Alpha-amylase production from catabolite derepressed Bacillus subtilis KCC103 utilizing sugarcane bagasse hydrolysate

A catabolite derepressed Bacillus subtilis strain KCC103 was used to produce alpha-amylase in medium containing sugarcane bagasse hydrolysate (SBH). Addition of SBH (1% reducing sugar (w/v)) to the nutrient medium supported maximum alpha-amylase production of 67.4 Um l(-1). HPLC analysis of SBH showed the presence of glucose, xylose and arabinose in the ratio of 0.9:1.0:0.16 (w/w/w). In SBH-medium glucose and xylose were consumed completely while arabinose remained unutilized. Uptake rate of glucose was 2-folds higher than xylose but rate of alpha-amylase production with xylose was 1.5-folds higher than glucose. Arabinose had no effect on growth and alpha-amylase synthesis. Further, alpha-amylase production in SBH-medium was enhanced to 144.5 Um l(-1) (2.2-fold) by response surface methodology where the levels of SBH, and other media components were varied. The modified medium consisted of (in gl(-1)) SBH: 24; peptone: 17.43; yeast extract: 1.32 and beef extract: 1.82. High level of SBH showed no significant inhibition of alpha-amylase synthesis. The derepressed strain KCC103 is useful to produce alpha-amylase economically in short time (30-36 h).     

Effect of L-arabinose and sucrose on the biosynthesis of heliomycin by its producer Streptomyces olivocinereus 11-98

When Streptomyces olivocinereus 11-98 MFU was grown in media containing L-arabinose or sucrose there was observed a converse relation between the culture growth and heliomycin biosynthesis. In media with two carbon sources: L-arabinose and glycerol or sucrose and glycerol at first L-arabinose or sucrose was consumed while the level of glycerol consumption remained low as compared to the control. After exhaustion of the first carbon source there was observed increased consumption of the second one i.e. glycerol. While the medium contained L-arabinose or sucrose the culture growth was mainly provided by these carbon sources and biosynthesis of heliomycin was inhibited. The culture started biosynthesis of heliomycin when L-arabinose or sucrose in the medium was exhausted. Probably control of heliomycin biosynthesis by L-arabinose or sucrose is achieved by catabolic type carbon regulation known as the general mechanism regulating biosynthesis of various antibiotics.     

Carbohydrate utilization patterns and substrate preferences in Bacteroides thetaiotaomicron

Specific growth rates of Bacteroides thetaiotaomicron NCTC 10582 with either glucose, arabinose, mannose, galactose or xylose as sole carbon sources were 0.42/h, 0.10/h, 0.38/h, 0.38/h and 0.16/h respectively, suggesting that hexose metabolism was energetically more efficient than pentose fermentation in this bacterium. Batch culture experiments to determine whether carbohydrate utilization was controlled by substrate-induced regulatory mechanisms demonstrated that mannose inhibited uptake of glucose, galactose and arabinose, but had less effect on xylose. Arabinose and xylose were preferentially utilized at high dilution rates (D > 0.26/h) in carbon-limited continuous cultures grown on mixtures of arabinose, xylose, galactose and glucose. When mannose was also present, xylose was co-assimilated at all dilution rates. Under nitrogen-limited conditions, however, mannose repressed uptake of all sugars, showing that its effect on xylose utilization was strongly concentration dependent. Studies with individual D-ZU-14C]-labelled substrates showed that transport systems for glucose, galactose, xylose and mannose were inducible. Measurements to determine incorporation of these sugars into trichloroacetic acid-precipitable material indicated that glucose and mannose were the principal precursor monosaccharides. Xylose was only incorporated into intracellular macromolecules when it served as growth substrate. Phosphoenolpyruvate:phosphotransferase systems were not detected in preliminary experiments to elucidate the mechanisms of sugar uptake, and studies with inhibitors of carbohydrate transport showed no consistent pattern of inhibition with glucose, galactose, xylose and mannose. These results indicate the existence of a variety of different systems involved in sugar transport in B. thetaiotaomicron.     

