Production of fructooligosaccharides by crude enzyme preparations of β-fructofuranosidase from <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis>

Fructooligosaccharides (FOS) were produced from sucrose by using crude enzyme preparations of β-fructofuranosidases (FFases) obtained from sucrose-cultured cells of Aureobasidium pullulans DSM 2404. When the preparation mainly consisted of FFase I, that has high transfructosylating activity, the FOS yield was 62%. When the reaction was carried out with additional commercial glucose isomerase (GI) at an activity ratio of FFase and GI of 1:2, the maximum FOS yield reached 69%. This value was higher than those obtained previously using other Aureobasidium spp. (53–59%).     

Rapid evaluation of 1-kestose producing β-fructofuranosidases from Aspergillus species and enhancement of 1-kestose production using a PgsA surface-display system

1-Kestose is a key prebiotic fructooligosaccharide (FOS) sugar. Some β-fructofuranosidases (FFases) have high transfructosylation activity, which is useful for manufacturing FOS. Therefore, obtaining FFases that produce 1-kestose efficiently is important. Here, we established a rapid FFase evaluation method using Escherichia coli that display different FFases fused to a PgsA anchor protein from Bacillus subtilis. E. coli cell suspensions expressing the PgsA-FFase fusion efficiently produce FOS from sucrose. Using this screening technique, we found that the E. coli transformant expressing Aspergillus kawachii FFase (AkFFase) produced a larger amount of 1-kestose than those expressing FFases from A. oryzae and A. terreus. Saturation mutagenesis of AkFFase was performed, and the mutant G85W was obtained. The E. coli transformant expressing AkFFase G85W markedly increased production of 1-kestose. Our results indicate that the surface display technique using PgsA is useful for screening of FFases, and AkFFase G85W is likely to be suitable for 1-kestose production.

Abbreviations: AkFFase: Aspergillus kawachii FFase; AoFFase: Aspergillus oryzae FFase; AtFFase: Aspergillus terreus FFase; FFase: β-fructofuranosidase; FOS: fructooligosaccharide; fructosylnystose: 1^F-β-fructofuranosylnystose     

Mannitol and Fructose Catabolic Pathways of Pseudomonas aeruginosa Carbohydrate-Negative Mutants and Pleiotropic Effects of Certain Enzyme Deficiencies

Mutant strains of Pseudomonas aeruginosa PAO were isolated on the basis of their inability to utilize mannitol as sole carbon source for growth. Four linkage groups (I through IV) among these mutant strains were resolved by two-factor crosses using the general transducing phage F116, and the strains appeared to contain point mutations as evidenced by ability to give rise to spontaneous revertants with wild phenotype on mannitol minimal agar. Group I strains were affected only in ability to grow on mannitol; all were deficient in inducible mannitol dehydrogenase activity, and all but one were deficient in inducible mannitol transport activity. Fructokinase was induced in group I strains and in wild-type bacteria during growth in the presence of mannitol but not fructose, indicating the presence of a pathway specific for endogenously generated fructose. Cells grown on fructose contained phosphoenolpyruvate:fructose-1-phosphotransferase activity, and mannitol-grown cells contained a lower level of this activity. Group II mutants were deficient in constitutive phosphoglucoisomerase, failed to grow on mannitol, grew very slowly on glycerol and fructose, but grew normally on glucose and gluconate. Group III strains were deficient in both nicotinamide adenine dinucleotide- and nicotinamide adenine dinucleotide phosphate-linked glucose-6-phosphate dehydrogenase activities that reside in a single enzyme species. 6-Phosphogluconate appeared to be the inductive effector for this enzyme, which was not required for aerobic growth on glucose or gluconate. A single mannitol-negative mutant in group IV also failed to grow on glycerol and glucose, but no biochemical lesion was identified.     

