Sugar cane bagasse as a possible source of fermentable carbohydrates. II. Optimization of the xylose isomerase reaction for isomerization of xylose as well as sugar cane bagasse hydrolyzate to xylulose in laboratory-scale units

Both the forward and backward reactions of xylose isomerase (Sweetzyme Q) with xylose and glucose as substrates have been studied in terms of kinetics and thermodynamics. The relationship between the two reactions can thus be determined. Much attention has been given to the reaction with xylose as substrate. The optimal conditions of the xylose reaction in terms of pH, buffer, metal ions, substrate concentration, temperature, and ionic strength have been determined. These findings did not differ much from those reported for the glucose reaction. Equilibrium constants for the aldose to ketose conversion were more favorable in the case of glucose. The results obtained with continuous isomerization of xylose in columns packed with either Sweetzyme Q or Taka-Sweet were very similar to those obtained from batch isomerization processes. Particle size had a definite effect on reaction rate, which indicates that diffusion limitations do occur with the immobilized enzyme particles. Heat stability of Sweetzyme Q was good with t(1/2) of 118, 248, and 1200 h at 70, 55, and 40 degrees C, respectively. A novel method for the separation of xylose-xylulose mixtures with water as eluant on a specially prepared Dowex 1 x 8 column was developed. This technique has the capability of producing pure xylulose for industrial or research applications. A writ for a patent regarding this technique is at present prepared.     

Fermentation of sugar cane bagasse hemicellulose hydrolysate to l(+)-lactic acid by a thermotolerant acidophilic Bacillus sp.**

Sugar cane bagasse hemicellulose, hydrolyzed by dilute H2SO4, supplemented with mineral salts and 0.5% corn steep liquor, was fermented to L(+)-lactic acid using a newly isolated strain of Bacillus sp. In batch fermentations at 50 degrees C and pH 5, over 5.5% (w/v) L(+)-lactic acid was produced (89% theoretical yield; 0.9 g lactate per g sugar) with an optical purity of 99.5%.     

Simultaneous saccharification and co-fermentation of crystalline cellulose and sugar cane bagasse hemicellulose hydrolysate to lactate by a thermotolerant acidophilic Bacillus sp

Polylactides produced from renewable feedstocks, such as corn starch, are being developed as alternatives to plastics derived from petroleum. In addition to corn, other less expensive biomass resources can be readily converted to component sugars (glucose, xylose, etc.) by enzyme and/or chemical treatment for fermentation to optically pure lactic acid to reduce the cost of lactic acid. Lactic acid bacteria used by the industry lack the ability to ferment pentoses (hemicellulose-derived xylose and arabinose), and their growth and fermentation optima also differ from the optimal conditions for the activity of fungal cellulases required for depolymerization of cellulose. To reduce the overall cost of simultaneous saccharification and fermentation (SSF) of cellulose, we have isolated bacterial biocatalysts that can grow and ferment all sugars in the biomass at conditions that are also optimal for fungal cellulases. SSF of Solka Floc cellulose by one such isolate, Bacillus sp. strain 36D1, yielded l(+)-lactic acid at an optical purity higher than 95% with cellulase (Spezyme CE; Genencor International) added at about 10 FPU/g cellulose, with a product yield of about 90% of the expected maximum. Volumetric productivity of SSF to lactic acid was optimal between culture pH values of 4.5 and 5.5 at 50 degrees C. At a constant pH of 5.0, volumetric productivity of lactic acid was maximal at 55 degrees C. Strain 36D1 also co-fermented cellulose-derived glucose and sugar cane bagasse hemicellulose-derived xylose simultaneously (SSCF). In a batch SSCF of 40% acid-treated hemicellulose hydrolysate (over-limed) and 20 g/L Solka Floc cellulose, strain 36D1 produced about 35 g/L lactic acid in about 144 h with 15 FPU of Spezyme CE/g cellulose. The maximum volumetric productivity of lactic acid in this SSCF was 6.7 mmol/L (h). Cellulose-derived lactic acid contributed to about 30% of this total lactic acid. These results show that Bacillus sp. strain 36D1 is well-suited for simultaneous saccharification and co-fermentation of all of the biomass-derived sugars to lactic acid.     