Transport and metabolism of glucose and arabinose in Bifidobacterium breve

Glucose was required for the transport of arabinose into Bifidobacterium breve. The non-metabolisable glucose analogue 2-deoxy-d-glucose (2-DG) did not facilitate assimilation of arabinose. Studies using d-[U-¹⁴C]-labelled arabinose showed that it was fermented to pyruvate, formate, lactate and acetate, whereas the principal metabolic products of d-[U-¹⁴C]-labelled glucose were acetate and formate. In contrast to glucose, arabinose was not incorporated into cellular macromolecules. A variety of metabolic inhibitors and inhibitors of sugar transport (proton ionophores, metal ionophores, compounds associated with electron transport) were used to investigate the mechanisms of sugar uptake. Only NaF, an inhibitor of substrate level phosphorylation, and 2-DG inhibited glucose assimilation. 2-DG had no effect on arabinose uptake, but NaF was stimulatory. High levels of phosphorylation of glucose and 2-DG by PEP and to a lesser degree, ATP were seen in phosphoenolpyruvate: phosphotransferase (PEP:PTS) assays. These data together with strong inhibition of glucose uptake by NaF suggest a role for phosphorylation in the transport process. Arabinose uptake in B. breve was not directly dependent on phosphorylation or any other energy-linked form of transport but may be assimilated by glucose-dependent facilitated diffusion.Abbreviations (2,4-DNP) 2,4-dinitrophenol - (2,4-DNP) carbonylcyanide m-chlorophenylhydrazone - (CCCP) (phosphoenolpyruvate phosphotransferase system) - PEP: PTS trichloroacetic acid - (TCA) 2-deoxy-d-glucose - (2-DG) 2-deoxy-d-glucose     

Fermentation performance of Candida guilliermondii for xylitol production on single and mixed substrate media

Semidefined media fermentation simulating the sugar composition of hemicellulosic hydrolysates (around 85 g l^-1 xylose, 17 g l^-1 glucose, and 9 g l^-1 arabinose) was investigated to evaluate the glucose and arabinose influence on xylose-to-xylitol bioconversion by Candida guilliermondii. The results revealed that glucose reduced the xylose consumption rate by 30%. Arabinose did not affect the xylose consumption but its utilization by the yeast was fully repressed by both glucose and xylose sugars. Arabinose was only consumed when it was used as a single carbon source. Xylitol production was best when glucose was not present in the fermentation medium. On the other hand, the arabinose favored the xylitol yield (which attained 0.74 g g^-1 xylose consumed) and it did not interfere with xylitol volumetric productivity (Q _P=0.85 g g^-1), the value of which was similar to that obtained with xylose alone.     

Induction and glucose repression of xylanase from a color variant strain ofAureobasidum pullulans

Summary Xylose, xylobiose and arabinose were identified as natural and direct inducers of xylanase from a color variant strain ofAureobasidium pullulans. Arabinose, in contrast to xylose, xylobiose and xylan, induced only the major isozyme of xylanase. Xylanase induction was subject to glucose repression.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U. S. Department of Agriculture over other firms or similar products not mentioned.     

Kinetic, structural and molecular docking studies on the inhibition of tyrosinase induced by arabinose

Tyrosinase plays a central role in biological pigment formation, and hence knowledge of tyrosinase catalytic mechanisms and regulation may have medical, cosmetic, and agricultural applications. We found in this study that arabinose significantly inhibited tyrosinase, and this was accompanied by conformational changes in enzyme structure. Kinetic analysis showed that arabinose-mediated inactivation followed first-order kinetics, and single and multiple classes of rate constants were measured. Arabinose displayed a mixed-type inhibitory mechanism with K(i)=0.22±0.07 mM. Measurements of intrinsic and ANS-binding fluorescence showed that arabinose induced tyrosinase to unfold and expose inner hydrophobic regions. We simulated the docking between tyrosinase and arabinose (binding energies were -26.28 kcal/mol for Dock6.3 and -2.02 kcal/mol for AutoDock4.2) and results suggested that arabinose interacts mostly with His61, Asn260, and Met280. The present strategy of predicting tyrosinase inhibition by simulation of docking by hydroxyl groups may prove useful in screening for potential tyrosinase inhibitors, as shown here for arabinose.     

