Methods 31 1
1. Suspend the starch well in demineralized water. Maximum starch concentration recommended for this procedure is 2-5 by dry weight see Notes - and - . 2. Add approx 100 ppm of calcium see Note 9 . 3. Adjust the pH to 6.6. Take care to keep the starch granules in suspension, and allow up to 15 s in a well-mixed system for the pH to come into equilibrium see Note . 4. Heat the starch suspension from room temperature to 100 C in about 0.5 h under continuous stirring. Mix especially well when the...
Materials 21 Ghz
1. Potato or tapioca starch, which can be obtained from one of the many starch producers like Avebe Veendam, The Netherlands or Emsland St rke Emlichheim, Experimental setup for preparing gelatinized starch. Experimental setup for preparing gelatinized starch. Germany , or from suppliers like Sigma. Starch may be stored at room temperature. There are differences between starches from different botanical sources see Note . 2. Heat-stable a-amylase from Bacillus licheniformis stored at 4 C see...
References Nmx
1. Bartolom , B., Faulds, C. B., Kroon, P. A., Waldron, K. W., Gilbert, H. J., et al. 1997 An Aspergillus niger esterase FAE-III and a recombinant Pseudomonas fluorescens subsp. cellulosa esterase XYLD release a 5-5' ferulic dehydrodimer diferulic acid from barley and wheat cell walls. Appl. Environ. Microbiol. 63, 208-212. 2. Saulnier, L., Vigouraux, J., and Thibault, J.-F. 1995 Isolation and partial characterization of feruloylated oligosaccharides from maize bran. Carbohydr. Res. 272,...
Analysis of Phenolics
1. Add 10 nmol of a suitable phenolic acid to the sample 0.4 mL as an internal standard. Cinnamic acid 1 mL of a 10 nmol mL stock solution in water is recommended since this phenolic is well separated from the common cell wall phenolics under the HPLC conditions described here. 2. Add 100 L of glacial acetic acid to the sample and vortex mix. The pH of the solution should be about 3.0 and should be tested with pH paper. The low pH ensures that phenolics are in the acid form and will be...
Notes Tvs
1. Sucrose and extracts of chicory or Jerusalem artichoke may also be used as useful carbon source for endoinulinase production from Pseudomonas sp. 2. The endoinulinase preparation can be utilized for IOS production, achieving a satisfactory IOS yield max. 83 . Thus, it is not a prerequisite to purify the endoinulinase for IOS production. 3. Various kinds of ion exchangers, which are inexpensive and commercially available, can be used successfully for endoinulinase immobilization. An anionic...
Notes
1. The sterilization of YM-T medium must be carried out as follows The solution containing the carbon source must be sterilized apart from the solution containing salts both solutions have to be concentrated to obtain the required concentration of nutrients when they are mixed . It is also necessary to sterilize all the Erlenmeyer flasks that are going to be employed and some tips of different pipettes. After sterilization, the carbon source and the other nutrients have to be mixed in a...
Principle of the Method
A combined sequential use of glycosidase together with glycosyltransferase, including a cofactor regeneration, transforms complicated multistep reactions into a one-pot reaction 5 ,thus avoiding laborious purifications of the intermediates. This reduces considerably the costs of the procedure, using a cheaper glycosidase, and a cheap substrate as well. Reversed hydrolysis of the disaccharide intermediate is blocked by its further glycosyltransferase-mediated sialylation, thus forming an...
Introduction Exm
The filamentous growing fungus Schizophyllum commune ATCC 38548 1 secretes a neutral homoglucan trivial name Schizophyllan that consists of a backbone chain of 1,3-P-d-glucopyranose units linked with single 1,6-bonded P-d-glucopyranoses at about every third glucose molecule in the basic chain Fig. 1 . The molecular weight varies between 6 and 12106 g mol 2 . Schizophyllan dissolves in water as a triple helix with protruding pendant P-1,6-linked d-glucose units originating from the outside of...
