| 1. |
- Aidemark, Mari, et al.
(författare)
-
Trichoderma viride cellulase induces resistance to the antibiotic pore-forming peptide alamethicin associated with changes in the plasma membrane lipid composition of tobacco BY-2 cells
- 2010
-
Ingår i: Bmc Plant Biology. - : Springer Science and Business Media LLC. - 1471-2229. ; 10
-
Tidskriftsartikel (refereegranskat)abstract
- Background: Alamethicin is a membrane-active peptide isolated from the beneficial root-colonising fungus Trichoderma viride. This peptide can insert into membranes to form voltage-dependent pores. We have previously shown that alamethicin efficiently permeabilises the plasma membrane, mitochondria and plastids of cultured plant cells. In the present investigation, tobacco cells (Nicotiana tabacum L. cv Bright Yellow-2) were pre-treated with elicitors of defence responses to study whether this would affect permeabilisation. Results: Oxygen consumption experiments showed that added cellulase, already upon a limited cell wall digestion, induced a cellular resistance to alamethicin permeabilisation. This effect could not be elicited by xylanase or bacterial elicitors such as flg22 or elf18. The induction of alamethicin resistance was independent of novel protein synthesis. Also, the permeabilisation was unaffected by the membrane-depolarising agent FCCP. As judged by lipid analyses, isolated plasma membranes from cellulase-pretreated tobacco cells contained less negatively charged phospholipids ( PS and PI), yet higher ratios of membrane lipid fatty acid to sterol and to protein, as compared to control membranes. Conclusion: We suggest that altered membrane lipid composition as induced by cellulase activity may render the cells resistant to alamethicin. This induced resistance could reflect a natural process where the plant cells alter their sensitivity to membrane pore-forming agents secreted by Trichoderma spp. to attack other microorganisms, and thus adding to the beneficial effect that Trichoderma has for plant root growth. Furthermore, our data extends previous reports on artificial membranes on the importance of lipid packing and charge for alamethicin permeabilisation to in vivo conditions.
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| 2. |
- Alvfors, Per, 1954-, et al.
(författare)
-
Research and development challenges for Swedish biofuel actors – three illustrative examples : Improvement potential discussed in the context of Well-to-Tank analyses
- 2010
-
Rapport (övrigt vetenskapligt/konstnärligt)abstract
- Currently biofuels have strong political support, both in the EU and Sweden. The EU has, for example, set a target for the use of renewable fuels in the transportation sector stating that all EU member states should use 10% renewable fuels for transport by 2020. Fulfilling this ambition will lead to an enormous market for biofuels during the coming decade. To avoid increasing production of biofuels based on agriculture crops that require considerable use of arable area, focus is now to move towards more advanced second generation (2G) biofuels that can be produced from biomass feedstocks associated with a more efficient land use. Climate benefits and greenhouse gas (GHG) balances are aspects often discussed in conjunction with sustainability and biofuels. The total GHG emissions associated with production and usage of biofuels depend on the entire fuel production chain, mainly the agriculture or forestry feedstock systems and the manufacturing process. To compare different biofuel production pathways it is essential to conduct an environmental assessment using the well-to-tank (WTT) analysis methodology. In Sweden the conditions for biomass production are favourable and we have promising second generation biofuels technologies that are currently in the demonstration phase. In this study we have chosen to focus on cellulose based ethanol, methane from gasification of solid wood as well as DME from gasification of black liquor, with the purpose of identifying research and development potentials that may result in improvements in the WTT emission values. The main objective of this study is thus to identify research and development challenges for Swedish biofuel actors based on literature studies as well as discussions with the the researchers themselves. We have also discussed improvement potentials for the agriculture and forestry part of the WTT chain. The aim of this study is to, in the context of WTT analyses, (i) increase knowledge about the complexity of biofuel production, (ii) identify and discuss improvement potentials, regarding energy efficiency and GHG emissions, for three biofuel production cases, as well as (iii) identify and discuss improvement potentials regarding biomass supply, including agriculture/forestry. The scope of the study is limited to discussing the technologies, system aspects and climate impacts associated with the production stage. Aspects such as the influence on biodiversity and other environmental and social parameters fall beyond the scope of this study. We find that improvement potentials for emissions reductions within the agriculture/forestry part of the WTT chain include changing the use of diesel to