Control of starch and exocellular polysaccharides biosynthesis by gibberellic acid with cells of sweet potato cultured in vitro

The regulation of starch synthesis and exocellular polysaccharide synthesis by GA₃ was studied with cells of sweet potato grown as suspension in glycerol medium. In the presence of GA₃, and under normal cell growth, starch formation was inhibited. The incorporation activity (starch synthesis) from ADP-[¹⁴C] glucose or UDP-[¹⁴C] glucose with GA₃ treated cells was reduced. On the other hand, the synthesis of exocellular polysaccharides composed of glucose, galactose, mannose and arabinose etc., was stimulated and a clear increase of the Man/Ara ratio was observed in the presence of GA₃. These results may indicate that GA₃ affects the regulation of starch synthesis and exocellular polysaccharide synthesis.     

The effects of isoniazid on the carbohydrates of Mycobacterium tuberculosis BCG

1. Mycobacterium tuberculosis BCG was usually grown in glycerol-asparagine-casein hydrolysate medium. A soluble fraction was obtained from the cells with aq. 50% ethanol; unbound lipids were then removed and the cells were treated with dilute alkali to give, after acidification, an alkali-extractable fraction and an insoluble fraction. On occasion, lipopolysaccharides were obtained by extracting with phenol or dimethyl sulphoxide instead of alkali. The soluble fraction contained, particularly after long extraction, polysaccharide containing mainly glucose, in addition to trehalose and monosaccharides and their derivatives. The alkali-extractable fraction contained polysaccharides containing mannose, glucose, arabinose, galactose and 6-O-methylglucose. These could be resolved into three fractions of markedly different molecular size. It is argued that the high-molecular-weight materials originated from the outside of the cell envelope and the medium-molecular-weight materials from a middle layer of the envelope. 2. Exposure of the growing cells to isoniazid, usually at 1 or 10mug/ml for 6-12h, increased the total cell carbohydrate, mainly due to an increase in trehalose and in insoluble glucan. It also facilitated the extraction of polysaccharide into the medium and the soluble fraction. This produced about a 25% decrease in the amount of carbohydrate in the alkaline-extractable fraction, mainly due to a fall in glucose, arabinose and 6-O-methylglucose. The decrease was confined to polysaccharides of large and medium molecular weight. When intact lipopolysaccharides were extracted, their amount was also decreased by isoniazid. 3. Substitution of ammonium sulphate for asparagine and casein hydrolysate in the medium, so that glycerol was the sole carbon source, decreased the carbohydrate accumulation brought about by isoniazid but did not alter its effect on polysaccharide extraction. 4. Growth with (14)C-labelled substrates showed that glycerol provided two to four times as much of the cell carbon as did asparagine, when both were present. Under these conditions isoniazid inhibited the incorporation of carbon atoms from asparagine into the cells, but had little effect on the total incorporation from glycerol. These experiments also showed that the effect of isoniazid on alkali-extractable polysaccharides was due to their loss to the soluble fraction and external medium. 5. It is suggested that isoniazid inhibits a pathway (probably the synthesis of mycolic acid) involved in the formation of the cell envelope, and that this inhibition results in some re-channelling of intermediates into carbohydrate synthesis and in some loss of polysaccharides through damage to the envelope.     

A mutagen assay detecting forward mutations in an arabinose-sensitive strain of Salmonella typhimurium.

Strain SV3 of Salmonella typhimurium is sensitive to arabinose, that is, unable to grow in a medium containing arabinose plus glycerol as carbon source. Arabinose resistance is the consequence of the mutational inactivation of one of at least three different genes. The selection of arabinose-resistant mutants provides a simple and sensitive assay for the detection of weak mutagens and for refined quantitative studies of strong ones. The assay is not influenced by experimental artifacts derived from physiological or lethal effects or from differences in plating density. Such artifacts are common with other bacterial mutagen assays, including those using strains analogous to SV3. As practical examples, the assay was used with N-methyl-N'-nitro-N-nitrosoguanidine and the fungicide captafol.     

