In vitro evaluation of fibrolytic enzymes as additives for maize (Zea mays L.) silage - III. Comparison of enzymes derived from psychrophilic, mesophilic or thermophilic sources

A completely randomised study was completed to examine the influence of fibrolytic enzymes derived from psychrophilic, (F), mesophilic, (L) or thermophilic (Ta) sources, applied at ensiling, on the chemical characteristics and in vitro rumen fermentation of maize silage, assessed using the Reading Pressure Technique (RPT). Treatments, all in triplicate, consisted of untreated maize forage or treated with preparations F, L, Ta or a mixture (1: 1, v/v) of F and L (FL), at two levels each, and ensiled for 210 days in plastic mini-silos. Addition of enzymes L decreased (P < 0.05) silage pH relative to the control, whereas enzyme Ta tended (P < 0.10) to reduce it. Preparations F, L and Ta tended to reduce (P < 0.10) the fibre contents of the silages, with effects being attributable to a decrease in the cellulose fraction. Starch contents were reduced (P < 0.05) in the treatments including enzyme F. End-point (96 h) gas production (GP) values did not differ among treatments, suggesting that enzymes did not change the total amount of fermentable substrate. However, consistent with the decrease in starch contents, adding enzyme F reduced (P < 0.05) GP at most incubation times. Addition of enzymes increased (P < 0.05) the initial (6 h) organic matter degradation (OMD) levels in all but one treatment (F), with increases of 14, 19, and 26% for preparations L, Ta, and FL, respectively, averaged across levels. Furthermore, the addition of enzymes increased (P < 0.05) the soluble OM losses, however, these increases did not fully account for the initial increase in OMD. The latter suggests that enzymes increased solubility and also altered silage structure, making it more amenable to degradation by ruminal microorganisms. As a result of the increase in OMD, without a concomitant increase in GP, the fermentation efficiency was greatly increased (P < 0.05) in enzyme treatments. Addition of enzymes to maize at ensiling, particularly those from the mesophilic and thermophilic sources used here, have the potential to increase the initial rate of silage OMD. (C) 2003 Elsevier B.V. All rights reserved.

Influence of fibrolytic enzymes on the hydrolysis and fermentation of pure cellulose and xylan by mixed ruminal microorganisms in vitro

A series of in vitro studies was, conducted to determine the effects of adding a commercial enzyme product on the hydrolysis and fermentation of cellulose, xylan, and a mixture (1:1 wt/wt) of both. The enzyme product (Liquicell 2500, Specialty Enzymes and Biochemicals, Fresno, CA) was derived from Trichoderma reesei and contained mainly xylanase and cellulase activities. Addition of enzyme (0.5, 2.55 and 5.1 muL/g of DM) in the absence of ruminal fluid increased (P < 0.001) the release of reducing sugars from xylan and the mixture after 20 h of incubation at 20degreesC. Incubations with ruminal fluid showed that enzyme (0.5 and 2.55 muL/g of DM) increased (P < 0.05) the initial (up to 6 h) xylanase, endoglucanase, and beta-D-glucosidase activities in the liquid fraction by an average of 85%. Xylanase and endoglucanase activities in the solid fraction also were increased (P < 0.05) by enzyme addition, indicating an increase in fibrolytic activity due to ruminal microbes. Gas production over 96 h of incubation was determined using a gas pressure measurement technique. Incremental levels of enzyme increased (P < 0.05) the rate of gas production of all substrates, suggesting that fermentation of cellulose and xylan was enzyme-limited. However, adding the enzyme at levels higher than 2.55 muL/g of DM failed to further increase the rate of gas production, indicating that the maximal level of stimulation was already achieved at lower enzyme concentrations. It was concluded that enzymes enhanced the fermentation of cellulose and xylan by a combination of pre- and postincubation effects (i.e., an increase in the release of reducing sugars during the pretreatment phase and an increase in the hydrolytic activity of the liquid and solid fractions of the ruminal fluid), which was reflected in a higher rate of fermentation.

