Two mannanases from Sclerotium rolfsii in total chlorine free bleaching of softwood kraft pulp

The plant pathogenic basidiomycete Sclerotium rolfsii produces a wide range of extracellular hemicellulolytic enzymes. To study the effect of β-mannanases in total chlorine free bleaching of softwood pulp, two purified β -mannanases from S. rolfsii, with molecular masses of 42 and 61 kDa, a xylanase preparation from S. rolfsii and combinations of these were tested in a O(QX)P bleaching sequence (O = oxygen delignification, X = treatment with enzymes, Q = chelation of metals, P = treatment with hydrogen peroxide in alkaline solution). A brightness increase of 1.6 and 1.9% ISO was obtained with the 42 and 61 kDa mannanase and a combination of each of these enzymes with xylanases gave a brightness increase of 2.5 and 2.8% ISO, respectively. The effect of mannanases and xylanases was nearly additive. Both mannanases alone caused a lower decrease of the kappa number as compared to xylanases. The mannanases differed in their ability to release oligosaccharides from different mannans. The 61 kDa mannanase liberated larger fragments and caused rapid depolymerisation of mannans, which seems to promote the bleaching of pulp.     

Hemicellulases of Bacillus species: preliminary comparative studies on production and properties of mannanases and galactanases

A range of Bacillus subtilis strains and other Bacillus species were screened for mannanase, β-mannosidase and galactanase activities. Maximum mannanase activity, 106.2 units/ml, was produced by B. subtilis NRRL 356. β-Mannosidase and galactanase activities from all strains were relatively low. The effect of carbon and nitrogen source on mannanase and galactanase production by B. brevis ATCC 8186, B. licheniformis ATCC 27811, B. polymyxa NRRL 842 and B. subtilis NRRL 356 was investigated. Highest mannanase production was observed in the four strains tested when the mannan substrate, locust bean gum, was used as carbon source. Induction was most dramatic in the case of B. subtilis NRRL 356 where only basal enzyme levels were produced in the presence of other carbon sources. β-Mannosidase was induced in the four Bacillus cultures by locust bean gum. Results indicated that galactose acted as an inducer for production of galactanase. Organic and inorganic nitrogen sources resulted in induction of high mannanase titres in B. subtilis. Highest galactanase activity was produced by each organism in media containing sodium nitrate as nitrogen source. Mannanases from B. brevis, B. licheniformis, B. polymyxa and B. subtilis retained 100% residual activity after a 3 h incubation at 65°C, 65°C, 60°C and 55°C respectively. Galactanases retained more than 95% activity at 55°C after 3 h. The pH optima of mannanases ranged from 6.5–6.8 whereas galactanases ranged from 5.1 in the case of B. brevis to 7.0 for B. polymyxa.     

Cellulose degrading enzymes and their potential industrial applications

Bioconversion of cellulose to soluble sugars and glucose is catalyzed by a group of enzymes called cellulases. Microorganisms including fungi, bacteria and actinomycetes produce mainly three types of cellulase components—endo-1,4-β-D-glucanase, exo-1,4-β-D-glucanase and β-glucosidase—either separately or in the form of a complex. Over the last several decades, cellulases have become better understood at a fundamental level; nevertheless, much remains to be learnt. The tremendous commercial potential of cellulases in a variety of applications remains the driving force for research in this area. This review summarizes the present state of knowledge on microbial cellulases and their applications.     

Mutation of fungal endoglucanases into glycosynthases and characterization of their acceptor substrate specificity

