clostridium thermocellum

Cellobiose phosphorylase was purified 111-fold from a cell extract of Clostridium thermocellum ATCC 27405, with a yield of 31.4%, to electrophoretic and column chromatographic homogenity. The molecular weight of the enzyme was estimated to be 150,000 by gel filtration and 85,000 by SDS-PAGE, suggesting that it consisted of two identical subunits. It was suggested by spectrophotometric and chemical analyse that the enzyme contained no pyridoxal phosphate. The enzyme was inactivated by N-ethylmaleimide and activated by dithiothreitol, indicating that the exposed thiol group(s) was important for the enzymatic activity. The enzyme could utilize, so far as examined, d-glucose, d-xylose, 2-deoxy-d-glucose, and d-mannose, as acceptors of glucose in the synthetic reaction of disaccharides. The enzyme could to a low degree utilize d-arabinose and d-fucose, as acceptors.     

Purification and characterization of 1-aminocyclopropane-1-carboxylate synthase from apple fruits

1-Aminocyclopropane-1-carboxylate (ACC) synthase, a key enzyme in ethylene biosynthesis, was isolated and partially purified from apple (Malus sylvestris Mill.) fruits. Unlike ACC synthase isolated from other sources, apple ACC synthase is associated with the pellet fraction and can be solubilized in active form with Triton X-100. Following five purification steps, the solubilized enzyme was purified over 5000-fold to a specific activity of 100 micromoles per milligram protein per hour, and its purity was estimated to be 20 to 30%. Using this preparation, specific monoclonal antibodies were raised. Monoclonal antibodies against ACC synthase immunoglobulin were coupled to protein-A agarose to make an immunoaffinity column, which effectively purified the enzyme from a relatively crude enzyme preparation (100 units per milligram protein). As with the tomato enzyme, apple ACC synthase was inactivated and radiolabeled by its substrate S-adenosyl-l-methionine. Apple ACC synthase was identified to be a 48-kilodalton protein based on the observation that it was specifically bound to immunoaffinity column and it was specifically radiolabeled by its substrate S-adenosyl-l-methionine.     

Three‐Enzyme Phosphorylase Cascade for Integrated Production of Short‐Chain Cellodextrins

Cellodextrins are linear β‐1,4‐gluco‐oligosaccharides that are soluble in water up to a degree of polymerization (DP) of ≈6. Soluble cellodextrins have promising applications as nutritional ingredients. A DP‐controlled, bottom‐up synthesis from expedient substrates is desired for their bulk production. Here, a three‐enzyme glycoside phosphorylase cascade is developed for the conversion of sucrose and glucose into short‐chain (soluble) cellodextrins (DP range 3–6). The cascade reaction involves iterative β‐1,4‐glucosylation of glucose from α‐glucose 1‐phosphate (αGlc1‐P) donor that is formed in situ from sucrose and phosphate. With final concentration and yield of the soluble cellodextrins set as targets for biocatalytic synthesis, three major factors of reaction efficiency are identified and partly optimized: the ratio of enzyme activity, the ratio of sucrose and glucose, and the phosphate concentration used. The efficient use of the phosphate/αGlc1‐P shuttle for cellodextrin production is demonstrated and the soluble product at 40 g L^?1 is obtained under near‐complete utilization of the donor substrate offered (88 mol% from 200 mm sucrose). The productivity is 16 g (L h)^?1. Through a simple two‐step route, the soluble cellodextrins are recovered from the reaction mixture in ≥95% purity and ≈92% yield. Overall, this study provides the basis for their integrated production.     

Marine bacterial proteases. I. Characterization of an endopeptidase produced by Vibrio B-30

A purification procedure is described for a highly active endopeptidase produced by a marine bacterium (Vibrio B-30). The purified enzyme was shown to be homogeneous by ion-exchange chromatography, gel filtration, and electrophoresis. A crystalline preparation was obtained. The pH optimum of the enzyme was about 7.0, and it was stable in the pH range of 5.0–8.5. Using hemoglobin as the substrate, a K_m of 0.095 mm was obtained. The temperature optimum of the enzyme was 40 ° in the absence of calcium and about 50 ° in the presence of 10⁻³ m calcium. Calcium both activated and stabilized the enzyme against thermal denaturation. The enzyme was shown to be a serine protease which was irreversibly inhibited by certain metal-complexing agents. Gel filtration studies revealed that Vibrio B-30 endopeptidase had a molecular weight of 49,000 ± 5,000 but it rapidly autolyzed during the second and third passage through a gel column. Removal of a metal ion (probably calcium) resulted in the formation of a high-molecular-weight, enzymatically inactive component and a low-molecular-weight, partially active component.     