References Wug
1. Kikumoto, S., Miyajima, T., Yoshizumi, S., Fujimoto, S., and Kimura, K. 1970 Polysaccharide produced by Schizophyllum commune. I. Formation and some properties of an extracellular polysaccharide. Nippon Nogei Kagaku Kaishi 44, 337-342. 2. Rau, U., M ller, R.-J., Cordes, K., and Klein, J. 1990 Process and molecular data of branched 1,3- -d-glucans in comparison with Xanthan. Bioprocess Eng. 5, 89-93. 3. Norisuye, T. Yanaki, T., and Fujita, H. 1980 Triple helix of a Schizophyllum commune...
References Sqm
1. Rastall, R. A. and Bucke, C. 1992 Enzymatic synthesis of oligosaccharides. Biotechnol. Genet. Eng. Rev. 10, 253-281. 2. Hedbys, L., Johansson, E., Mosbach, K., and Larsson, P-O. 1989 Synthesis of by the sequential use of B-d-galactosidases from bovine testes and Escherichia coli. Carbohydr. Res. 186, 217-223. 3. Johansson, E., Hedbys, L., Mosbach, K., Larsson, P-O., and Gunnarsson, A. and Svensson, S. 1989 Studies of the reversed a-mannosidase reaction at high concentrations of mannose. Enz....
Allomorph Characterization by FTIR and FTRaman Spectroscopy
The FTIR spectrum for the synthetic cellulose, for the region 4000-2200 cm-1 is given in Fig. 4 and for the region 2000-650 cm-1 in Fig. 5. In each case the precise frequency of absorption is also given for each detected peak. The corresponding Raman spectrum for this material is given in Fig. 6 again with the precise Raman shift frequencies given at the top of the spectrum. These spectra are compared with those obtained for pulp and Viscose in Fig. 7 FTIR spectra and Fig. 8 FT-Raman spectra...
Contributors
Alain Arnaud Chaire de Microbiologie Industrielle et de G netique des Micro-organismes, Ecole Nationale Sup rieure Agronomique de Montpellier, Montpellier, France Bego a Bartolome Biochemistry Department, Institute of Food Research, Norwich Research Park, Norwich, UK Emmanuel Boures ULICE, Zac Les Portes de Riom, Riom, France Marie-Pierre Bousquet Centre de Bioing nierie Gilbert Durand, D partement de G nie Biochemique et Alimentaire, INSA, UMR CNRS, LA INRA, Complexe Scientifique de Rangueil,...
References Snj
1. Desai, J. D. and Desai, A. J. 1993 Production of biosurfactants, in Biosurfactants Production, Properties, Applications, Surfactant Science Series, vol. 48 Kosaric, N., ed. , Marcel Dekker, New York, pp. 65-97. 2. Lang, S. and Wagner, F. 1993 Bioconversion of oils and sugars to glycolipids, in Biosurfactants Production, Properties, Applications, Surfactant Science Series, vol. 48 Kosaric, N., ed. , Marcel Dekker, New York, pp. 205-227. 3. Wagner, F. and Lang, S. 1996 Microbial and enzymatic...
Method 1 Microbial Production of Lactonic Sophorose Lipids
1. Sterilize all media at 121 C and 1 bar overpressure for 20 min. 2. For maintaining C. bombicola ATCC 22214 in your laboratory, incubate the yeast strain on agar slants at 27 C for 48 h, and deposit at 4 C. 3. Transfer a loopful of cells into a 500-mL Erlenmeyer shake flask with two inserts containing 100 mL of seed culture medium inoculate five flasks in parallel. After culturing for 36 h at 27 C on a rotary shaker 100 rpm , transfer 50-mL portions into 10 500-mL cultures placed in 2-L...