The effect of human placental lactogen upon the metabolism of rat and human adipose tissue.

The metabolic effects of human placental lactogen (HPL) on rat and human white fat were tested in vitro. When tested against rat tissue, HPL resembled insulin in stimulating uptake of glucose and incorporation of [14C] glucose into CO2, triglyceride and glycogen, but differed from insulin in stimulating glycerol release and in failing to stimulate the incorporation of [14C] The stimulation of [14C] glucose incorporation and the inhibition of glycerol release by insulin were antagonized by HPL. The effects of HPL on human white fat resembled those on rat white fat,except that glycerol release was not stimulated in human tissue. The possible role of HPL in causing the diabetogenic stress of pregnancy is discussed in the light of these findings.     

Kinetic aspects of the relation of the growth of the producer and the biosynthesis of heliomycin depending on the carbon source in the medium

Kinetic parameters of Streptomyces olivocinereus 11-98 growth and biosynthesis of heliomycin were studied. It was shown that carbon sources such as glycerol, mannitol and ramnose were the most favourable for the antibiotic biosynthesis. These carbon sources belonged to the group of substances providing high growth rates of the culture. Ranging of the culture growth rates and antibiotic production levels revealed a set of carbon sources providing a converse relationship between the growth rate and antibiotic biosynthesis i.e. L-arabinose, potassium gluconate, raffinose and sucrose. It was suggested that these compounds were catabolic type regulators of heliomycin biosynthesis.     

Phospholipid biosynthesis is required for stalk elongation in Caulobacter crescentus.

Membrane phospholipid synthesis was inhibited in Caulobacter crescentus by growth of a glycerol auxotroph in the absence of glycerol or by treatment with the antibiotic cerulenin. It was observed that the final step in the swarmer cell-to-stalked cell transition, stalk elongation, was inhibited under these conditions. Since an early effect of inhibiting phospholipid synthesis in C. crescentus is the termination of deoxyribonucleic acid (DNA) replication (I. Contreras, R. Bender, A. Weissborn, K. Amemiya J. D. Mansour, S. Henry, and L. Shapiro, J. Mol. Biol. 138:401-410, 1980), we questioned whether the inhibition of stalk formation was due directly to the inhibition phospholipid synthesis or secondarily to the inhibition of DNA synthesis. Under conditions which inhibited DNA synthesis but permitted phospholipid synthesis, i.e., growth of a temperature-sensitive DNA elongation mutant at the restrictive temperature or treatment with hydroxy-urea, stalk elongation occurred normally. Therefore phospholipid synthesis is required for stalk elongation in C. crescentus.     

Elaboration of the method of "quiescent cells" for the study of heliomycin-synthesizing system of Streptomyces olivocinereus

A resting cell procedure was developed for S. olivocinereus. Washed mycelium of S. olivocinereus produced heliomycin for a short incubation period of 1.5 hours in a nitrogen-free medium containing a buffer solution, salts, a source of carbon and an inhibitor of protein synthesis. With the developed procedure production of heliomycin in the system of resting cells was investigated. For this purpose mycelium collected during various phases of S. olivocinereus development in batch cultures was used. It was found that in the batch cultures the rate of heliomycin production by the 24th hour of the development was comparable with that of the antibiotic accumulation in the resting cell system. After that period it markedly decreased by the 48th hour. This deviation in the dynamics of heliomycin production in batch cultures and the resting cell system can serve as a basis for further studies on heliomycin biosynthesis control by the carbon source.     