In vitro evaluation of fibrolytic enzymes as additives for maize (Zea mays L.) silage - I. Effects of ensiling temperature, enzyme source and addition level

A series of experiments was completed to investigate the impact of addition of enzymes at ensiling on in vitro rumen degradation of maize silage. Two commercial products, Depot 40 (D, Biocatalysts Ltd., Pontypridd, UK) and Liquicell 2500 (L, Specialty Enzymes and Biochemicals, Fresno, CA, USA), were used. In experiment 1, the pH optima over a pH range 4.0-6.8 and the stability of D and L under changing pH (4.0, 5.6, 6.8) and temperature (15 and 39 degreesC) conditions were determined. In experiment 2, D and L were applied at three levels to whole crop maize at ensiling, using triplicate 0.5 kg capacity laboratory minisilos. A completely randomized design with a factorial arrangement of treatments was used. One set of treatments was stored at room temperature, whereas another set was stored at 40 degreesC during the first 3 weeks of fermentation, and then stored at room temperature. Silages were opened after 120 days. Results from experiment I indicated that the xylanase activity of both products showed an optimal pH of about 5.6, but the response differed according to the enzyme, whereas the endoglucanase activity was inversely related to pH. Both products retained at least 70% of their xylanase activity after 48 h incubation at 15 or 39 degreesC. In experiment 2, enzymes reduced (P < 0.05) silage pH, regardless of storage temperature and enzyme level. Depol 40 reduced (P < 0.05) the starch contents of the silages, due to its high alpha-amylase activity. This effect was more noticeable in the silages stored at room temperature. Addition of L reduced (P < 0.05) neutral detergent fiber (NDF) and acid detergent fiber (ADF) contents. In vitro rumen degradation, assessed using the Reading Pressure Technique (RPT), showed that L increased (P < 0.05) the initial 6 h gas production (GP) and organic matter degradability (OMD), but did not affect (P > 0.05) the final extent of OMD, indicating that this preparation acted on the rumen degradable material. In contrast, silages treated with D had reduced (P < 0.05) rates of gas production and OMD. These enzymes, regardless of ensiling temperature, can be effective in improving the nutritive quality of maize silage when applied at ensiling. However, the biochemical properties of enzymes (i.e., enzymic activities, optimum pH) may have a crucial role in dictating the nature of the responses. (C) 2003 Elsevier B.V. All rights reserved.

In vitro evaluation of fibrolytic enzymes as additives for maize (Zea mays L.) silage - II. Effects on rate of acidification, fibre degradation during ensiling and rumen fermentation

A study was carried out to determine the influence of fibrolytic enzymes derived from mesophilic or thermophilic fungal sources, added at ensiling, on time-course fermentation characteristics and in vitro rumen degradation of maize silage. The mesophilic enzyme was a commercial product derived from Trichodenna reesei (L), whereas the thermophilic enzyme was a crude extract produced from Thermoascus aurantiacus (Ta) in this laboratory. The fungus was cultured using maize cobs as a carbon source. The resulting fermentation extract was deionised to remove sugars and characterised for its protein concentration, main and side enzymic activities, optimal pH, protein molecular mass and isoelectric point. In an additional study, both enzymes were added to maize forage (333.5 g DM/kg, 70.0, 469.8, 227.1 and 307.5 g/kg DM of CP, NDF, ADF and starch, respectively) at two levels each, normalized according to xylanase activity, and ensiled in 0.5 kg capacity laboratory minisilos. Duplicate silos were opened at 2, 4, 8, 15, and 60 days after ensiling, and analysed for chemical characteristics. Silages from 60 days were bulked and in vitro gas production (GP) and organic matter degradability (OMD) profiles evaluated using the Reading Pressure Technique (RPT), in a completely randomised design. The crude enzyme extract contained mainly xylanase and endoglucanase activities, with very low levels of exoglucanase, which probably limited hydrolysis of filter paper. The extract contained three major protein bands of between 29 and 55 kDa, with mainly acidic isoelectric points. Ensiling maize with enzymes lowered (P < 0.05) the final silage pH, with this effect being observed throughout the ensiling process. All enzyme treatments reduced (P < 0.05) ADF contents. Treatments including Ta produced more gas (P < 0.05) than the controls after 24 h incubation in vitro, whereas end point gas production at 96 h was not affected. Addition of Ta increased (P < 0.01) OMD after 12 h (410 and 416 g/kg versus 373 g/kg), whereas both L and Ta increased (P < 0.05) OMD after 24 h. Addition of enzymes from mesophilic or thermophilic sources to maize forage at ensiling increased the rate of acidification of the silages and improved in vitro degradation kinetics, suggesting an improvement in the nutritive quality. (C) 2003 Elsevier B.V All rights reserved.