The phosphotransferase system of Corynebacterium glutamicum: features of sugar transport and carbon regulation

In this review, we describe the phosphotransferase system (PTS) of Corynebacterium glutamicum and discuss genes for putative global carbon regulation associated with the PTS. C. glutamicum ATCC 13032 has PTS genes encoding the general phosphotransferases enzyme I, HPr and four enzyme II permeases, specific for glucose, fructose, sucrose and one yet unknown substrate. C. gluamicum has a peculiar sugar transport system involving fructose efflux after hydrolyzing sucrose transported via sucrose EII. Also, in addition to their primary PTS, fructose and glucose are each transported by a second transporter, glucose EII and a non-PTS permease, respectively. Interestingly, C. glutamicum does not show any preference for glucose, and thus co-metabolizes glucose with other sugars or organic acids. Studies on PTS-mediated sugar uptake and its related regulation in C. glutamicum are important because the production yield of lysine and cell growth are dependent on the PTS sugars used as substrates for fermentation. In many bacteria, the PTS is also involved in several regulatory processes. However, the detailed molecular mechanism of global carbon regulation associated with the PTS in this organism has not yet been revealed.     

Production of ethanol by filamentous and yeast-like forms of <Emphasis Type="Italic">Mucor indicus</Emphasis> from fructose,glucose, sucrose,and molasses

The fungus Mucor indicus is found in this study able to consume glucose and fructose, but not sucrose in fermentation of sugarcane and sugar beet molasses. This might be an advantage in industries which want to selectively remove glucose and fructose for crystallisation of sucrose present in the molasses. On the other hand, the fungus assimilated sucrose after hydrolysis by the enzyme invertase. The fungus efficiently grew on glucose and fructose and produced ethanol in synthetic media or from molasses. The cultivations were carried out aerobically and anaerobically, and manipulated toward filamentous or yeast-like morphology. Ethanol was the major metabolite in all the experiments. The ethanol yield in anaerobic cultivations was between 0.35 and 0.48 g/g sugars consumed, depending on the carbon source and the growth morphology, while a yield of as low as 0.16 g/g was obtained during aerobic cultivation. The yeast-like form of the fungus showed faster ethanol production with an average productivity of 0.90 g/l h from glucose, fructose and inverted sucrose, than the filamentous form with an average productivity of 0.33 g/l h. The biomass of the fungus was also analyzed with respect to alkali-insoluble material (AIM), chitin, and chitosan. The biomass of the fungus contained per g maximum 0.217 g AIM and 0.042 g chitosan in yeast-like cultivation under aerobic conditions.     

Fermentations of fructo-oligosaccharides and their components by Bifidobacterium infantis ATCC 15697 on batch culture in semi-synthetic medium

AIMS: To compare the physiological behaviour of Bifidobacterium infantis ATCC 15697 growing on synthetic oligofructose or its components. METHODS AND RESULTS: The studies were carried out in regulated or non-regulated batch cultures on semi-synthetic media. Differences between the carbohydrate utilization patterns with glucose, fructose, sucrose and fructo-oligosaccharides (FOS) were determined. Glucose was the preferred substrate for growth and biomass production, whereas fructose was the best for lactate and acetate production. With sucrose, biomass production reached the level obtained with glucose, whereas with FOS, more metabolites were produced, as with fructose. In a mixture of FOS, the shorter saccharides were used first and fructose was released in the medium. Fructofuranosidase, an enzyme necessary to hydrolyse FOS, was inducible by fructose. CONCLUSION: Glucose contained in FOS and sucrose might sustain growth and cell production, while fructose might enable the production of major metabolites. SIGNIFICANCE AND IMPACT OF THE STUDY: A better understanding of the bifidogenic nature of oligofructose has been gained.     

Initial reactions in the fungal degradation of guaiacylglycerol-β-coniferyl ether,a lignin substructure model

Fusarium solani M-13-1 was shake-cultured in a medium containing guaiacylglycerol-β-coniferyl ether (I), a model compound representing the arylglycerol-β-aryl ether linkage in lignin, as sole carbon source. From the culture filtrate guaiacylglycerol-β-coniferyl aldehyde ether (II) and guaiacylglycerol-β-ferulic acid ether (III) were isolated as metabolic products. Incubation with (III) resulted in formation of guaiacylglycerol-β-vanillin ether (IV), which was further metabolized to guaiacyglycerol-β-vanillic acid ether (V). The results indicate that the cinnamyl alcohol group of (I) is initially oxidized to an aldehyde group, which is further oxidized to a carboxyl group, yielding (II) and (III). Compound (III) is converted to (IV) by the release of a C₂ fragment, and the aldehyde group of (IV) is further oxidized to a carboxyl group, giving (V). In the pathway from (I) to (V), neither oxidation of the benzylic secondary alcohol to ketone nor cleavage of the arylglycerol-β-aryl ether linkage was observed. The fungus was found to attack both erythro and threo form without distinction.     