low-CO2-emitting fuels, changing to more fuel-efficient tractors, more efficient cultivation and manufacture of fertilizers (commercial nitrogen fertilizer can be produced in plants which have nitrous oxide gas cleaning) as well as improved fertilization strategies (more precise nitrogen application during the cropping season). Furthermore, the cultivation of annual feedstock crops could be avoided on land rich in carbon, such as peat soils and new agriculture systems could be introduced that lower the demand for ploughing and harrowing. Other options for improving the WTT emission values includes introducing new types of crops, such as wheat with higher content of starch or willow with a higher content of cellulose. From the case study on lignocellulosic ethanol we find that 2G ethanol, with co-production of biogas, electricity, heat and/or wood pellet, has a promising role to play in the development of sustainable biofuel production systems. Depending on available raw materials, heat sinks, demand for biogas as vehicle fuel and existing 1G ethanol plants suitable for integration, 2G ethanol production systems may be designed differently to optimize the economic conditions and maximize profitability. However, the complexity connected to the development of the most optimal production systems require improved knowledge and involvement of several actors from different competence areas, such as chemical and biochemical engineering, process design and integration and energy and environmental systems analysis, which may be a potential barrier.
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| 3. |
- Alvfors, Per, et al.
(författare)
-
Research and development challenges for Swedish biofuel actors – three illustrative examples
- 2010
-
Rapport (övrigt vetenskapligt/konstnärligt)abstract
- Currently biofuels have strong political support, both in the EU and Sweden. The EU has, for example, set a target for the use of renewable fuels in the transportation sector stating that all EU member states should use 10% renewable fuels for transport by 2020. Fulfilling this ambition will lead to an enormous market for biofuels during the coming decade. To avoid increasing production of biofuels based on agriculture crops that require considerable use of arable area, focus is now to move towards more advanced second generation (2G) biofuels that can be produced from biomass feedstocks associated with a more efficient land use.Climate benefits and greenhouse gas (GHG) balances are aspects often discussed in conjunction with sustainability and biofuels. The total GHG emissions associated with production and usage of biofuels depend on the entire fuel production chain, mainly the agriculture or forestry feedstock systems and the manufacturing process. To compare different biofuel production pathways it is essential to conduct an environmental assessment using the well-to-tank (WTT) analysis methodology. In Sweden the conditions for biomass production are favourable and we have promising second generation biofuels technologies that are currently in the demonstration phase. In this study we have chosen to focus on cellulose based ethanol, methane from gasification of solid wood as well as DME from gasification of black liquor, with the purpose of identifying research and development potentials that may result in improvements in the WTT emission values. The main objective of this study is thus to identify research and development challenges for Swedish biofuel actors based on literature studies as well as discussions with the the researchers themselves. We have also discussed improvement potentials for the agriculture and forestry part of the WTT chain. The aim of this study is to, in the context of WTT analyses, (i) increase knowledge about the complexity of biofuel production, (ii) identify and discuss improvement potentials, regarding energy efficiency and GHG emissions, for three biofuel production cases, as well as (iii) identify and discuss improvement potentials regarding biomass supply, including agriculture/forestry. The scope of the study is limited to discussing the technologies, system aspects and climate impacts associated with the production stage. Aspects such as the influence on biodiversity and other environmental and social parameters fall beyond the scope of this study. We find that improvement potentials for emissions reductions within the agriculture/forestry part of the WTT chain include changing the use of diesel to low-CO2-emitting fuels, changing to more fuel-efficient tractors, more efficient cultivation and manufacture of fertilizers (commercial nitrogen fertilizer can be produced in plants which have nitrous oxide gas cleaning) as well as improved fertilization strategies (more precise nitrogen application during the cropping season). Furthermore, the cultivation of annual feedstock crops could be avoided on land rich in carbon, such as peat soils and new agriculture systems could be introduced that lower the demand for ploughing and harrowing. Other options for improving the WTT emission values includes introducing new types of crops, such as wheat with higher content of starch or willow with a higher content of