Modification of hemicellulose content by antisense down-regulation of UDP-glucuronate decarboxylase in tobacco and its consequences for cellulose extractability

Extractability and recovery of cellulose from cell walls influences many industrial processes and also the utilisation of biomass for energy purposes. The utility of genetic manipulation of lignin has proven potential for optimising such processes and is also advantageous for the environment. Hemicelluloses, particularly secondary wall xylans, also influence the extractability of cellulose. UDP-glucuronate decarboxylase produces UDP-xylose, the precursor for xylans and the effect of its down-regulation on cell wall structure and cellulose extractability in transgenic tobacco has been investigated. Since there are a number of potential UDP-glucuronate decarboxylase genes, a 490 bp sequence of high similarity between members of the family, was chosen for general alteration of the expression of the gene family. Sense and antisense transgenic lines were analysed for enzyme activity using a modified and optimised electrophoretic assay, for enzyme levels by western blotting and for secondary cell wall composition. Some of the down-regulated antisense plants showed high glucose to xylose ratios in xylem walls due to less xylose-containing polymers, while arabinose and uronic acid contents, which could also have been affected by any change in UDP-xylose provision, were unchanged. The overall morphology and stem lignin content of the modified lines remained little changed compared with wild-type. However, there were some changes in vascular organisation and reduction of xylans in the secondary walls was confirmed by immunocytochemistry. Pulping analysis showed a decreased pulp yield and a higher Kappa number in some lines compared with controls, indicating that they were less delignified, although the level of residual alkali was reduced. Such traits probably indicate that lignin was less available for removal in a reduced background of xylans. However, the viscosity was higher in most antisense lines, meaning that the cellulose was less broken-down during the pulping process. This is one of the first studies of a directed manipulation of hemicellulose content on cellulose extractability and shows both positive and negative outcomes.

Modification of hemicellulose content by antisense down-regulation of UDP-glucuronate decarboxylase in tobacco and its conscequence for cellulose extractability

Extractability and recovery of cellulose from cell walls influences many industrial processes and also the utilisation of biomass for energy purposes. The utility of genetic manipulation of lignin has proven potential for optimising such processes and is also advantageous for the environment. Hemicelluloses, particularly secondary wall xylans, also influence the extractability of cellulose. UDP-glucuronate decarboxylase produces UDP-xylose, the precursor for xylans and the effect of its down-regulation on cell wall structure and cellulose extractability in transgenic tobacco has been investigated. Since there are a number of potential UDP-glucuronate decarboxylase genes, a 490 bp sequence of high similarity between members of the family, was chosen for general alteration of the expression of the gene family. Sense and antisense transgenic lines were analysed for enzyme activity using a modified and optimised electrophoretic assay, for enzyme levels by western blotting and for secondary cell wall composition. Some of the down-regulated antisense plants showed high glucose to xylose ratios in xylem walls due to less xylose-containing polymers, while arabinose and uronic acid contents, which could also have been affected by any change in UDP-xylose provision, were unchanged. The overall morphology and stem lignin content of the modified lines remained little changed compared with wild-type. However, there were some changes in vascular organisation and reduction of xylans in the secondary walls was confirmed by immunocytochemistry. Pulping analysis showed a decreased pulp yield and a higher Kappa number in some lines compared with controls, indicating that they were less delignified, although the level of residual alkali was reduced. Such traits probably indicate that lignin was less available for removal in a reduced background of xylans. However, the viscosity was higher in most antisense lines, meaning that the cellulose was less broken-down during the pulping process. This is one of the first studies of a directed manipulation of hemicellulose content on cellulose extractability and shows both positive and negative outcomes.