β-Fructofuranosidase production by repeated batch fermentation with immobilized <Emphasis Type="Italic">Aspergillus japonicus</Emphasis>

The fungus Aspergillus japonicus ATCC 20236 was immobilized in vegetal fiber and used in repeated batch fermentations of sucrose (200 g/l) for the production of β-fructofuranosidases (FFase). The assays were performed during eight consecutive cycles that were completed in a total period of 216 h. After each 24-h cycle of fermentation (except for the first cycle, which lasted 48 h), the fermented broth was replaced by fresh medium, and the FFase activity was determined in the replaced medium. The average value of FFase activity was a constant 40.6 U/ml at the end of the initial seven cycles, but had decreased by 22% at the end of the eighth cycle. Concurrent with these high and constant FFase values, the hydrolyzing activity of this enzyme increased during the cycles, while the transfructosylating activity decreased. As a consequence, the maximum production of fructooligosaccharides of 134.60 g/l observed in the initial 30 h of fermentation (first cycle) had gradually decreased by the end of the subsequent cycles, reaching approximately 23% of this value during cycles 4–8. Based on these results, we conclude that the present immobilization system has a great potential for application in a semi-continuous process for the production of FFase, but further studies are necessary to maintain the FFase transfructosylation activity at high levels during the overall process.     

Succinate production from different carbon sources under anaerobic conditions by metabolic engineered Escherichia coli strains

Succinic acid has drawn much interest as a precursor of many industrially important chemicals. Using a variety of feedstocks for the bio-production of succinic acid would be economically beneficial to future industrial processes. Escherichia coli SBS550MG is able to grow on both glucose and fructose, but not on sucrose. Therefore, we derived a SBS550MG strain bearing both the pHL413 plasmid, which contains Lactococcus lactis pycA gene, and the pUR400 plasmid, which contains the scrK, Y, A, B, and R genes for sucrose uptake and catalyzation. Succinic acid production by this modified strain and the SBS550pHL413 strain was tested on fructose, sucrose, a mixture of glucose and fructose, a mixture of glucose, fructose and sucrose, and sucrose hydrolysis solution. The modified strain can produce succinic acid efficiently from all combinations of different carbon sources tested with minimal byproduct formation and with high molar succinate yields close to that of the maximum theoretic values. The molar succinic acid yield from fructose was the highest among the carbon sources tested. Using the mixture of glucose and fructose as the carbon source resulted in slightly lower yields and much higher productivity than using fructose alone. Fermenting sucrose mixed with fructose and glucose gave a 1.76-fold higher productivity than that when sucrose was used as the sole carbon source. Using sucrose pretreated with sulfuric acid as carbon source resulted in a similar succinic acid yield and productivity as that when using the mixture of sucrose, fructose, and glucose. The results of the effect of agitation rate in aerobic phase on succinate production showed that supplying large amount of oxygen in aerobic phase resulted in higher productions of formate and acetate, and therefore lower succinate yield. This study suggests that fructose, sucrose, mixture of glucose and fructose, mixture of glucose, fructose and sucrose, or sucrose hydrolysis solution could be used for the economical and efficient production of succinic acid by our metabolic engineered E. coli strain.     

Lysine and glutamate production by Corynebacterium glutamicum on glucose, fructose and sucrose: roles of malic enzyme and fructose-1,6-bisphosphatase

In the biotechnological production of L-lysine and L-glutamate by Corynebacterium glutamicum media based on glucose, fructose or sucrose are typically used. Glutamate production by C. glutamicum was very similar on glucose, fructose, glucose plus fructose and sucrose. In contrast, lysine production of genetically defined C. glutamicum strains was significantly higher on glucose than on the other carbon sources. To test whether malic enzyme or fructose-1,6-bisphosphatase might limit growth and lysine on fructose, glucose plus fructose or sucrose, strains overexpressing either malE which encodes the NADPH-dependent malic enzyme or the fructose-1,6-bisphosphatase gene fbp were generated. Overexpression of malE did not improve lysine production on any of the tested carbon sources. Upon overexpression of fbp lysine yields on glucose and/or fructose were unchanged, but the lysine yield on sucrose increased twofold. Thus, fructose-1,6-bisphosphatase was identified as a limiting factor for lysine production by C. glutamicum with sucrose as the carbon source.     