Introduction Iww
Starch mainly consists of amylopectin 70-80 w w and amylose 20-30 w w . Amylopectin is a branched macromolecule mol wt 107-5.108 of which on average 1 in 20-25 a- 1,4 -bond glucose units is branched by an a- 1,6 -d-glucosidic bond. Amylose mol wt 105-107 consists of much longer linear chains of which on average one in several hundred a- 1,4 -bond glucose units is branched by an a- 1,6 -d-glucosidic bond 1 . The hydrolysis of starch to low-mol-wt products catalyzed by an a-amylase is one of the...
Introduction Rbf
This chapter demonstrates a practical approach to a rather sophisticated multienzymatic one-pot reaction in glycoscience. The first applications using the multienzyme system in glycosylation were mostly targeted to generation or regeneration of the activated glycosyl donors in situ kinases, phosphorylation sequences . Recently, more sophisticated approaches emerged, e.g., sequential use of more glycosyltransferases, including cofactor regeneration 1-4 , or even sequential use of glycosidases...
References Djn
1. Jeanloz, R. W. 1970 Mucopolysaccharides in higher animals, in The Carbohydrates, Pigman, W., Horton, D., and Herp, A., eds. , Academic, New York pp. 589-625, vol. IIB, 2. Lindahl, U. and Backstrom, G. 1972 Biosynthesis of l-iduronic acid in heparin Epimerization and d-glucuronic acid on the polymer level. Biochem. Biophys. Res. Commun. 46, 985-991. 3. Larsen, B. and Haug, A. 1971 Biosynthesis of alginate. Part I. Composition and structure of alginate produced by Azotobacter vinelandii Lipman...
Cibacron Blue Agarose Preparation
1. About 100 g of agarose 6B Sigma shipped in 20 EtOH should be loaded into a column and washed well with distilled water to remove EtOH. 2. Suspend the gel in 350 mL O in a 1000-mL conical flask. 3. Add a solution of Cibacron blue F3G-A Fluka 1 g 100 mL O, and mix the slurry slowly on the rotary or reciprocal shaker not the magnetic one-bead disruption for 5 min. 4. Add 50 mL of 20 w v NaCl, and stir the mixture for another 30 min. 5. Add 2.5 mL of 5 N NaOH, and continue the mixing for 3 d. 6....
Enzyme Fractionation
The global procedure for enzyme fractionation see Note 9 and concentration is summarized in Fig. 1. 1. Harvesting when the optical density stabilizes around 18-20, after 7 h of culture, the culture medium is cooled to 4 C and the pH adjusted to 5.6 to favor enzyme stability. 2. Centrifugation the two forms of L. mesenteroides NRRL B-1299 dextransucrase are separated by centrifugation of 500 mL of culture broth at 7000g 4 C, for 30 min. 3. Dextransucrase insoluble preparation IGT the pellet,...
Notes Tgm
1. Although fermentation parameters of aeration, agitation, pH, and temperature should be established for each microorganism, the general conditions for producing fructosyltransferase by growing cultures of organisms are well established. For example, sucrose is the best carbon source for both cell growth and enzyme activity, maintaining pH above 5.5 is important, and the optimum temperature for growth is 30 C. Intracellular fructosyltransferase activity can be greatly enhanced by increasing...
References Gli
1. Banks, W. and Greenwood, C. T. 1975 Starch and Its Components. Edinburgh University Press, Edinburgh. 2. Nigam, P. and Singh, D. 1995 Enzyme and microbial systems involved in starch processing. EnzymeMicrob. Technol. 17, 770-778. 3. Schenck, F. W. and Hebeda, R. E. 1992 Starch Hydrolysis Products. VCH, New York. 4. Bertoft, E. 1991 Investigation of the fine structure of alpha-dextrins derived from amylopectin and their relation to the structure of waxy-maize starch. Carbohydr. Res. 212,...