Humicola insolens mutant Cel7B E197A is a powerful endo-glycosynthase displaying an acceptor substrate specificity restricted to β-d-glucosyl, β-d-xylosyl, β-d-mannosyl and β-d-glucosaminyl in +1 subsite. Our aim was to extend this substrate specificity to β-d-N-acetylglucosaminyl, in order to get access to a wider array of oligosaccharidic structures obtained through glycosynthase assisted synthesis. In a first approach a trisaccharide bearing a β-d-N-acetylglucosaminyl residue was docked at the +1 subsite of H. insolens Cel7B, indicating that the mutation of only one residue, His209, could lead to the expected wider acceptor specificity. Three H. insolens Cel7B glycosynthase mutants (H209A, H209G and H209A/A211T) were produced and expressed in Aspergillus oryzae. In parallel, sequence alignment investigations showed that several cellulases from family GH7 display an alanine residue instead of histidine at position 209. Amongst them, Trichoderma reesei Cel7B, an endoglucanase sharing the highest degree of sequence identity with Humicola Cel7B, was found to naturally accept a β-d-N-acetylglucosaminyl residue at +1 subsite. The T. reesei Cel7B mutant nucleophile E196A was produced and expressed in Saccharomyces cerevisiae, and its activity as glycosynthase, together with the H. insolens glycosynthase mutants, was evaluated toward various glycosidic acceptors.     

Compatibility testing and enhancing the pulp bleaching process by hydrolases of the newly isolated thermophilic Isoptericola variabilis strain UD-6

Abstract

In the present study, Isoptericola variabilis strain UD-6 isolated from alkaline hot spring of Unapdev, Maharashtra, India was assessed for its biobleaching activity by hydrolytic enzymes on rice straw pulp. Results of primary and secondary screening manifested that it was a multi-enzyme producer, competent to produce amylase, cellulase, mannanase, pectinase, and xylanase at 9.73, 4.11, 6.26, 8.42, and 6.61?IU?ml^?1 in fermentation conditions, respectively. Maximum activity of all enzymes was gained at thermal temperature (50–55?°C), alkaline condition (pH 8–9), under 5?mM KCl and 5?mM NaCl salt concentration. In compatibility testing, activities of all enzymes were spectacularly reduced when they utilized with chemicals of pulp bleaching. Results of rice straw pulp bleaching was effectual when pulp was initially bleached with mannanase, pectinase, and xylanase enzymes (Es) for 90?min and then with diluted chemicals (DC) for further 90?min instead of their separate use. Treatment of rice straw pulp with Es?+?DC, enhanced the release of reducing sugars, hydrophobic compounds, and phenolic compounds, whereas Kappa number was reduced. Overall, the results of the present study indicated that pre-bleaching of pulp with hydrolytic enzymes obtained from I. variabilis strain UD-6 helps to minimize chemicals used in the bleaching process and make it more sustainable for pulp and paper industries as well as for the environment.     

Production of a heterologous endo-1,4-beta-xylanase in the yeast Pichia stipitis with an O(2)-regulated promoter

The Cryptococcus albidus XLN-gene (encoding endo-1,4-β-xylanase) was expressed in the yeast Pichia stipitis under the control of the PsADH2-promoter, which is activated under O₂ limitation. The resulting transformant produced endo-1,4-β-xylanase after a shift to anoxic conditions. Endo-1,4-β-xylanase production was enhanced by limited aeration after the shift.     

Opportunities for avoidance of land‐use change through substitution of soya bean meal and cereals in European livestock diets with bioethanol coproducts

An analysis is presented which quantifies the potential for distillers dried grains with solubles (DDGS, a coproduct of wheat bioethanol production) to replace soya bean meal (SBM) and cereals in livestock rations. A major proportion of the SBM imported into Europe as a protein‐rich feedstuff for livestock comes from South America, where land‐use change (LUC) is associated with high carbon emissions. Production of DDGS can therefore reduce LUC in South America by substitution of SBM in animal feed. The analysis indicates that a single bioethanol distillery processing 1 million tonnes of wheat, and producing ca. 330 000 tonnes of DDGS per annum, would substitute at least 136 493 tonnes of whole soya beans grown on 47 725 ha of land, and save greenhouse gas emissions equivalent to 0.63 million tonnes CO₂ per annum. By growing sugar beet and wheat in an average ratio of 0.06 : 0.94 on 1 ha of land in Europe, the net area of agricultural land required to produce feed ingredients equivalent to 6.08 t of sugar beet pulp (SBP) and 1.72 t of DDGS associated with 2363 L of bioethanol, is reduced to 0.40 ha. This accounts for 0.42 ha of soya that is not required when DDGS displaces SBM, and 0.18 ha of wheat that is not required when DDGS and SBP displace wheat in livestock rations.     