cellulose. From the case study on lignocellulosic ethanol we find that 2G ethanol, with co-production of biogas, electricity, heat and/or wood pellet, has a promising role to play in the development of sustainable biofuel production systems. Depending on available raw materials, heat sinks, demand for biogas as vehicle fuel and existing 1G ethanol plants suitable for integration, 2G ethanol production systems may be designed differently to optimize the economic conditions and maximize profitability. However, the complexity connected to the development of the most optimal production systems require improved knowledge and involvement of several actors from different competence areas, such as chemical and biochemical engineering, process design and integration and energy and environmental systems analysis, which may be a potential barrier. Three important results from the lignocellulosic ethanol study are: (i) the production systems could be far more complex and intelligently designed than previous studies show, (ii) the potential improvements consist of a large number of combinations of process integration options wich partly depends on specific local conditions, (iii) the environmental performance of individual systems may vary significantly due to systems design and local conditons.From the case study on gasification of solid biomass for the production of biomethane we find that one of the main advantages of this technology is its high efficiency in respect to converting biomass into fuels for transport. For future research we see a need for improvements within the gas up-grading section, including gas cleaning and gas conditioning, to obtain a more efficient process. A major challenge is to remove the tar before the methanation reaction. Three important results from the biomethane study are: (i) it is important not to crack the methane already produced in the syngas, which indicates a need for improved catalysts for selective tar cracking, (ii) there is a need for new gas separation techniques to facilitate the use of air oxidation agent instead of oxygen in the gasifier, and (iii) there is a need for testing the integrated process under realistic conditions, both at atmospheric and pressurized conditions. From the case study on black liquor gasification for the production of DME we find that the process has many advantages compared to other biofuel production options, such as the fact that black liquor is already partially processed and exists in a pumpable, liquid form, and that the process is pressurised and tightly integrated with the pulp mill, which enhances fuel production efficiency. However, to achieve commercial status, some challenges still remain, such as demonstrating that materials and plant equipment meet the high availability required when scaling up to industrial size in the pulp mill, and also proving that the plant can operate according to calculated heat and material balances. Three important results from the DME study are: (i) that modern chemical pulp mills, having a potential surplus of energy, could become important suppliers of renewable fuels for transport, (ii) there is a need to demonstrate that renewable DME/methanol will be proven to function in large scale, and (iii) there is still potential for technology improvements and enhanced energy integration. Although quantitative improvement potentials are given in the three biofuel production cases, it is not obvious how these potentials would affect WTT values, since the biofuel production processes are complex and changing one parameter impacts other parameters. The improvement potentials are therefore discussed qualitatively. From the entire study we have come to agree on the following common conclusions: (i) research and development in Sweden within the three studied 2G biofuel production technologies is extensive, (ii) in general, the processes, within the three cases, work well at pilot and demonstration scale and are now in a phase to be proven in large scale, (iii) there is still room for improvement although some processes have been known for decades, (iv) the biofuel production processes are complex and site specific and process improvements need to be seen and judged from a broad systems perspective (both within the production plant as well as in the entire well-to-tank perspective), and (v) the three studied biofuel production systems are complementary technologies. Futher, the process of conducting this study is worth mentioning as a result itself, i.e. that many different actors within the field have proven their ability and willingness to contribute to a common report, and that the cooperation climate was very positive and bodes well for possible future collaboration within the framework of the f3 center. Finally, judging from the political ambitions it is clear that the demand for renewable fuels will significantly increase during the coming decade. This will most likely result in opportunities for a range of biofuel options. The studied biofuel options all represent 2G biofuels and they can all be part of the solution to meet the increased renewable fuel demand.
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| 4. |
- Alvors, Per, et al.
