Monocomponent Enzymes Research Articles

Confirmation of Cage Effect and Prebiotic Production Potential of a β-Mannanase, with SBM as Substrate Using Microscopy and Wet Chemistry

In vitro assays were carried out to investigate the solubilization of cell walls and generation of mannan oligosaccharides of a b-mannanase-containing commercial product on SBM. Using commercial dosages of the b-mannanase (500 g per ton of feed) cell wall degradation of mannan in SBM cell walls was visualized and an increase in reducing ends (0.12±0.02 mg/mL) and the generation of mannan oligosaccharides of degree of polymerization 2 and 4 (22.9±3.2 mg/L and 398.8±25.4 mg/L) were also measured using HPLC. Mannan, which is H-bonded to cellulose and xyloglucan, was solubilized using a single monocomponent enzyme, allowing for visualization of the disintegration of the entire SBM cell wall structure. This work is the first of its kind using strictly commercial dosage levels of enzyme for evaluating efficacy of the same microscopically. These data confirm the hypothesis that there most likely is a need for only a single relevant NSP enzyme targeting its specific substrate, independent of the concentration of the latter within the complex polysaccharide matrix in the plant cell wall to experience the beneficial effects of the enzyme both in vitro and in vivo. An analogy to compare our data would be destruction of the foundation (mannan) of a building or a bridge (soybean cell wall) which would inevitably lead to dismantling or demolition the entire building or bridge.

Exploring the flexibility of cellulase cocktail obtained from mutant UV-8 of Talaromyces verruculosus IIPC 324 in depolymerising multiple agro-industrial lignocellulosic feedstocks

Effective management and the valorization of agro-industrial lignocellulosic feedstocks can only be realized if a versatile cellulase cocktail is developed that can release glucose at affordable cost irrespective of biomass type. In the present study the flexibility of using cellulase cocktail obtained from mutant UV-8 of Talaromyces verruculosus IIPC 324 in depolymerizing multiple agro-industrial lignocellulosic feedstocks was explored.Five different dilute acid pretreated biomasses were evaluated and cellulase loading was done at 25 mg protein/g cellulose content. After 72 h of hydrolysis at 55 °C and pH 4.5, corn cob and rice straw emerged as the easiest and toughest substrates with saccharification yield of 83.9 ± 1.17 and 35.5 ± 1.16% respectively from their cellulose fraction. Addition of PEG 6000 could retain >65% of all mono-component enzymes present in cellulase cocktail.Structural elucidation of biomasses gave an insight about key features responsible for variable recalcitrance in the different agro-industrial feedstock. Cellulose hydrolysis showed a significant negative correlation in the order of Cr I > S/G ratio > ash content. The chemical composition of lignin had a major impact on enzyme-lignin interactions. Higher H lignin content and lower S/G ratio promoted enzyme desorption, thereby increasing the likelihood of their recycling and reuse.

Pa-225 モノコンポーネント酵素を組み合わせた高性能バイオマス糖化酵素の調製 : 東大・バイオエタノール革新技術研究組合草本系バイオマスからのエタノール一貫生産技術開発(3)(2.技術,ポスターセッション1)

Pa-225 モノコンポーネント酵素を組み合わせた高性能バイオマス糖化酵素の調製 : 東大・バイオエタノール革新技術研究組合草本系バイオマスからのエタノール一貫生産技術開発(3)(2.技術,ポスターセッション1)

Development of enzyme cocktails for complete saccharification of chitin using mono-component enzymes from Serratia marcescens

Abstract One potential strategy for biorefining of chitin-rich biomass entails enzymatic saccharification, which, so far, has been scarcely explored. Here, saccharification of chitin was explored using response surface methodology available in the MODDE ® 10 software, to develop optimal cocktails of five mono-component enzymes from Serratia marcescens , three chitinases, Sm ChiA, Sm ChiB, Sm ChiC, a lytic polysaccharide monooxygenase, Sm LPMO10A (or “CBP21”), and a beta- N -acetylhexosaminidase, Sm CHB (“chitobiase”). These five enzymes were recombinantly produced in Escherichia coli . For both shrimp and crab chitins, Sm ChiA was the most abundant (40% and 38%, respectively) in the optimized cocktails, whereas Sm ChiB, Sm ChiC and Sm LPMO10A were present at 30% and 26%, 15% and 23%, and 3% and 2%, respectively. Saccharification yields were 70%–75%, whereas a “minimal” cocktail of Sm ChiA and Sm CHB gave only 40% saccharification. These results show that enzymatic saccharification of chitin requires multiple enzyme activities applied at dosages similar to those used for saccharification of cellulose.