The effect of dietary faba bean and non-starch polysaccharide degrading enzymes on the growth performance and gut physiology of young turkeys

The aim of this study was to investigate the effect of dietary replacement of soya bean meal (SBM) with faba bean (FB) and a blend of non-starch polysaccharide (NSP) degrading enzymes on the gastrointestinal function, growth performance and welfare of young turkeys (1 to 56 days of age). An experiment with a 2×2 factorial design was performed to compare the efficacy of four diets: a SBM-based diet and a diet containing FB, with and without enzyme supplementation (C, FB, C_E and FB_E, respectively). In comparison with groups C, higher dry matter content and lower viscosity of the small intestinal digesta were noted in groups FB. The content of short-chain fatty acids (SCFAs) in the small intestinal digesta was higher in groups FB, but SCFA concentrations in the caecal digesta were comparable in groups C and FB. In comparison with control groups, similar BW gains, higher feed conversion ratio (FCR), higher dry matter content of excreta and milder symptoms of footpad dermatitis (FPD) were noted in groups FB. Enzyme supplementation increased the concentrations of acetate, butyrate and total SCFAs, but it did not increase the SCFA pool in the caecal digesta. The enzymatic preparation significantly improved FCR, reduced excreta hydration and the severity of FPD in turkeys. It can be concluded that in comparison with the SBM-based diet, the diet containing 30% of FB enables to achieve comparable BW gains accompanied by lower feed efficiency during the first 8 weeks of rearing. Non-starch polysaccharide-degrading enzymes can be used to improve the nutritional value of diets for young turkeys, but more desirable results of enzyme supplementation were noted in the SBM-based diet than in the FB-based diet.     

Family-10 and Family-11 xylanases differ in their capacity to enhance the bleachability of hardwood and softwood paper pulps

Enzyme-aided bleaching of softwood and hardwood kraft pulps by glycosyl hydrolase family-10 and -11 xylanases and a family-26 mannanase was investigated. The ability to release reducing sugar from pulp xylan and to enhance bleachability is not a characteristic shared by all xylanases. Of the six enzymes tested, two xylanases belonging to family 11 were most effective at increasing bleachability and improving final paper brightness. None of the enzymes had a deleterious effect on pulp fibre integrity. The efficiency of individual xylanases as bleach enhancers was not dependent on the source microorganism, and could not be predicted solely on the basis of the quantity or nature of products released from pulp xylan. Cooperative interactions between xylanase/xylanase and xylanase/mannanase combinations, during the pretreatment of softwood and hardwood pulps, were investigated. Synergistic effects on reducing-sugar release and kappa number reduction were elicited by a combination of two family-10 xylanases. Pretreatment of kraft pulp with mannanase A from Pseudomonas fluorescens subsp. cellulosa and any one of a number of xylanases resulted in increased release of reducing sugar and a larger reduction in kappa number than obtained with the xylanases alone, confirming the beneficial effects of family-26 mannanases on enzyme-aided bleaching of paper pulp. Received: 6 January 1997 / Received revision: 10 April 1997 / Accepted: 19 April 1997     

Extracellular mannanases and galactanases from selected fungi

Summary Eight thermophilic fungi were tested for production of mannanases and galactanases. Highest mannanase activities were produced byTalaromyces byssochlamydoides andTalaromyces emersonii. Mannanases from all strains tested were induced by locust bean gum except in the case ofThermoascus aurantiacus, where mannose had a greater inducing effect. Locust bean gum was also the best inducer of -mannosidase and galactanase except in the case ofT. emersonii where galactose was a better inducer of both these enzymes. Highest mannanase activity was produced byTalaromyces species when peptone was used as nitrogen source whereas sodium nitrate promoted maximum production of this enzyme byThielavia terrestris andT. aurantiacus. The pH optima of mannanases from the thermophilic fungi were in the range 5.0–6.6 and contrasted with the low pH optimum (3.2) of the enzyme fromAspergillus niger. Galactanases had pH optima in the range 4.3–5.8. The mannanase fromT. emersonii and the galactanase fromT. terrestris were most thermostable, each retaining 100% activity for 3 h at 60°C.     