Soluble Cell Wall Polysaccharides Released from Pea Stems by Centrifugation : II. EFFECT OF ETHYLENE

The effect of ethylene on cell wall metabolism in sections excised from etiolated pea stems was studied. Ethylene causes an inhibition of elongation and a pronounced radial expansion of pea internodes as shown by an increase in the fresh weight of excised, 1-cm sections. Cell wall metabolism was studied using centrifugation to remove the cell wall solution from sections. The principal neutral sugars in the cell wall solution extracted with H₂O are arabinose, xylose, galactose, and glucose. Both xylose and glucose decline relative to controls in air within 1 hour of exposure to ethylene. Arabinose and galactose levels are not altered by ethylene until 8 hours of treatment, whereupon they decline in controls in air relative to ethylene treatment. When alcohol-insoluble polymers are fractionated into neutral and acidic polysaccharides, xylose and glucose predominate in the neutral fraction and arabinose and galactose in the acidic fraction. Ethylene depresses the levels of xylose and glucose in the neutral fraction and elevates arabinose and galactose in the acidic fraction. Ethylene treatment does not affect the level of uronic acids extracted with H₂O; however, the level of hydroxyproline-rich proteins in this water-extracted cell wall solution is increased by ethylene. Extraction of sections with CaCl₂ results in an increase in the levels of neutral sugars particularly arabinose. Ethylene depresses the yield of arabinose in calcium-extracted solution relative to controls in air. Similarly, extraction with CaCl₂ increases the yield of extracted hydroxyproline in ethanol-insoluble polymers and ethylene depresses its level relative to controls. Metabolism of uronic acids and neutral sugars and growth in response to ethylene treatment contrast markedly with auxin-induced polysaccharide metabolism and growth. With auxin, sections increase mostly in length not radius, and this growth form is associated with an increase in the levels of xylose, glucose, and uronic acids. With ethylene, on the other hand, stem elongation is suppressed and expansion is promoted, and this growth pattern is associated with a decrease in xylose and glucose in the ethanol-insoluble polysaccharides.     

Inhibition of Acid-Enhanced Elongation of Zea mays Root Segments by Galactose

The effect of sugars and metabolic inhibitors on the elongation of Zea mays root segments was analyzed by a rhizometer which records the elongation of each of 32 root segments at the same time. Galactose suppressed the acid-enhanced rapid elongation after a lag period of 1.5 hours, but it did not inhibit the slow elongation at pH 7. Mannose was less inhibitory than galactose. Arabinose, xylose, glucose, sucrose, mannitol, and sorbitol caused no inhibition. When galactose was removed after a 1-hour treatment, the elongation was partially recovered. Cycloheximide and 2-deoxyglucose suppressed acid-enhanced elongation when these were applied at the same time as acid treatments, whereas cordycepin (3′-deoxyadenosine) inhibited elongation only if it was applied prior to acid treatment. Over the 9-hour period of elongation studied, the inhibition by galactose was comparable to that of cycloheximide. Since galactose has been reported to suppress the sugar metabolism necessary for the cell wall synthesis, the later phase of acid-enhanced elongation of root segments may at least partially depend on the synthesis or metabolism of cell wall components. The inhibition of root growth by galactose may be partially ascribed to a direct effect on the elongation process in roots, an effect that is enhanced by the acidification of the cell walls.     

Zinc inhibition of hepatic fructose metabolism in rats

The ability of Zn to modulate key metabolic processes was investigated in a study of gluconeogenesis in isolated hepatocytes from fasted rats. Zn (100 μM) inhibited glucose production from fructose by 41%, sorbitol by 28%; glycerol by 17%, and glyceraldehyde by 26%. Maximum inhibition of gluconeogenesis from fructose occurred at 25 μM Zn. Zn inhibited the rate of lactate production from fructose by 24% but not from sorbitol, glycerol, or glyceraldehyde. Fructose uptake by hepatocytes was not affected by Zn. A positive linear relationship (r=0.994) was obtained between inhibition by Zn of glucose and lactate production, indicating that a common step in both pathways is inhibited by Zn. The effect of Zn on fructokinase, aldolase-B, and triokinase activities was determined on semipurified rat liver enzyme preparations. Zn had no affect on triokinase activity but inhibited the two other enzymes in a dose-dependent manner, with the inhibition of aldolase-B being much greater than of fructokinase for concentrations of Zn between 2.5 and 20 μM. Zn increased the intracellular concentration of fructose-1-P in hepatocytes incubated with fructose, indicating a more potent Zn inhibition of aldolase-B than fructokinase. In addition, hepatocytes treated with Zn had decreased ATP and ADP concentrations, but had normal energy charge, suggesting an effect of Zn on adenine nucleotide degradation or synthesis. The demonstration that Zn inhibits two enzymes in fructose metabolism adds to the growing list of metabolic pathways that are catalyzed by enzymes that are sensitive to Zn.     