Characterization of an archaeal multidrug transporter with a unique amino acid composition

The Smr family of multidrug transporters consists of small membrane proteins that extrude various drugs in exchange with protons rendering cells resistant to these drugs. Smr proteins identified to date have been found only in Eubacteria. In this work we present the cloning and characterization of an Smr protein from the archaeon Halobacterium salinarum, the first Smr in the archaeal kingdom. The protein, named Hsmr, was identified through sequence similarity to the Smr family, and the DNA sequence was cloned into an Escherichia coli expression system. Hsmr is heterologously expressed in a functional form despite the difference in lipid composition of the membrane and the lower salt in the cell and its environment. Cells harboring the Hsmr plasmid transport ethidium bromide in an uncoupler-sensitive process and gain resistance to ethidium bromide and acriflavine. Hsmr binds tetraphenylphosphonium (TPP(+)) with a relatively low affinity (K(D) approximately 200 nm) at low salt concentration that increases (K(D) approximately 40 nm) upon the addition of 2 m of either NaCl or KCl. The Hsmr protein contains many of the signature sequence elements of the Smr family and also a high content of negative residues in the loops, characteristic of extreme halophiles. Strikingly, Hsmr is composed of over 40% valine and alanine residues. These residues are clustered at certain regions of the protein in domains that are not important for activity, as judged from lack of conservation and from previous studies with other Smr proteins. We suggest that this high content of alanine and valine residues is a reflection of a "natural" alanine and valine scanning necessitated by the high GC content of the gene. This phenomenon reveals significant sequence elements in small multidrug transporters.     

The quantitative variability and monosaccharide composition of sediment carbohydrates associated with intertidal diatom assemblages

The extracellular secretions of epipelic diatoms (Bacillariophyceae) axe an important source of carbohydrates on intertidal sediments. For analytical purposes, sediment carbohydrates have been operationally separated into colloidal and bulk fractions that are often assumed to be similar in their chemical properties. However, there has been little investigation into the nature of the two fractions. In this study, carbohydrate fractions were sampledin situ, isolated, purified and biochemically characterised using gas chromatography-mass spectrometry (GC-MS). Both carbohydrate fractions were found to contain similar sugars although in different proportions. Glucose represented more than 80% of the monosaccharides identified in the colloidal carbohydrate fraction while only 37% of monosaccharides present in the bulk carbohydrate fraction. Colloidal carbohydrate concentrations showed short-term variability and were correlated with diatom biomass (as chlorophylla) suggesting the colloidal fraction is labile and may be of recent origin, perhaps representing diatom activity. Concentrations of the bulk carbohydrate fraction did not show significant short-term variation and was therefore more refractory. This combination of biochemical and field data suggested that the bulk and colloidal carbohydrate fractions were chemically and physically different. These findings have relevance to studies of estuarine carbon cycling.Abbreviations LTSEM Low-temperature scanning electron microscopy - GC-MS Gas chromatography-mass spectroscopy - EPS Extracellular polymeric substances     

Isolation and amino acid composition of human angiotensin I

1. Angiotensin, the most powerful pressor substance known, suspected to be a causal substance in renal hypertension and previously isolated from animal sources, has now been isolated from human sources and the amino acid composition was analysed. 2. The procedures followed in the successful isolation of human angiotensin include: (a) preparation of stable materials to obtain maximum formation of human angiotensin; (b) a relatively selective adsorption of the formed angiotensin on Dowex 50W (X2); (c) gel filtration through Sephadex G-25; (d) cation-exchange chromatography on CM-cellulose; (e) anion-exchange gel filtration on DEAE-Sephadex A-25; (f) molecular-sieve chromatography through Bio-Gel P-2. 3. The homogeneity of the human angiotensin isolated was confirmed by paper and thin-layer chromatography and paper electrophoresis. 4. The biological activity observed indicates the substance isolated to be human angiotensin I. 5. The amino acid analysis suggested the following proportional composition: Asp, 1; Pro, 1; Val, 1; Ile, 1; Leu, 1; Tyr, 1; Phe, 1; His, 2; Arg, 1. This composition is similar to that of horse angiotensin I, i.e. isoleucine(5)-angiotensin I.     