Efficacy of β-mannanase supplementation to corn–soya bean meal-based diets on growth performance,nutrient digestibility,blood urea nitrogen,faecal coliform and lactic acid bacteria and faecal noxious gas emission in growing pigs

A study was conducted to determine the efficacy of β-mannanase supplementation to a diet based on corn and soya bean meal (SBM) on growth performance, nutrient digestibility, blood urea nitrogen (BUN), faecal coliforms and lactic acid bacteria, and noxious gas emission in growing pigs. A total of 140 pigs [(Landrace × Yorkshire) × Duroc; average body weight 25 ± 3 kg] were randomly allotted to a 2 × 2 factorial arrangement with dietary treatments consisting of hulled or dehulled SBM without or with supplementation of 400 U β-mannanase/kg. During the 6 weeks of experimental feeding, β-mannanase supplementation had no effect on body weight gain, feed intake and gain:feed (G:F) ratio. Compared with dehulled SBM, feeding hulled SBM caused an increased feed intake of pigs in the entire trial (p = 0.05). The G:F ratio was improved in pigs receiving dehulled SBM (p < 0.05). Dietary treatments did not influence the total tract digestibility of dry matter, nitrogen and gross energy. Enzyme supplementation reduced (p < 0.05) the population of faecal coliforms and tended to reduce the NH₃ concentration after 24 h of fermentation in a closed box containing faecal slurry. Feeding hulled SBM tended to reduce NH₃ emission on days 3 and 5 of fermentation. In conclusion, mannanase supplementation had no influence on growth performance and nutrient digestibility but showed a positive effect on reducing coliform population and tended to reduce NH₃ emission. Dehulled SBM increased G:F ratio and hulled SBM tended to reduce NH₃ emission.     

Lupin as primary protein source in young broiler chicken diets: Effect of enzymes preparations catalyzing degradation of non-starch polysaccharides or phytates

This work examined the effects of three enzyme preparations (A,B,C) directed towards degradation of Non Starch Polysaccharides (NSP) and one targeting phytates (D) on performance traits in broilers fed maize meal basal diets containing 400 g/kg of yellow lupine seeds (LM). A soybean meal (SBM) based diet served as a reference control. Growth rate, coefficients of total tract apparent digestibility (CTTAD) of organic matter, protein and energy, as well as morphometric measurements of selected sections of gastrointestinal tract (GIT) were determined. In comparison to chickens fed the SBM diet, chickens fed the LM diet consumed less feed, had considerably lower body weight gain, as well as lower CTTAD of measured nutrients and energy. Also the GIT relative weight and length were increased within the group fed the LM diet. Addition of each NSP degrading enzymes (A,B,C) to the LM diet increased feed intake and decreased size of GIT organs (all p < 0.05). Addition of enzymes A or B increased (p < 0.05) growth rate of chicks, whereas only enzyme B increased fed efficiency (p < 0.05) and tended to slightly improve CTTAD of nutrients. The addition of enzyme D did not have any effect on feed intake, growth rate or CTTAD. This study indicates that a diet containing high levels of LM is detrimental to feed intake and condition of the digestive tract of young broilers, and thus affects their performance. However, when the LM diet is supplemented with suitable enzyme preparations, performance parameters are not different from those obtained with SBM.     

Changes in wall-bound polysaccharidase activities during the culture cycle of a Rubus fruticosus cell suspension

Several hydrolytic enzyme activities were detected in the wall of developing cells of Rubus truticosus in suspension culture. The corresponding substrates of the enzymes are mostly polysaccharide wall constituents, except for chitinase activity. The activities measured when the enzymes were in the free state or wall-bound showed the positive influence of the cell wall micro-environment. Changes in the activities during a cell culture cycle demonstrated that those enzymes acting on xyloglucans behaved differently from the others, and suggest that xyloglucans undergo modifications in vivo over a longer period of time during the exponential growth phase. The same activities were identified in the culture medium. Endo-1,4-β-d-glucanase activities which depolymerized car☐ymethylcellulose (CMC) and xyloglucans (XG) were assayed viscosimetrically. It was found that XG oligosaccharides exhibited an inhibitory effect on the depolymerization of xyloglucans but not on that of CMC. This suggests that true xyloglucanases are present in the culture of Rubus cells.     