(författare)
-
Research and development challenges for Swedish biofuel actors – three illustrative examples : Improvement potential discussed in the context of Well-to-Tank analyses
- 2010
-
Rapport (övrigt vetenskapligt/konstnärligt)abstract
- Currently biofuels have strong political support, both in the EU and Sweden. The EU has, for example, set a target for the use of renewable fuels in the transportation sector stating that all EU member states should use 10% renewable fuels for transport by 2020. Fulfilling this ambition will lead to an enormous market for biofuels during the coming decade. To avoid increasing production of biofuels based on agriculture crops that require considerable use of arable area, focus is now to move towards more advanced second generation (2G) biofuels that can be produced from biomass feedstocks associated with a more efficient land use.Climate benefits and greenhouse gas (GHG) balances are aspects often discussed in conjunction with sustainability and biofuels. The total GHG emissions associated with production and usage of biofuels depend on the entire fuel production chain, mainly the agriculture or forestry feedstock systems and the manufacturing process. To compare different biofuel production pathways it is essential to conduct an environmental assessment using the well-to-tank (WTT) analysis methodology.In Sweden the conditions for biomass production are favourable and we have promising second generation biofuels technologies that are currently in the demonstration phase. In this study we have chosen to focus on cellulose based ethanol, methane from gasification of solid wood as well as DME from gasification of black liquor, with the purpose of identifying research and development potentials that may result in improvements in the WTT emission values. The main objective of this study is thus to identify research and development challenges for Swedish biofuel actors based on literature studies as well as discussions with the the researchers themselves. We have also discussed improvement potentials for the agriculture and forestry part of the WTT chain. The aim of this study is to, in the context of WTT analyses, (i) increase knowledge about the complexity of biofuel production, (ii) identify and discuss improvement potentials, regarding energy efficiency and GHG emissions, for three biofuel production cases, as well as (iii) identify and discuss improvement potentials regarding biomass supply, including agriculture/forestry. The scope of the study is limited to discussing the technologies, system aspects and climate impacts associated with the production stage. Aspects such as the influence on biodiversity and other environmental and social parameters fall beyond the scope of this study.We find that improvement potentials for emissions reductions within the agriculture/forestry part of the WTT chain include changing the use of diesel to low-CO2-emitting fuels, changing to more fuel-efficient tractors, more efficient cultivation and manufacture of fertilizers (commercial nitrogen fertilizer can be produced in plants which have nitrous oxide gas cleaning) as well as improved fertilization strategies (more precise nitrogen application during the cropping season). Furthermore, the cultivation of annual feedstock crops could be avoided on land rich in carbon, such as peat soils and new agriculture systems could be introduced that lower the demand for ploughing and harrowing. Other options for improving the WTT emission values includes introducing new types of crops, such as wheat with higher content of starch or willow with a higher content of cellulose.From the case study on lignocellulosic ethanol we find that 2G ethanol, with co-production of biogas, electricity, heat and/or wood pellet, has a promising role to play in the development of sustainable biofuel production systems. Depending on available raw materials, heat sinks, demand for biogas as vehicle fuel and existing 1G ethanol plants suitable for integration, 2G ethanol production systems may be designed differently to optimize the economic conditions and maximize profitability. However, the complexity connected to the development of the most optimal production systems require improved knowledge and involvement of several actors from different competence areas, such as chemical and biochemical engineering, process design and integration and energy and environmental systems analysis, which may be a potential barrier.Three important results from the lignocellulosic ethanol study are: (i) the production systems could be far more complex and intelligently designed than previous studies show, (ii) the potential improvements consist of a large number of combinations of process integration options wich partly depends on specific local conditions, (iii) the environmental performance of individual systems may vary significantly due to systems design and local conditons.From the case study on gasification of solid biomass for the production of biomethane we find that one of the main advantages of this technology is its high efficiency in respect to converting biomass into fuels for transport. For future research we see a need for improvements within the gas up-grading section, including gas cleaning and gas conditioning, to obtain a more efficient process. A major challenge is to remove the tar before the methanation reaction.Three important results from the biomethane study are: (i) it is important not to crack the methane already produced