Fiber entrapment of naringinase from Penicillium sp. and application to fruit juice debittering

Naringinase from Penicillium sp. was immobilized on cellulose triacetate by the fiber entrapment method. Although the optimum pH (3.7) and optimum temperature (55°C) of the fiber-entrapped enzyme were similar to those of the native form, the immobilized enzyme had better heat stability. Kinetic studies showed that the immobilized enzyme had higher K_m values than its native form. When this immobilized naringinase was successively used in a column reactor for the hydrolysis of ρ-nitrophenyl-α-l-rhamnoside or naringin in a simulated fruit juice system or grapefruit juice, the enzyme column could be operated with satisfactory stability. In addition, when the natural grapefruit juice was recycled through the column reactor, no column blocking or filtering action of the catalyst bed was observed.     

Purification and characterization of glyoxalase II from Hansenula mrakii

Glyoxalase II [S-(2-hydroxyacyl)glutathione hydrolase], one of the components of the glyoxalase system, catalyzes the hydrolysis of S-lactoylglutathione to glutathione and d-lactic acid. The enzyme was partially purified from the yeast Hansenula mrakii IFO 0895 by successive column chromatographies and polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 22,000 daltons by gel-filtration of Sephadex G-150 column chromatography and 24,000 daltons by SDS-polyacrylamide gel electrophoresis. The enzyme was specific to S-lactoyglutathione and S-acetylglutathione. The activity of the enzyme was strongly inhibited by Cu²⁺, p-chloromercuribenzoate and HgCl₂. The enzyme activity was also inhibited by hemimercaptal, a non-enzymatic condensation product between glutathione and methylglyoxal.     

Studies on the boronation of methyl-β-d-cellobioside—a cellulose model

The conversion of phenylboronic acid (PBA) with methyl-β-d-cellobioside (Me-β-d-clb) and cellodextrins (DP_w 12) was investigated to gain a basic understanding of the interactions of boric acid derivatives with oligo- and polyglucans. By means of MS and NMR experiments, it was possible to show a first stage formation of a six-membered ring at C-4 and C-6 of the non-reducing glucose occurs as in the case of monosaccharides. If the amount of reagent is increased the formation of seven-membered rings at the secondary OH moieties is observed. Even the existence of two of these large ring-systems in the direct neighborhood was found. Application of an excess of boronation reagent led to dimerization reactions of Me-β-d-clb via the primary reducing glucose residue as confirmed by DOSY NMR studies. Preliminary ¹³C NMR studies for the interaction of cellodextrins with PBA in DMSO solution confirmed a functionalization at the trans-1,2-diol moieties of these oligomers. The amount of reagent applied may either was shown to lead to soluble products or to insoluble cross-linked material.     

Nonradioactive enzyme measurement by high-performance liquid chromatography of partially purified sugar-1-kinase (glucuronokinase) from pollen of Lilium longiflorum

Here we present a highly sensitive and simple high-performance liquid chromatography (HPLC) method that enables specific quantification of glucuronokinase activity in partially purified extracts from pollen of Lilium longiflorum without radioactive labeled substrates. This assay uses a recombinant UDP-sugar pyrophosphorylase with broad substrate specificity from Pisum sativum (PsUSP) or Arabidopsis thaliana (AtUSP) as a coupling enzyme. Glucuronokinase was partially purified on a DEAE-sepharose column. Kinase activity was measured by a nonradioactive coupled enzyme assay in which glucuronic acid-1-phosphate, produced in this reaction, is used by UDP-sugar pyrophosphorylase and further converted to UDP-glucuronic acid. This UDP-sugar, as well as different by-products, is detected by HPLC with either a strong anion exchange column or a reversed phase C18 column at a wavelength of 260 nm. This assay is adaptive to different kinases and sugars because of the broad substrate specificity of USP. The HPLC method is highly sensitive and allows measurement of kinase activity in the range of pmol min^-1. Furthermore, it can be used for determination of pure kinases as well as crude or partially purified enzyme solutions without any interfering background from ATPases or NADH oxidizing enzymes, known to cause trouble in different photometric assays.     