Xylanase and Mannanase enzymes from Streptomyces galbus NR and their use in biobleaching of softwood kraft pulp

Enzymatic pretreatment of softwood kraft pulp was investigated using xylanase and mannanase, singly or in combination, either sequentially or simultaneously. Enzymes were obtained from Streptomyces galbus NR that had been cultivated in a medium, containing either xylan of sugar cane bagasse or galactomannan of palm-seeds, when they were used as sole carbon sources from local wastes in fermentation media. No cellulase activity was detected. Incubation period, temperature, initial pH values and nature of nutritive constituents were investigated. Optimum production of both enzymes was achieved after 5 days incubation on a rotary shaker (200 rpm) at 35 degrees C and initial pH 7.0. Partial purification of xylanase and mannanase in the cultures supernatant were achieved by salting out at 40-60 and 60-80% ammonium sulphate saturation with a purification of 9.63- and 8.71-fold and 68.80 and 62.79% recovery, respectively. The xylanase and mannanase from S. galbus NR have optimal activity at 50 and 40 degrees C, respectively. Both enzymes were stable at a temperature up to 50 degrees C. Xylanase and mannanase showed highest activity at pH 6.5 and were stable from 5.0 to 8.0 and from 5.5 to 7.5, respectively. The partial purified enzymes preparations of xylanase and mannanase enzymes showed high bleaching activity, which is an important consideration for industry. Xylanase was found to be more effective for paper-bleaching than mannanase. When xylanase and mannanase were dosed together (simultaneously), both enzymes were able to enhance the liberation of reducing sugars and improve pulp bleachability, possibly as a result of nearly additive interactions. The simultaneous addition of both enzymes was more effective in pulp treatment than their sequential addition.     

Recombinant expression, characterization, and pulp prebleaching property of a Phanerochaete chrysosporium endo-β-1,4-mannanase

A Phanerochaete chrysosporium cDNA predicted to encode endo-1,4-β-d-mannanase, man5D, was cloned and expressed in Aspergillus niger. The coding region of the gene man5D was predicted to contain, in order from the N-terminal: a secretory signal peptide, cellulose-binding domain, linker region, and glycosyl hydrolase family 5 catalytic site. The enzyme was purified from culture filtrate of A. niger transformants that carried the recombinant man5D. Recombinant Man5D had an apparent molecular size of about 65 kDa by SDS-PAGE, and optimal activity at pH 4.0–6.0 and 60 °C. It was stable from pH 4.0 to 8.0 and up to 60 °C. The enzyme showed affinity for Avicel cellulose, suggesting that the predicted cellulose-binding domain is biologically functional. The specific activities of Man5D on mannan, galactomannan, and glucomannan at pH 5 and 60 °C ranged from 160 to 460 μmol/(min mg), with apparent K_m values from 0.54 to 2.3 mg/mL. Product analysis results indicated that Man5D catalyzes endo-cleavage, and appears to have substantial transglycosylase activity. When used to treat softwood kraft pulp, Man5D hydrolyzed mainly glucomannan and exhibited a positive effect as a prebleaching agent. Compared to a commercial prebleaching with xylanase, the prebleaching effect of Man5D was weaker but with reduced loss of fibre yield as determined by the release of solubilized sugars.     

Immobilization of fungus Aspergillus sp. by a novel cryogel technique for production of extracellular hydrolytic enzymes

Aerobic cells of a fungus isolate Aspergillus sp. CX-1 have been immobilized in macroporous cryoPAG and in different composite cryoPAGs — fibrous adjunct carriers. The productivity of the extracellular enzymes (exo-1.4-β-glucanase, endo-1.4-β-glucanase, β-glucosidase and xylanase), and the viability, growth and ultrastructure of the immobilized fungus have been studied. The enzyme activities and stability during long-term repeated batch cultivation in the immobilized fungus were higher than in free mycelia when batch cultivated. The fungus immobilized in the composite cryoPAG, containing polypropylene non-woven fabric, possessed the highest exo-1.4-β-glucanase activity, the longest durability of enzyme production (85 days) and the most reliable mechanical strength. The fungus immobilized in porous composite cryogel possessed a variety of advantages including easy control of cryogel porosity, improved mechanical strength and durability, simplicity of construction, high enzyme productivity and high stability.     