Method 3 Microbial Production of Mannosylerythritol Lipids
1. Microorganism C. antarctica CBS 6821, available from the Centralbureau voor Schimmelcultures, Julianalaan 67, 2628 BC Delft, The Netherlands. 2. Medium for agar slants potato-dextrose agar Difco . Use immediately. 3. Seed culture medium 60 g L glucose, 1 g L NH4NO3, 0.5 g L KH2PO4, 0.5 g L MgSO4 7 H2O, 1 g L yeast extract, and tap water. Use immediately. 4. Fermentation medium 80 mL L soybean oil, 2 g L NaNO3, 0.2 g L KH2PO4, 0.2 g L MgSO4 7 H2O, 1 g L yeast extract, and tap water. Use...
Introduction Czc
The enzymatic interconversions of the various hexoses found in polysaccharides and glycoproteins normally take place either on the unsubstituted sugars, their phosphate esters, or at the sugar nucleotide levels. In a few cases, however, epimerization reactions take place after the polysaccharides are formed. This type of postpolymerization epimerization is a feature of several uronic-acid containing polysaccharides found both in eukaryotes and in bacteria. These reactions are exemplified by C-5...
Introduction Gcd
Cellulose, a homopolysaccharide whose glucose units are connected through 13 1 -4 glycosidic linkages, has been of great interest in many scientific and application fields as polymeric drugs and new biomaterials 1 . So far, highly pure samples of cellulose and its derivatives have been produced for functional macromolecules, specimen for basic crystallographical research, enzyme substrates, and so on starting from various kinds of naturally occurring celluloses. The elongation of cellulose...
Introduction
Manufacture of high-molecular-weight compounds with thickener properties has been traditionally related to plants, seeds, and seaweeds. These compounds have been named gums. The rheological properties of their solutions show important alterations depending on uncontrolled variables such as weather, and their manual-collection labor cost can often influence their market price. Production of molecules with thickener properties from microorganisms was an important advance. This production is made...
Spectrophotometric Method
The only satisfactory spectrophotometric method available for alginate measurement was originally developed by Kennedy and Bradshaw 14 to assay alginate from brown seaweeds and, subsequently, modified to measure bacterial alginates 3,15 . The following procedure, adapted from Brivonese and Sutherland 3 , is used 1. Add 1 cm3 of 0.3 PHMBH CL- in 1 sodium acetate to 0.5 cm3 of culture supernatant eventually diluted to obtain a final alginate concentration less than 3 g.dm-3 into 1.5-cm3 Eppendorf...
Analytical Methods
To check the state of the fermentation, some analytical techniques must be used. The main components to be analyzed are biomass, xanthan, carbon source sucrose , and nitrogen source ammonium concentrations. Dissolved oxygen is also a very important component to consider, but it is usually monitored on line by means of an electrode. Biomass concentration is obtained by means of the measurement of the optical density at 540 nm of the diluted broth. Biomass concentration can be determined...
Inoculum Buildup
The size of the inoculum to be employed is around 5-10 of the total fermentation volume. The microorganism state to be used for production has a great influence on overall process evolution rate. In xanthan fermentation this step is really important, because xanthan is the bacterial capsule of X. campestris, and the gum is produced when the microorganism is growing. The xanthan gum produced during this stage is not welcome because of its great resistance to nutrient uptake by the cells. The...
Cannon-fenske Viscometer Tube
Alginates are a group of polysaccharides occurring as structural components or as capsular materials in the cell wall of the brown seaweeds or soil bacteria, respectively 1 . About 30,000 metric tons of sodium alginates per year are currently used in the food, pharmaceutical, textile and paper industries as thickening, stabilising and jellifying agents 2 . Since only a few of the many species of brown algae are suitable and are limited in abundance and location for commercial alginate...
Xanthan Production
The production stage can be divided into three steps fermentor sterilization and preparation, inoculation, and fermentation itself. Fermentor Sterilization and Preparation 1. The concentrated medium without carbon source is sterilized in the vessel and the concentrated solution of the carbon source sucrose is sterilized separately and afterwards introduced into the 2. The pH electrode must be calibrated before fermentor sterilization. 3. All the exits, inputs and electrodes of the fermentor...