Lignin-derived inhibition of monocomponent cellulases and a xylanase in the hydrolysis of lignocellulosics

Non-productive enzyme binding onto lignin is the major inhibitory mechanism, which reduces hydrolysis rates and yields and prevents efficient enzyme recycling in the hydrolysis of lignocellulosics. The detailed mechanisms of binding are still poorly understood. Enzyme-lignin interactions were investigated by comparing the structural properties and binding behaviour of fungal monocomponent enzymes, cellobiohydrolases TrCel7A and TrCel6A, endoglucanases TrCel7B and TrCel5A, a xylanase TrXyn11 and a β-glucosidase AnCel3A, onto lignins isolated from steam pretreated spruce and wheat straw. The enzymes exhibited decreasing affinity onto lignin model films in the following order: TrCel7B>TrCel6A>TrCel5A>AnCel3A>TrCel7A>TrXyn11. As analysed in Avicel hydrolysis, TrCel6A and TrCel7B were most inhibited by lignin isolated from pretreated spruce. This could be partially explained by adsorption of the enzyme onto the lignin surface. Enzyme properties, such as enzyme surface charge, thermal stability or surface hydrophobicity could not alone explain the adsorption behaviour.

A commercial GH 11 xylanase mediates xylan solubilisation and degradation in wheat, rye and barley as demonstrated by microscopy techniques and wet chemistry methods

Xylanases in animal feed are primarily utilized to alleviate the antinutritional effects of arabinoxylan, a predominant polymer in cereal cell walls consisting of a β-1,4-xylose polymer, substituted with various sugars. Although xylanases efficiently hydrolyze unsubstituted and substituted arabinoxylans, currently available enzymes are not always able to access highly decorated forms of the polysaccharide, such as arabinoxylans (AX) that contain arabinofuranose substitutions. The highly substituted regions are enriched in both 2- and 3-disubstituted xylose residues. Wheat AX contains very small amounts of 2-monosubstituted xylose residues, whereas barley AXs are characterised by a larger amount of 2-monosubstitution. The structure of rye AX is similar to that of wheat AX, but differences in the abundances of the 2- monosubstituted and 2-and 3- disubstituted xylose residues have been reported. In the current study the efficiency of a monocomponent xylanase was demonstrated on structurally different arabinoxylans present in wheat, rye and barley kernels using microscopic visualization, viscosity and arabinoxylan solubilisation measured as the sum of arabinose and xylose in an analysis of the non-starch polysaccharide (NSP) constituents, as well as xylose alone. Degradation of arabinoxylan components upon exposure to the enzyme was visualized by scanning electron microscopy, autofluorescence and with fluorescence immunocytochemistry and confocal microscopy using commercial antibodies recognizing arabinoxylan in the cereal cell walls. Viscosity data clearly demonstrated effective depolymerisation of arabinoxylans in water extracts from wheat and rye and thereby hydrolytic capacity of the monocomponent enzyme on structurally diverse arabinoxylans. The viscosity reducing effect observed in barley was limited, most likely due to the presence of highly viscous mixed linked ß-d-glucans. Wet chemistry analyses of the complete solubilisation of arabinoxylan (oligomers and polysaccharides) using a standardized xylose assay as well as NSP analyses (measuring polysaccharides, DP>10) on wheat, rye and barley, provided additional and complementary information to the microscopy conducted on the same cereal samples.The study has shown that microscopy techniques visualizing cell wall solubilising effects of xylanase can be useful for understanding enzyme mode of action and efficacy on different feed ingredients.