Cellulose microfibril angle in the cell wall of wood fibres

The term microfibril angle (MFA) in wood science refers to the angle between the direction of the helical windings of cellulose microfibrils in the secondary cell wall of fibres and tracheids and the long axis of cell. Technologically, it is usually applied to the orientation of cellulose microfibrils in the S2 layer that makes up the greatest proportion of the wall thickness, since it is this which most affects the physical properties of wood. This review describes the organisation of the cellulose component of the secondary wall of fibres and tracheids and the various methods that have been used for the measurement of MFA. It considers the variation of MFA within the tree and the biological reason for the large differences found between juvenile (or core) wood and mature (or outer) wood. The ability of the tree to vary MFA in response to environmental stress, particularly in reaction wood, is also described. Differences in MFA have a profound effect on the properties of wood, in particular its stiffness. The large MFA in juvenile wood confers low stiffness and gives the sapling the flexibility it needs to survive high winds without breaking. It also means, however, that timber containing a high proportion of juvenile wood is unsuitable for use as high-grade structural timber. This fact has taken on increasing importance in view of the trend in forestry towards short rotation cropping of fast grown species. These trees at harvest may contain 50% or more of timber with low stiffness and therefore, low economic value. Although they are presently grown mainly for pulp, pressure for increased timber production means that ways will be sought to improve the quality of their timber by reducing juvenile wood MFA. The mechanism by which the orientation of microfibril deposition is controlled is still a matter of debate. However, the application of molecular techniques is likely to enable modification of this process. The extent to which these techniques should be used to improve timber quality by reducing MFA in juvenile wood is, however, uncertain, since care must be taken to avoid compromising the safety of the tree.

Cellulose microfibril angle in the cell wall of wood fibres

The term microfibril angle (MFA) in wood science refers to the angle between the direction of the helical windings of cellulose microfibrils in the secondary cell wall of fibres and tracheids and the long axis of cell. Technologically, it is usually applied to the orientation of cellulose microfibrils in the S2 layer that makes up the greatest proportion of the wall thickness, since it is this which most affects the physical properties of wood. This review describes the organisation of the cellulose component of the secondary wall of fibres and tracheids and the various methods that have been used for the measurement of MFA. It considers the variation of MFA within the tree and the biological reason for the large differences found between juvenile (or core) wood and mature (or outer) wood. The ability of the tree to vary MFA in response to environmental stress, particularly in reaction wood, is also described. Differences in MFA have a profound effect on the properties of wood, in particular its stiffness. The large MFA in juvenile wood confers low stiffness and gives the sapling the flexibility it needs to survive high winds without breaking. It also means, however, that timber containing a high proportion of juvenile wood is unsuitable for use as high-grade structural timber. This fact has taken on increasing importance in view of the trend in forestry towards short rotation cropping of fast grown species. These trees at harvest may contain 50% or more of timber with low stiffness and therefore, low economic value. Although they are presently grown mainly for pulp, pressure for increased timber production means that ways will be sought to improve the quality of their timber by reducing juvenile wood MFA. The mechanism by which the orientation of microfibril deposition is controlled is still a matter of debate. However, the application of molecular techniques is likely to enable modification of this process. The extent to which these techniques should be used to improve timber quality by reducing MFA in juvenile wood is, however, uncertain, since care must be taken to avoid compromising the safety of the tree.