Metabolism of cyclic adenosine 3'',5''-monophosphate and induction of tryptophanase in Escherichia coli.

The relationship between cyclic adenosine 3',5'-monophosphate (cyclic AMP) metabolism and the induction of tryptophanase and beta-galactosidase was studied in several strains of Escherichia coli grown with succinate, acetate, glycerol, or glucose as the carbon source. No consistent relationship between the intracellular concentration of cyclic AMP in the several strains cultured and the various carbon sources was discerned. In E. coli K-12-1 the induction of tryptophanase was found to vary in the order: succinate greater than acetate greater than glycerol greater than glucose, and that of beta-galactosidase was found in the order: glycerol greater than acetate greater than succinate greater than glucose. Rate of accumulation of cyclic AMP in the culture filtrate was in the order: succinate greater than acetate greater than glycerol greater than glucose. The addition of glycerol to E. coli K-12-1 grown in acetate caused inhibition of tryptophanase and slight inhibition of accumulation of extracellular cyclic AMP. These same conditions caused beta-galactosidase induction to be stimulated. The addition of exogenous cyclic AMP to cultures grown with four different carbon sources had an effect characteristic for each of the two enzymes studied as well as each individual carbon source. The results suggest that there are control elements distinct from cyclic AMP and its receptor protein which respond to the catabolic situation of the cell.     

Inhibitory effect of glucose and adenosine 3',5'-monophosphate on the synthesis of inducible N-acetylglucosamine catabolic enzymes in yeast

Glucose can block the utilization of N-acetylglucosamine in Saccharomyces cerevisiae, a facultative aerobe, but not in Candida albicans, an obligatory aerobe. Furthermore, glucose represses the synthesis of the enzymes of the N-acetylglucosamine catabolic pathway in S. cerevisiae, but not in C. albicans. The results suggest that catabolite repression is present in S. cerevisiae, but not in C. albicans. Cyclic AMP added to S. cerevisiae cells maintained in a glucose medium cannot bring about their release from catabolite repression. On the contrary, the synthesis of inducible enzymes of N-acetylglucosamine pathway was inhibited by cyclic AMP in both the yeasts. This seems to indicate that cyclic AMP can penetrate into the yeast cells. Furthermore, cyclic AMP inhibits protein synthesis, suggesting that protein synthesis in yeast is under cyclic AMP control.     

Potentiation by ammonia of the metabolic effects of pent-4-enoate in isolated rat hepatocytes.

The metabolic effects of pent-4-enoate were studied in isolated rat hepatocytes; 1 mM-pent-4-enoate did not significantly inhibit gluconeogenesis from lactate, alanine and glycerol, but significantly decreased glucose synthesis from pyruvate. The addition of 1 mM-NH4Cl led to a drastic inhibition of glucose synthesis from all these substrates. In hepatocytes incubated with 10 mM-alanine and 1 mM-oleate, pent-4-enoate at 0.05-1 mM slightly inhibited glucose synthesis and ketogenesis. The addition of ammonia resulted in a dramatic potentiation of the metabolic effects of pent-4-enoate. Half-maximum effect of ammonia was observed at 0.2 mM concentration. Concomitant cellular concentrations of ATP and acetyl-CoA were also decreased by the addition of ammonia, as were lactate/pyruvate ratio and beta-hydroxybutyrate/acetoacetate ratio. These data suggest that ammonia seriously interferes with the cellular metabolism of pent-4-enoate and leads to a dramatic potentiation of its effects.