Toward optimal value creation through the application of exogenous mono-component protease in the diets of non-ruminants

Exogenous protease has formed part of several commercial enzyme admixtures for the past 15–20 years but has become available as a mono-component enzyme only in the last 5–10 years (Cowieson and Adeola, 2005; Fru-Nji et al., 2011). As part of an enzyme admixture (often also containing various carbohydrases and/or phytase) it is difficult to separate the contributions of the component parts and the efficacy of each has implicit rather than explicit value. However, the effectiveness of mono-component enzymes can be readily assessed under different production and nutritional circumstances and their complementarity with alternative enzymes is readily rendered transparent. The motivation for the use of exogenous protease in non-ruminant nutrition is typically the reduction of feed cost at constant animal performance. This objective is usually achieved via displacement of expensive protein/amino acid sources in the diet through the assignment of a nutrient matrix on the enzyme in least cost formulation. Recently attention has moved from this classical approach to protease application to so-called ‘extra-proteinaceous’ effects including environmental benefits, litter management, enteric resilience, uniformity and microbial stability. Whilst these ‘secondary’ effects may not be the principal motivator for the use of proteases in animal production they are increasingly visible and require explanation. Furthermore, these responses, if understood, allow positive translation to production metrics such as reduced FCR and improved weight gain. It is the purpose of this review to briefly summarise the current state of the art in mono-component exogenous protease use in non-ruminants and to suggest mechanisms whereby the ‘extra-proteinaceous’ effects may be explained. Finally, key diet/animal factors that may promote beneficial effects of exogenous protease on live performance will be discussed.

Enzymatic generation of galactose-rich oligosaccharides/oligomers from potato rhamnogalacturonan I pectic polysaccharides

Potato pulp by-product rich in galactan-rich rhamnogalacturonan I (RG I) was investigated as a new source of oligosaccharides with potential prebiotic properties. The efficiency of selected monocomponent enzymes and multi-enzymatic preparations to generate oligosaccharides/oligomers from potato RG I was evaluated. These overall results of yield were dependent on the activity profile of the multi-enzymatic preparations. Highest oligo-RG I yield of 93.9% was achieved using multi-enzymatic preparation (Depol 670L) with higher hydrolytic activity toward side chains of RG I as compared to its backbone. Main oligo-RG I products were oligosaccharides with DP of 2–12 (79.8–100%), while the oligomers with DP of 13–70 comprised smaller proportion (0.0–20.2%). Galactose (58.9–91.2%, w/w) was the main monosaccharide of oligo-RG I, while arabinose represented 0.0–12.1%. An understanding of the relationship between the activity profile of multi-enzymatic preparations and the yield/DP of oligo-RG I was achieved. This is expected to provide the capability to generate galacto- and galacto(arabino) oligosaccharides and their corresponding oligomers from an abundant by-product.

Differences in the adsorption of enzymes onto lignins from diverse types of lignocellulosic biomass and the underlying mechanism

BackgroundNon-productive cellulase adsorption onto lignin has always been deemed to negatively affect the enzymatic hydrolysis of lignocellulosic feedstocks. Therefore, understanding enzyme-lignin interactions is essential for the development of enzyme mixtures, the processes of lignocellulose hydrolysis, and the genetic modification of lignocellulosic biomass and enzymes. In this work, we examined the properties of six lignins from diverse types of lignocellulosic biomass (aspen, pine, corn stover, kenaf, and two Arabidopsis lines, wild-type and SALK mutant of fah1) to determine the mechanism of differences in their adsorption of enzymes.ResultsWe found that lignin sources affected enzyme adsorption using structural features, such as functional groups and lignin composition. Guaiacyl (G) lignin had a higher adsorption capacity on enzymes than syringyl (S) lignin. The low S/G ratio and high uniform lignin fragment size had good correlations with high adsorption capacity. A higher content of phenolic hydroxyl groups and a lower content of carboxylic acid groups resulted in stronger adsorption affinity for corn stover lignin (CL) than for kenaf lignin (KL) and aspen lignin (AL). The lower amount of aliphatic hydroxyls that reduced hydrophobic interactions could explain the higher adsorption capacity of pine lignin (PL) than CL. Enzyme activity assays, as well as the hydrolysis of Avicel, phosphoric acid-swollen cellulose (PASC), and holocellulose, were performed to study the behaviors of mono-component enzymes that resulted in adsorption. We found that cellobiohydrolase (CBH) and xylanase were adsorbed the most by all lignins, endoglucanase (EG) showed less inhibition, and β-glucosidase (BG) was the least affected by lignins, indicating the important role of carbohydrate-binding module (CBM) in protein adsorption.ConclusionLignin sources affect enzyme adsorption using structural features and lignin composition, such as S/G ratio, carboxylic acid, aliphatic hydroxyl, and phenolic hydroxyl. For mono-component enzymes, the adsorption capacity decreased in the order CBH, xylanase > EG > BG. These investigations revealed the difference in lignin properties between diverse biomass and adsorption capacity of enzymes to lignins, and the possible underlying mechanism. The results can also serve as a reference for the genetic modification of lignocellulosic biomass and enzymes.