in the syngas, which indicates a need for improved catalysts for selective tar cracking, (ii) there is a need for new gas separation techniques to facilitate the use of air oxidation agent instead of oxygen in the gasifier, and (iii) there is a need for testing the integrated process under realistic conditions, both at atmospheric and pressurized conditions.From the case study on black liquor gasification for the production of DME we find that the process has many advantages compared to other biofuel production options, such as the fact that black liquor is already partially processed and exists in a pumpable, liquid form, and that the process is pressurised and tightly integrated with the pulp mill, which enhances fuel production efficiency. However, to achieve commercial status, some challenges still remain, such as demonstrating that materials and plant equipment meet the high availability required when scaling up to industrial size in the pulp mill, and also proving that the plant can operate according to calculated heat and material balances. Three important results from the DME study are: (i) that modern chemical pulp mills, having a potential surplus of energy, could become important suppliers of renewable fuels for transport, (ii) there is a need to demonstrate that renewable DME/methanol will be proven to function in large scale, and (iii) there is still potential for technology improvements and enhanced energy integration.Although quantitative improvement potentials are given in the three biofuel production cases, it is not obvious how these potentials would affect WTT values, since the biofuel production processes are complex and changing one parameter impacts other parameters. The improvement potentials are therefore discussed qualitatively. From the entire study we have come to agree on the following common conclusions: (i) research and development in Sweden within the three studied 2G biofuel production technologies is extensive, (ii) in general, the processes, within the three cases, work well at pilot and demonstration scale and are now in a phase to be proven in large scale, (iii) there is still room for improvement although some processes have been known for decades, (iv) the biofuel production processes are complex and site specific and process improvements need to be seen and judged from a broad systems perspective (both within the production plant as well as in the entire well-to-tank perspective), and (v) the three studied biofuel production systems are complementary technologies. Futher, the process of conducting this study is worth mentioning as a result itself, i.e. that many different actors within the field have proven their ability and willingness to contribute to a common report, and that the cooperation climate was very positive and bodes well for possible future collaboration within the framework of the f3 center.Finally, judging from the political ambitions it is clear that the demand for renewable fuels will significantly increase during the coming decade. This will most likely result in opportunities for a range of biofuel options. The studied biofuel options all represent 2G biofuels and they can all be part of the solution to meet the increased renewable fuel demand.
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| 5. |
- Wang, Yang
(författare)
-
Exploring glycoside hydrolase family 5 (GH5) enzymes
- 2013
-
Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In 1990, the classification of carbohydrate-active enzymes (CAZymes) was introduced by the scientist Bernard Henrissat. According to sequence similarity, these enzymes were separated into families with conserved structures and reaction mechanisms. One interesting class of CAZymes is the group of glycoside hydrolases (GHs) containing more than 138000 modules divided into 131 families as of February 2013. One of the most versatile and the largest of these GH families, containing enzymes with numerous biomass-deconstructing activities, is glycoside hydrolase family 5 (GH5). However, for large and diverse families like the GH5 family, another layer of classification is required to get a better understanding of the evolution of diverse enzyme activities. In Paper I, a new subfamily classification of GH5 is presented in order to sort the family members into distinct groups with predictive power. In total, 51 subfamilies were defined. Despite the fact that several hundred GH5 enzymes have been characterized, 20 subfamilies lacking biochemically characterized enzymes and 38 subfamilies without structural data were identified. These highlighted subfamilies contain interesting targets for future investigation.The GH5 family includes endo-β-mannanases catalyzing the hydrolysis of the β-1,4-linked backbone of mannan polysaccharides, which are common hemicelluloses found as storage and structural polymers in plant cell walls. Mannans are commonly utilized as raw biomaterials in food, feed, paper, textile and cosmetic industries, and mannanases are often applied for modifying and controlling the property of mannan polysaccharides in such applications. The overwhelming majority of characterized mannanases are from microbial origin. The situation for plant mannanases is quite different, as the catalytic properties for only a handful have been determined. Paper II describes the first characterization of a heterologously expressed Arabidopsis β-mannanase.
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| 6. |
- Ademark, Pia, et al.