Affinity chromatography of Pseudomonas salicylate hydroxylase

In order to facilitate the purification of salicylate hydroxylase (salicylate 1-monooxygenase, EC 1.14.13.1) from Pseudomonas sp. RPP (ATCC 29351), an affinity chromatography procedure was developed employing immobilized salicylate as the affinity ligand. The immobilization was achieved by reacting p-aminosalicylate with the N-hydroxysuccinimide ester of Sepharose 4B-6-aminohexanoic acid. When the bacterial crude extract was chromatographed with this affinity column, salicylate hydroxylase was absorbed to the gel while the bulk of protein freely passed through. The absorbed enzyme was subsequently eluted from the affinity column by applying a 0–60 mm sodium salicylate gradient. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzymatically most active fraction of the affinity effluent revealed salicylate hydroxylase was by far the most predominant protein but there were also small amounts of contaminating proteins. However, a virtually homogeneous enzyme preparation was obtained when the crude extract was first fractionated with a DE-52 anion-exchange column followed by the affinity step. The enzyme preparation obtained by this two-step procedure showed a specific activity of 14.9 units/mg and an A₄₅₀:A₃₇₂:A₂₈₀ of 1.01:1:10.23. Because most of the enzymes belonging to the class of external flavoprotein monooxygenase utilize salicylate analogs as substrates and share many other common properties, there is a strong possibility that the salicylate column may be useful for the purification of other member monooxygenases.     

Isolation of a Cellodextrinase from Bacteroides succinogenes

An enzyme which released the cellobiose group from p-nitrophenyl cellobioside was isolated from the periplasmic space of Bacteroides succinogenes grown on Avicel crystalline cellulose in a continuous cultivation system and separated from endoglucanases by column chromatography. The molecular weight of the enzyme was approximately 40,000, as estimated by gel filtration. The enzyme has an isoelectric point of 4.9. The enzyme exhibited low hydrolytic activity on acid-swollen cellulose and practically no activity on carboxymethyl cellulose, Avicel cellulose, and cellobiose, but it hydrolyzed p-nitrophenyl lactoside and released cellobiose from cellotriose and from higher cello-oligosaccharides. These data demonstrate that the enzyme is a cellodextrinase with an exotype of function.     

The Unique Binding Mode of Cellulosomal CBM4 from Clostridium thermocellum Cellobiohydrolase A

The crystal structure of the carbohydrate-binding module (CBM) 4 Ig fused domain from the cellulosomal cellulase cellobiohydrolase A (CbhA) of Clostridium thermocellum was solved in complex with cellobiose at 2.11 Å resolution. This is the first cellulosomal CBM4 crystal structure reported to date. It is similar to the previously solved noncellulosomal soluble oligosaccharide-binding CBM4 structures. However, this new structure possesses a significant feature—a binding site peptide loop with a tryptophan (Trp118) residing midway in the loop. Based on sequence alignment, this structural feature might be common to all cellulosomal clostridial CBM4 modules. Our results indicate that C. thermocellum CbhA CBM4 also has an extended binding pocket that can optimally bind to cellodextrins containing five or more sugar units. Molecular dynamics simulations and experimental binding studies with the Trp118Ala mutant suggest that Trp118 contributes to the binding and, possibly, the orientation of the module to soluble cellodextrins. Furthermore, the binding cleft aromatic residues Trp68 and Tyr110 play a crucial role in binding to bacterial microcrystalline cellulose (BMCC), amorphous cellulose, and soluble oligodextrins. Binding to BMCC is in disagreement with the structural features of the binding pocket, which does not support binding to the flat surface of crystalline cellulose, suggesting that CBM4 binds the amorphous part or the cellulose “whiskers” of BMCC. We propose that clostridial CBM4s have possibly evolved to bind the free-chain ends of crystalline cellulose in addition to their ability to bind soluble cellodextrins.