Comparative Analysis of End Point Enzymatic Digests of Arabino-Xylan Isolated from Switchgrass (Panicum virgatum L) of Varying Maturities using LC-MSn.

Switchgrass (Panicum virgatum L., SG) is a perennial grass presently used for forage and being developed as a bioenergy crop for conversion of cell wall carbohydrates to biofuels. Up to 50% of the cell wall associated carbohydrates are xylan. SG was analyzed for xylan structural features at variable harvest maturities. Xylan from each of three maturities was isolated using classical alkaline extraction to yield fractions (Xyl A and B) with varying compositional ratios. The Xyl B fraction was observed to decrease with plant age. Xylan samples were subsequently prepared for structure analysis by digesting with pure endo-xylanase, which preserved side-groups, or a commercial carbohydrase preparation favored for biomass conversion work. Enzymatic digestion products were successfully permethylated and analyzed by reverse-phase liquid chromatography with mass spectrometric detection (RP-HPLC-MSn). This method is advantageous compared to prior work on plant biomass because it avoids isolation of individual arabinoxylan oligomers. The use of RP-HPLC- MSn differentiated 14 structural oligosaccharides (d.p. 3–9) from the monocomponent enzyme digestion and nine oligosaccharide structures (d.p. 3–9) from hydrolysis with a cellulase enzyme cocktail. The distribution of arabinoxylan oligomers varied depending upon the enzyme(s) applied but did not vary with harvest maturity.

Enzyme kinetics and identification of the rate-limiting step of enzymatic arabinoxylan degradation

This study investigated the kinetics of multi-enzymatic degradation of soluble wheat arabinoxylan by monitoring the release of xylose and arabinose during designed treatments with mono-component enzymes at different substrate concentrations. The results of different combinations of α-l-arabinofuranosidases (EC 3.2.1.55), one derived from Aspergillus niger (AFAn) and one from Bifidobacterium adolescentis (AFBa), respectively, a β-xylosidase (EC 3.2.1.37) from Trichoderma reesei, and an engineered D11F/R122D variant of Bacillus subtilis XynA endo-1,4-β-xylanase (EC 3.2.1.8) were examined. The two selected α-l-arabinofuranosidases catalyze liberation of arabinose residues linked 1→3 to singly (AFAn) or doubly (AFBa) substituted xyloses in arabinoxylan, respectively. When added to arabinoxylan at equimolar levels, the AFBa enzyme catalyzed the release of more arabinose, i.e. had a higher rate constant than AFAn, but with respect to the xylose release, AFAn – as expected – exhibited a better synergistic effect than AFBa with β-xylosidase. This synergistic effect with AFAn was estimated to increase the number of β-xylosidase catalyzed cuts from ∼3 (with β-xylosidase alone) to ∼7 in each arabinoxylan substrate molecule. However, the synergistic effects between β-xylosidase and the α-l-arabinofuranosidases on the xylose release were low as compared to the effect of xylanase addition with β-xylosidase, which increased the xylose release by ∼25 times in 30min, to a yield equivalent to ∼104 β-xylosidase catalyzed cuts in each arabinoxylan substrate molecule. At equimolar addition levels of the four enzymes, the xylanase activity was thus rate-limiting for the β-xylosidase catalyzed depolymerization to release xylose from arabinoxylan. The work provides clues to design efficient enzymatic degradation of arabinoxylan into fermentable monosaccharides.