(författare)
-
Cloning and characterisation of Aspergillus niger genes encoding an alpha-galactosidase and a beta-mannosidase involved in galactomannan degradation
- 2001
-
Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956. ; 268:10, s. 2982-2990
-
Tidskriftsartikel (refereegranskat)abstract
- α-Galactosidase (EC 3.2.1.22) and β-mannosidase (EC 3.2.1.25) participate in the hydrolysis of complex plant saccharides such as galacto(gluco)mannans. Here we report on the cloning and characterization of genes encoding an α-galactosidase (AglC) and a β-mannosidase (MndA) from Aspergillus niger. The aglC and mndA genes code for 747 and 931 amino acids, respectively, including the eukaryotic signal sequences. The predicted isoelectric points of AglC and MndA are 4.56 and 5.17, and the calculated molecular masses are 79.674 and 102.335 kDa, respectively. Both AglC and MndA contain several putative N-glycosylation sites. AglC was assigned to family 36 of the glycosyl hydrolases and MndA was assigned to family 2. The expression patterns of aglC and mndA and two other genes encoding A. nigerα-galactosidases (aglA and aglB) during cultivation on galactomannan were studied by Northern analysis. A comparison of gene expression on monosaccharides in the A. niger wild-type and a CreA mutant strain showed that the carbon catabolite repressor protein CreA has a strong influence on aglA, but not on aglB, aglC or mndA. AglC and MndA were purified from constructed overexpression strains of A. niger, and the combined action of these enzymes degraded a galactomanno-oligosaccharide into galactose and mannose. The possible roles of AglC and MndA in galactomannan hydrolysis is discussed.
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| 7. |
- Ademark, Pia, et al.
(författare)
-
Hydrolytic properties of a beta-mannosidase purified from Aspergillus niger. J. Biotechnol. 75: 281-289.
- 1999
-
Ingår i: Journal of Biotechnology. - 1873-4863. ; 75:2-3, s. 281-289
-
Tidskriftsartikel (refereegranskat)abstract
- A β-mannosidase was purified to homogeneity from the culture filtrate of Aspergillus niger. A specific activity of 500 nkat mg−1 and a 53-fold purification was achieved using ammonium sulfate precipitation, anion-exchange chromatography, and gel filtration. The isolated enzyme has an isoelectric point of 5.0 and appears to be a dimer composed of two 135-kDa subunits. It is a glycoprotein and contains 17% N-linked carbohydrate by weight. Maximal activity was observed at pH 2.4–5.0 and at 70°C. The β-mannosidase hydrolyzed β-1,4-linked manno-oligosaccharides of degree of polymerization (DP) 2–6 and also released mannose from polymeric ivory nut mannan and galactomannan. The Km and Vmax values for p-nitrophenyl-β-Image-mannopyranoside were 0.30 mM and 500 nkat mg−1, respectively. Hydrolysis of Image-galactose substituted manno-oligosaccharides showed that the β-mannosidase was able to cleave up to, but not beyond, a side group. An internal peptide sequence of 15 amino acids was highly similar to that of an Aspergillus aculeatus β-mannosidase belonging to family 2 of glycosyl hydrolases.
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| 8. |
- Ademark, Pia, et al.
(författare)
-
Multiple alpha-galactosidases from Aspergillus niger: purification, characterization, and substrate specificities
- 2001
-
Ingår i: Enzyme and Microbial Technology. - 0141-0229. ; 29:6-7, s. 441-448
-
Tidskriftsartikel (refereegranskat)abstract
- Enzymes with α-galactosidase activity are produced by many organisms, often in multiple forms. Here we compare the biochemical and hydrolytic properties of four major α-galactosidase forms (α-gal I-IV) that were purified from the culture filtrate of Aspergillus niger. α-Gal II, III and IV appear to be isoforms of the same enzyme, and N-terminal amino acid sequence data suggest that they are closely related or identical to A. niger AglB in family 27 of the glycosyl hydrolases. α-Gal I is a completely different enzyme that belongs to family 36. α-Gal I had an isoelectric point of 4.15 and appears to be a tetramer composed of four 94-kDa subunits. α-Gal II, III and IV were dimers with monomeric molecular masses of 64 kDa and isoelectric points of 4.5, 4.7 and 4.8, respectively. α-Gal II-IV were stable when incubated for 17 h at 50°C and pH 2–5, whereas α-gal I was most stable at pH 5–6. All enzymes had maximal catalytic activity at pH 4.5 and 60°C, and hydrolyzed melibiose, raffinose and stachyose. α-Gal II-IV also degraded galactomanno-oligosaccharides and released 66% of the galactose side groups from polymeric locust bean gum galactomannan. α-Gal I released galactose from locust bean gum only in combination with A. niger β-mannosidase. Kinetic experiments showed that α-gal I hydrolyzed p-nitrophenyl-α-Image-galactopyranoside and melibiose more efficiently than α-gal II-IV. The distinct hydrolytic and biochemical properties of α-gal I and α-gal II-IV further signifies the difference between α-galactosidases of family 27 and 36.