Purification and characterization of two minor endo-β-1,4-xylanases of Schizophyllum commune

Two minor extracellular endo-β-1,4-xylanases (XynB and XynC, EC 3.2.1.8) were purified from the culture filtrate of Schizophyllum commune grown on cellulose. The molecular mass of enzymes was estimated to be 30.5 kDa for XynB and 30 kDa for XynC according to SDS-PAGE. Both enzymes were acidic, with pI value 2.8 for XynB and 3.6 for XynC. The highest activities were achieved at 50 °C and pH 5.5 and enzymes were stable up to 40 °C in the pH range 5–7. A comparison of hydrolysis products of glucuronoxylan, rhodymenan and acetylxylan showed different mode of action of all three xylanases of S. commune. Known XynA generated products typical for family 11 of glycoside hydrolase – aldopentaouronic acid from glucuronoxylan and isomeric xylotetraose from rhodymenan. XynB released fragments by one xylopyranosyl unit shorter – aldotetraouronic acid MeGlcA1-2Xylβ1-4Xylβ1-4Xyl from glucuronoxylan and isomeric xylotriose from rhodymenan, products usually generated by xylanases from glycoside hydrolase family 10. XynC liberated aldotetraouronic acid Xylβ-1,4-(MeGlcA-1,2-)Xylβ-1,4-Xyl with glucuronoyl unit attached to the middle xylopyranosyl unit from glucuronoxylan and isomeric xylotetraose from rhodymenan. XynC was also able to release xylose from the reducing end of aldotetraouronic acid MeGlcA1-2Xylβ1-4Xylβ1-4Xyl.     

Biotransformation of steviol by cultured cells of eucalyptus perriniana and Coffea arabica

A new biotransformation product, steviol 19-β-gentiobiosyl ester, together with steviol 19-β-glucopyranosyl ester and steviol-13-O-β-glucopyranoside 19-β-glucopyranosyl ester (rubusoside), was isolated from Eucalyptus perriniana jar fermentor culture following the administration of steviol. Only rubusoside was isolated as a biotransforination product of steviol from Coffea arabica cell suspension culture.     

Specific Fusion of β-1,4-Endoglucanase and β-1,4-Glucosidase Enhances Cellulolytic Activity and Helps in Channeling of Intermediates

Identification and design of new cellulolytic enzymes with higher catalytic efficiency are a key factor in reducing the production cost of lignocellulosic bioalcohol. We report here identification of a novel β-glucosidase (Gluc1C) from Paenibacillus sp. strain MTCC 5639 and construction of bifunctional chimeric proteins based on Gluc1C and Endo5A, a β-1,4-endoglucanase isolated from MTCC 5639 earlier. The 448-amino-acid-long Gluc1C contained a GH superfamily 1 domain and hydrolyzed cellodextrin up to a five-sugar chain length, with highest efficiency toward cellobiose. Addition of Gluc1C improved the ability of Endo5A to release the reducing sugars from carboxymethyl cellulose. We therefore constructed six bifunctional chimeric proteins based on Endo5A and Gluc1C varying in the positions and sizes of linkers. One of the constructs, EG5, consisting of Endo5A-(G₄S)₃-Gluc1C, demonstrated 3.2- and 2-fold higher molar specific activities for β-glucosidase and endoglucanase, respectively, than Gluc1C and Endo5A alone. EG5 also showed 2-fold higher catalytic efficiency than individual recombinant enzymes. The thermal denaturation monitored by circular dichroism (CD) spectroscopy demonstrated that the fusion of Gluc1C with Endo5A resulted in increased thermostability of both domains by 5°C and 9°C, respectively. Comparative hydrolysis experiments done on alkali-treated rice straw and CMC indicated 2-fold higher release of product by EG5 than that by the physical mixture of Endo5A and Gluc1C, providing a rationale for channeling of intermediates. Addition of EG5 to a commercial enzyme preparation significantly enhanced release of reducing sugars from pretreated biomass, indicating its commercial applicability.