Tailored enzymatic production of oligosaccharides from sugar beet pectin and evidence of differential effects of a single DP chain length difference on human faecal microbiota composition after in vitro fermentation

Sugar beet pectin was degraded enzymatically and separated by ion exchange chromatography into series of highly purified homogalacturonides and rhamnogalacturonides. MALDI-TOF/TOF mass-spectrometry was used to determine sizes and structural features. The methodology was based on the sequential use of monocomponent enzymes that were selected to target specific substructures in the sugar beet pectin. Notably pectin lyase and rhamnogalacturonan I lyase were used, which allowed detection of the resulting cleavage products by UV spectroscopy. Seven different homogalacturonides (HG) with degrees of polymerization (DP) from 2 to 8 and six different rhamnogalacturonide (RGI) structures, ranging from DP4 to 6 with defined galactose substitutions were purified. Total recoveries of 200mg homogalacturonides and 67mg rhamnogalacturonides per gram sugar beet pectin were obtained. This integrated biorefining method provides an option for advanced upgrading of sugar beet pectin into HG and RGI oligosaccharides of defined size and structure. In vitro microbial fermentation by human faecal samples (n=9) showed a different response to the DP4 and DP5 HG structures on the ratio between Bacteroidetes and Firmicutes. This indicates that pectic oligosaccharides with only slightly different structures have significantly different biological effects. This is the first report of pectic oligosaccharide activity on gut bacterial populations related to the metabolic syndrome associated with obesity.

Adsorption of monocomponent enzymes in enzyme mixture analyzed quantitatively during hydrolysis of lignocellulose substrates

The adsorption of purified Trichoderma reesei cellulases (TrCel7A, TrCel6A and TrCel5A) and xylanase TrXyn11 and Aspergillus niger β-glucosidase AnCel3A was studied in enzyme mixture during hydrolysis of two pretreated lignocellulosic materials, steam pretreated and catalytically delignified spruce, along with microcrystalline cellulose (Avicel). The enzyme mixture was compiled to resemble the composition of commercial cellulase preparations. The hydrolysis was carried out at 35 °C to mimic the temperature of the simultaneous saccharification and fermentation (SSF). Enzyme adsorption was followed by analyzing the activity and the protein amount of the individual free enzymes in the hydrolysis supernatant. Most enzymes adsorbed quickly at early stages of the hydrolysis and remained bound throughout the hydrolysis, although the conversion reached was fairly high. Only with the catalytically oxidized spruce samples, the bound enzymes started to be released as the hydrolysis degree reached 80%. The results based on enzyme activities and protein assay were in good accordance.

Enzymatic Xylose Release from Pretreated Corn Bran Arabinoxylan: Differential Effects of Deacetylation and Deferuloylation on Insoluble and Soluble Substrate Fractions

In the present work enzymatic hydrolysis of arabinoxylan from pretreated corn bran (190 degrees C, 10 min) was evaluated by measuring the release of xylose and arabinose after treatment with a designed minimal mixture of monocomponent enzymes consisting of alpha-L-arabinofuranosidases, an endoxylanase, and a beta-xylosidase. The pretreatment divided the corn bran material approximately 50:50 into soluble and insoluble fractions having A:X ratios of 0.66 and 0.40, respectively. Addition of acetyl xylan esterase to the monocomponent enzyme mixture almost doubled the xylose release from the insoluble substrate fraction and gave release of 1 mol of xylose/mol of acetic acid released, whereas addition of feruloyl esterase promoted release of only approximately 0.4 mol of xylose/mol of ferulic acid released. For the soluble substrate fraction up to 36% of the xylose could be released by the enzymatic treatment. Acetyl xylan esterase addition on top of the minimal monocomponent enzyme mixture resulted in liberation of up to 0.5 mol of xylose/mol of acetic acid released, whereas feruloyl esterase addition released 1 mol of xylose/mol of ferulic acid released from the soluble substrate. The results imply that on the insoluble material the acetyl xylan esterase was more important for the enzymatic degradation than feruloyl esterase, whereas on soluble arabinoxylan the feruloyl esterase seemed to be more important for the release of xylose.