Effect of carbohydrates in the culture medium on amino acid composition of a cephalosporin C producer

The intracellular and extracellular amino acid composition of an auxotrophic methionine-deficient strain of Acremonium chrysogenum was studied in respect of the content of various carbohydrates in the fermentation medium. In the presence of glucose, an intensive involvement of exogenous DL-methionine into the cell metabolism was observed at earlier stages than in the presence of dextran or succrose. The total content of intracellular amino acids was lower in the cells grown on the medium with glucose. The production of cephalosporin C depended on the intracellular content of methionine, glutamic acid and lysine.     

Inability of Lactobacillus plantarum and other lactic acid bacteria to grow on D-ribose as sole source of fermentable carbohydrate

Lactobacillusplantarum NCIMB 8026, NCIMB 8026(s), NCIMB 8014, NCFB 1752, Lact. brevis NCIMB 4617, Leuconostoc mesenteroides NCIMB 8023, Streptococcus agalactiae NCFB 1348, Pediococcus acidilactici NCFB 1859 and Ped. pentosaceus NCFB 990 did not grow on D-ribose as the sole source of fermentable carbohydrate in a chemically defined medium but grew on D-ribose in the presence of glucose. Lactobacillus plantarum NCIMB 8026(s) also grew on D-xylose and L-arabinose in the presence but not in the absence of glucose. Enterococcus faecalis NCFB 581 grew with D-ribose as the sole fermentable carbohydrate. Leuconostoc mesenteroides NCIMB 8710 and Lactococcus lactis subsp. lactis NCFB 763 did not use ribose in the presence or absence of glucose. Lactobacillus plantarum NCIMB 8026(s) utilized ribose and glucose simultaneously in the proportion of approximately 1 ribose to 1 glucose, producing approximately 3 lactate to 1 acetate and similar yields of dry biomass from glucose and ribose. Growth of Lact. plantarum 8026(s) with glucose and excess D-ribose ceased when D-glucose was exhausted, but metabolism of D-ribose to lactic and acetic acids continued. The enzyme system for the metabolism of D-ribose in Lact. plantarum was inducible, requiring D-glucose and amino acids for adaptation.     

Rapid purification of porcine colostral whey lactoferrin by affinity chromatography on single-stranded DNA-agarose. Characterization, amino acid composition and N-terminal amino acid sequence

We have determined that the major iron-binding and DNA-binding protein in porcine colostral whey is lactoferrin. This lactoferrin was purified to homogeneity in one chromatographic step using immobilized single-stranded DNA-agarose. Although different in chromatographic behavior from human lactoferrin, the porcine lactoferrin purified in this manner was shown to be homogeneous by high-performance ion-exchange chromatography (Mono-S), immobilized metal ion (Cu2+) affinity chromatography, size-exclusion chromatography (TSK-4000SW), and reverse-phase (phenyl) chromatography. Electrophoresis on SDS-polyacrylamide gradient (10-20%) gels under reducing conditions showed the purified lactoferrin to be a single protein (silver-stained) of 78 kDa. Apolactoferrin purified in this manner bound iron and displayed a UV/VIS absorption spectrum indistinguishable from that of human lactoferrin. The molar absorption coefficient of hololactoferrin was 3.86 x 10(3) M-1 at 465 nm and 1.08 x 10(5) M-1 at 280 nm. Affinity elution analyses of the purified lactoferrin on immobilized DNA revealed that the affinity of this protein for DNA was independent of bound iron. Porcine lactoferrin was recognized by antibodies directed against human lactoferrin and bovine lactoferrin. The amino acid composition and N-terminal amino acid sequence analysis (30 residues) revealed a high degree of sequence homology with human, equine and bovine lactoferrin. These results demonstrate the effectiveness of immobilized DNA as a rapid and simple lactoferrin purification procedure and demonstrate the presence of a lactoferrin in porcine colostral whey with a high degree of sequence homology to human lactoferrin.     

Primary structure of a human IgA1 immunoglobulin. I. Isolation, composition, and amino acid sequence of the chymotryptic peptides.

As the initial phase of the determination of the complete covalent structure of a human immunoglobulin A, 52 chymotryptic peptides, ranging in length from 2 to 37 residues, were isolated and characterized from the reduced and carboxymethylated alpha1 heavy chain of the myeloma IgA protein Bur. The peptides were subjected to sequence analysis by the dansylation technique, manual and automatic Edman degradation, and carboxypeptidase digestion. The results, in conjunction with the data on the tryptic and thermolysin peptides and the cyanogen bromide fragments reported in the accompanying papers, established the complete primary structure of a human IgA chain.