Mechanical effects of plant cell wall enzymes on xyloglucan/cellulose composites

Xyloglucan-acting enzymes are believed to have effects on type I primary plant cell wall mechanical properties. In order to get a better understanding of these effects, a range of enzymes with different in vitro modes of action were tested against cell wall analogues (bio-composite materials based on Acetobacter xylinus cellulose and xyloglucan). Tomato pericarp xyloglucan endo transglycosylase (tXET) and nasturtium seed xyloglucanase (nXGase) were produced heterologously in Pichia pastoris. Their action against the cell wall analogues was compared with that of a commercial preparation of Trichoderma endo-glucanase (EndoGase). Both 'hydrolytic' enzymes (nXGase and EndoGase) were able to depolymerise not only the cross-link xyloglucan fraction but also the surface-bound fraction. Consequent major changes in cellulose fibril architecture were observed. In mechanical terms, removal of xyloglucan cross-links from composites resulted in increased stiffness (at high strain) and decreased visco-elasticity with similar extensibility. On the other hand, true transglycosylase activity (tXET) did not affect the cellulose/xyloglucan ratio. No change in composite stiffness or extensibility resulted, but a significant increase in creep behaviour was observed in the presence of active tXET. These results provide direct in vitro evidence for the involvement of cell wall xyloglucan-specific enzymes in mechanical changes underlying plant cell wall re-modelling and growth processes. Mechanical consequences of tXET action are shown to be complimentary to those of cucumber expansin.

Miscibility studies of the blends of chitosan with some cellulose ethers

The polymeric films have been prepared based on blends of chitosan with two cellulose ethers-hydroxypropylmethylcellulose and methylcellulose by casting from acetic acid solutions. The films were transparent and brittle in a dry state but an immersion of the samples in deionized water for over 24 h leads to their disintegration or partial dissolution. The miscibility of the polymers in the blends has been assessed by infrared spectroscopy, wide-angle X-ray diffraction, scanning electron microscopy and thermal gravimetric analysis. It was shown that although weak hydrogen bonding exists between the polymer functional groups the blends are not fully miscible in a dry state. (c) 2005 Elsevier Ltd. All rights reserved.

Design of mucoadhesive polymeric films based on blends of poly(acrylic acid) and (hydroxypropyl)cellulose

Mixing of aqueous solutions of poly(acrylic acid) and (hydroxypropyl) cellulose results in formation of hydrogen-bonded interpolymer complexes, which precipitate and do not allow preparation of homogeneous polymeric films by casting. In the present work the effect of pH on the complexation between poly(acrylic acid) and (hydroxypropyl)cellulose in solutions and miscibility of these polymers in solid state has been studied. The pH-induced complexation-miscibility-immiscibility transitions in the polymer mixtures have been observed. The optimal conditions for preparation of homogeneous polymeric films based on blends of these polymers have been found, and the possibility of radiation cross-linking of these materials has been demonstrated. Although the gamma-radiation treatment of solid polymeric blends was found to be inefficient, successful cross-linking was achieved by addition of N, N'- methylenebis(acrylamide). The mucoadhesive potential of both soluble and cross-linked films toward porcine buccal mucosa is evaluated. Soluble films adhered to mucosal tissues undergo dissolution within 30-110 min depending on the polymer ratio in the blend. Cross-linked films are retained on the mucosal surface for 10-40 min and then detach.

Synthesis of prebiotic galactooligosaccharides from lactose using bifidobacterial β-galactosidase (BbgIV) immobilised on DEAE-Cellulose, Q-Sepharose and amino-ethyl agarose

The bifidobacterial β-galactosidase BbgIV was immobilised on DEAE-Cellulose and Q-Sepharose via ionic binding and on amino-ethyl- and glyoxal-agarose via covalent attachment, and was then used to catalyse the synthesis of galactooligosaccharides (GOS). The immobilisation yield exceeded 90 % using ionic binding, while it was low using aminoethyl agarose (25 – 28 %) and very low using glyoxal agarose (< 3 %). This was due to the mild conditions and absence of chemical reagents in ionic binding, compared to covalent attachment. The maximum GOS yield obtained using DEAE-Cellulose and Q-Sepharose was similar to that obtained using free BbgIV (49 – 53 %), indicating the absence of diffusion limitation and mass transfer issues. For amino-ethyl agarose, however, the GOS yield obtained was lower (42 – 44 %) compared to that obtained using free BbgIV. All the supports tried significantly (P < 0.05) increased the BbgIV operational stability and the GOS synthesis productivity up to 55 °C. Besides, six successive GOS synthesis batches were performed using BbgIV immobilised on Q-Sepharose; all resulted in similar GOS yields, indicating the possibility of developing a robust synthesis process. Overall, the GOS synthesis operation performance using BbgIV was improved by immobilising the enzyme onto solid supports, in particular on Q-Sepharose