Hydrolysis of cellulose using mono-component enzymes shows synergy during hydrolysis of phosphoric acid swollen cellulose (PASC), but competition on Avicel

To study the synergy between the three groups of cellulolytic enzymes, 20 mixtures of different mole percentage of Humicola insolens Cel45A (EG V) and Cel6A (CBH II), and Penicillium brasilianum Cel3A (β-glucosidase) were used to hydrolyze Avicel and phosphoric acid swollen cellulose/Avicel (PASC). In contrast to previous studies, where β-glucosidase was either not added or added in excess, we here focus on engineering binary, as well as, ternary cellulase mixtures (including a range of different mol% of Cel3A) for maximal total sugar production. Precise hydrolysis pattern based on the concentration of soluble hydrolysis products (glucose to cellohexaose measured by HPLC) was determined. The importance of proper assay selection for hydrolysis products detection was illustrated. It was found that degree of synergy (DS) for degradation of PASC were generally larger than 1 (indicating cooperativity between the enzymes), increasing as the hydrolysis proceeded. DS of binary exo-/endo-glucanase mixtures, decreased as the mol% of Cel45A increased. In contrast to hydrolysis of PASC, DS values during degradation of Avicel were less then 1, indicating inhibition of the involved enzymes. Thus, our data point to competition for the same binding sites between endo- and exo-glucanases, and preferential absorbance of exo-glucanases on crystalline substrates.

Evaluation of Minimal Trichoderma reesei Cellulase Mixtures on Differently Pretreated Barley Straw Substrates

The commercial cellulase product Celluclast 1.5, derived from Trichoderma reesei (Novozymes A/S, Bagsvaerd, Denmark), is widely employed for hydrolysis of lignocellulosic biomass feedstocks. This enzyme preparation contains a broad spectrum of cellulolytic enzyme activities, most notably cellobiohydrolases (CBHs) and endo-1,4-beta-glucanases (EGs). Since the original T. reesei strain was isolated from decaying canvas, the T. reesei CBH and EG activities might be present in suboptimal ratios for hydrolysis of pretreated lignocellulosic substrates. We employed statistically designed combinations of the four main activities of Celluclast 1.5, CBHI, CBHII, EGI, and EGII, to identify the optimal glucose-releasing combination of these four enzymes to degrade barley straw substrates subjected to three different pretreatments. The data signified that EGII activity is not required for efficient lignocellulose hydrolysis when addition of this activity occurs at the expense of the remaining three activities. The optimal ratios of the remaining three enzymes were similar for the two pretreated barley samples that had been subjeced to different hot water pretreatments, but the relative levels of EGI and CBHII activities required in the enzyme mixture for optimal hydrolysis of the acid-impregnated, steam-exploded barley straw substrate were somewhat different from those required for the other two substrates. The optimal ratios of the cellulolytic activities in all cases differed from that of the cellulases secreted by T. reesei. Hence, the data indicate the feasibility of designing minimal enzyme mixtures for pretreated lignocellulosic biomass by careful combination of monocomponent enzymes. This strategy can promote both a more efficient enzymatic hydrolysis of (ligno)cellulose and a more rational utilization of enzymes.

Isolation, Characterization, and Pectin-Modifying Properties of a Thermally Tolerant Pectin Methylesterase fromCitrus sinensisVar. Valencia

The thermally tolerant pectin methylesterase (TT-PME) was isolated as a monocomponent enzyme from sweet orange fruit (Citrus sinensis var. Valencia). It was also isolated from flower and vegetative tissue. The apparent molecular weight of fruit TT-PME was 40800 by SDS-PAGE and the isoelectric point estimated as pI 9.31 by IEF-PAGE. MALDI-TOF MS identified no tryptic-peptide ions from TT-PME characteristic of previously described citrus PMEs. TT-PME did not absolutely require supplemented salt for activity, but salt activation and pH-dependent activity patterns were intermediate to those of thermolabile PMEs. Treatment of non-calcium-sensitive pectin with TT-PME (reducing the degree of methylesterification by 6%) increased the calcium-sensitive pectin ratio from 0.01 to 0.90, indicating a blockwise mode of action. TT-PME produced a significantly lower end-point degree of methylesterification at pH 7.5 than at pH 4.5. Extensive de-esterification with TT-PME did not reduce the pectin molecular weight or z-average radius of gyration, as determined by HPSEC.