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| 9. |
- Ademark, Pia, et al.
(författare)
-
Softwood hemicellulose-degrading enzymes from Aspergillus niger: Purification and properties of a β-mannanase
- 1998
-
Ingår i: Journal of Biotechnology. - 1873-4863. ; 63:3, s. 199-210
-
Tidskriftsartikel (refereegranskat)abstract
- The enzymes needed for galactomannan hydrolysis, i.e. β-mannanase, α-galactosidase and β-mannosidase, were produced by the filamentous fungus Aspergillus niger. The β-mannanase was purified to electrophoretic homogeneity in three steps using ammonium sulfate precipitation, anion-exchange chromatography and gel filtration. The purified enzyme had an isoelectric point of 3.7 and a molecular mass of 40 kDa. Ivory nut mannan was degraded mainly to mannobiose and mannotriose when incubated with the β-mannanase. Analysis by 1H NMR spectroscopy during hydrolysis of mannopentaose showed that the enzyme acts by the retaining mechanism. The N-terminus of the purified A. niger β-mannanase was sequenced by Edman degradation, and comparison with Aspergillus aculeatus β-mannanase indicated high identity. The enzyme most probably lacks a cellulose binding domain since it was unable to adsorb on cellulose.
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| 10. |
- Anderson, Lars, et al.
(författare)
-
Kinetics and stereochemistry of the Cellulomonas fimi beta-mannanase studied using H-1-NMR
- 2008
-
Ingår i: Biocatalysis and Biotransformation. - : Informa UK Limited. - 1024-2422 .- 1029-2446. ; 26:1-2, s. 86-95
-
Tidskriftsartikel (refereegranskat)abstract
- Endo-1,4-beta-mannanases (beta-mannanases) randomly hydrolyse the mannosidic bonds within the main chain of various mannans and heteromannans. Some of these polysaccharides are hemicelluloses, a major part of the plant cell-wall. The beta-mannanases have been assigned to family 5 and 26 of the glycoside hydrolase clan A. This work presents a detailed kinetic analysis of the family 26 beta-mannanase CfMan26A from the soil-bacterium Cellulomonas fimi. The full-length enzyme consists of five modules: a family 26 catalytic module, an immunoglobulin-like module, a mannan-binding module, a surface layer homology-module and a module of unknown function. A truncated variant consisting of the catalytic module and the immunoglobulin-like module was used in these studies. The degradation of mannotriose, mannotetraose and mannopentaose was studied by H-1-NMR. First, the mutarotation of one of the hydrolysis products (mannose) was determined to be 1.7 10(-5) s(-1) at 5 degrees C and pH 5.0. As expected for a family 26 glycoside hydrolase, the hydrolysis was shown to proceed with overall retention of the anomeric configuration. Many 'retaining' enzymes can perform transglycosylation reactions. However, no transglycosylation could be detected. Kinetic constants were calculated from progress curves using computer simulation. It was revealed that the -3 subsite had a greater impact on the apparent k(cat)/K-m ratio (the catalytic efficiency) than the +2 subsite. The beta-anomer of mannotriose was hydrolysed 1000-times more efficiently than the alpha-anomer indicating selectivity for the beta- over the alpha-anomer in the +1 subsite. With background information from the previous published 3D-structure of the truncated variant of Man26A, a structural explanation for the observations is discussed.
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