Effect of endo‐xylanase‐containing enzyme preparations and laccase on the solubility of rye bran arabinoxylan

AbstractThree commercial enzyme preparations with endo‐xylanase activity, namely Bio‐Feed Wheat, Bio‐Feed Plus and Grindamyl H 640, and laccase have been tested for their effects on the solubilisation of arabinoxylan (AX) in rye bran or autoclaved rye bran. Autoclaving efficiently increased the availability of AX for enzymatic degradation. Both Bio‐Feed Wheat (a monocomponent enzyme) and Bio‐Feed Plus (a multicomponent preparation with different enzymatic activities) efficiently degraded the autoclaved rye bran AX into lower‐molecular‐weight fragments. As much as 70% of the xylose residues and 58% of the arabinose residues in the autoclaved bran were soluble after treatment with Bio‐Feed Plus; the weight—average molecular weight of the detectable portion of these soluble polymers was 104 000 Da. Grindamyl H 640 solubilised only a small fraction of the AX in autoclaved rye bran; the molecular weight of these soluble fragments was higher than that of those released by the Bio‐Feed xylanases. Addition of laccase during treatment with Bio‐Feed Wheat or Grindamyl H 640 decreased the yield of water‐soluble AX.© 2003 Society of Chemical Industry

Cellulose Hydrolysis - The Role of Monocomponent Cellulases in Crystalline Cellulose Degradation

Changes in the molecular structure of cellulose during hydrolysis with four recombinant β-1,4-glycanases from the cellulolytic bacterium Cellulomonas fimi were assessed and compared in an attempt to elucidate the mechanism of crystalline cellulose degradation. It was apparent that the two endoglucanases, Cel6A and Cel5A, degraded sigmacell cellulose differently; Cel5A liberated more soluble sugars (cellobiose and cellotriose) and significantly altered the molecular weight distribution, while Cel6A had a limited effect on the polymer size and liberated primarily cellobiose and glucose. Additionally, both endoglucanases slightly increased the crystallinity of cellulose. In contrast, the cellobiohydrolases, Cel6B and Cel48A, had no effect on cellulose molecular weight and liberated only cellobiose and cellotriose. However, Cel48A was shown to be effective at reducing the crystallinity of the cellulosic substrate, while Cel6B increased the crystallinity index. Synergistic hydrolysis using combinations of the different enzymes showed that, although the cellulose was extensively hydrolysed, the molecular structure of the substrate was similar to the original material. This phenomenon suggests that the actions of individual monocomponent enzymes are offset by the concurrent modification by the complementing enzymes during synergistic hydrolysis.

PERFUSION CHROMATOGRAPHY SEPARATION OF THE TOMATO FRUIT-SPECIFIC PECTIN METHYLESTERASE FROM A SEMIPURIFIED COMMERCIAL ENZYME PREPARATION1

A rapid and simple method was developed, using perfusion chromatography media, to separate the fruit-specific pectin methylesterase (PME) isoform from the depolymerizing enzyme polygalacturonase (PG) and other contaminating pectinases present in a commercial tomato enzyme preparation. Pectinase activities were adsorbed onto a Poros HS (a strong cation exchanger) column in 20 M HEPES buffer at pH 7.5. The fruit-specific PME was eluted from the column with 80 mM NaCl, followed by a step to 300 mM NaCl to elute PG activity. Rechromatography of the PME activity peak with a linear gradient further resolved two PME isoenzymes and removed residual traces of PG activity. The PG activity peak was further treated with lectin affinity chromatography to provide purified PG enzyme, which was separated from a salt-dependent PME (tentatively identified as a “ubiquitous-type” isoform), and a pectin acetylesterase. The later enzyme has not been reported previously in tomato. This method provides monocomponent enzymes that will be useful for studying enzyme mechanisms and for modifying pectin structure and functional properties. Mention of brand or firm name does not constitute an endorsement by the U.S. Department of Agriculture over others of a similar nature not mentioned.