The Effect of Ethanol on the Growth of Dipodascus aggregatus

Ethanol (68.2 mM) did not appreciably affect the growth of Dipodascus aggregatus with glucose (55.5 mM] as carbon source. Growth with fructose was inhibited whereas growth with galactose was stimulated by ethanol in this concentration. The fungus could grow with ethanol as the sole carbon source. D. aggregatus did not grown with maltose as the sole carbon source. Growth with maltose + ethanol started much earlier than growth with ethanol alone. The maltose concentration of the medium did not measurably decrease during growth with maltose-n ethanol. D. aggregatus did not grow with sucrose as the sole carbon source     

Biochemical characterization and kinetic/thermodynamic study of Aspergillus tamarii URM4634 β-fructofuranosidase with transfructosylating activity

This study reports on the biochemical characterization as well as the kinetic and thermodynamic study of Aspergillus tamarii URM4634 β-fructofuranosidase (FFase) with transfructosylating activity. Conditions for FFase activity were optimized by means of a central composite rotational design using pH and temperature as the independent variables, while residual activity tests carried out in the temperature range of 45–65°C enabled us to investigate FFase thermostability and estimate the kinetic and thermodynamic parameters of enzyme denaturation. Optimal conditions for sucrose hydrolysis and fructosyl transfer catalyzed by crude FFase were 50°C, and pH 6.0 and 7.4, respectively. The thermodynamic properties of irreversible enzyme inactivation were found to be activation energy of 293.1 kJ mol⁻¹, and activation enthalpy, entropy, and Gibbs free energy in the ranges 290.3–290.4 kJ mol⁻¹, 568.7–571.0 J mol⁻¹ K⁻¹, and 97.9–108.8 kJ mol⁻¹, respectively. The results obtained in this study point out satisfactory enzyme activity and thermostability at temperatures commonly used for industrial fructo-oligosaccharide (FOS) synthesis; therefore, this novel FFase appears to be a promising biocatalyst with great potential for long-term FOS synthesis and invert sugar production. To the best of our knowledge, this is the first report on kinetic and thermodynamic parameters of an A. tamarii FFase.     

Candida glycerinogenes不同碳源代谢的初步研究

研究了不同碳源对Candidaglycerinogenes的菌体生长、发酵液pH值及代谢产物的影响,结果发现以葡萄糖、果糖等单糖为碳源时茵体生长较快,最终生物量比以蔗糖、麦芽糖等二糖为碳源时高20％～30％;导致发酵前12h发酵液pH值明显下降的主要因素是乳酸;与葡萄糖为碳源转化为甘油相比,果糖为碳源时更易累积乙醇;以蔗糖、麦芽糖为碳源时,用于转化生成甘油的碳源明显降低,碳源主要用于茵体自身生物合成及HMP途径,以蔗糖为碳源时,用于乳酸、丙酸及柠檬酸生物合成的碳源较麦芽糖明显提高,TCA途径代谢较为活跃。     

Electrophysiological response of honey bee labial palp contact chemoreceptors to sugars and electrolytes

ABSTRACT. The sensilla chaetica on segments II, III and IV of honey-bee labial palps were investigated electrophysiologically. The responses (spikes/s) correlated with the log of the concentrations of sucrose, glucose, fructose, NaCl, KCl and LiCl, but not with CaCl₂ or MgCl₂, which gave inconsistent responses. The firing rates were higher and thresholds lower to the sugars than to the electrolytes. The sensitivity of the segments fell in the order: III > II > IV for most of the stimulants, which elicited responses in the order: sucrose > glucose = fructose' KCl > LiCl > NaCl. The sensilla adapted logarithmically with time. No synergism of response was noted when mixed-sugar solutions were applied, but inhibition of response was seen when glucose–sucrose, fructose–sucrose, and glucose–fructose–sucrose mixtures were applied. None of the sensilla tested responded to water.     

Kinetics of Bifidobacterium longum ATCC 15707 fermentations: effect of the dilution rate and carbon source

The effect of the dilution rate on biomass and product synthesis in fermentations of glucose, fructose and a commercial mixture of fructooligosaccharides (FOS) by Bifidobacterium longum ATCC 15707 was studied. Kinetic parameters (maximum specific growth rate, Monod constant, maintenance, and yield coefficients) in the mathematical model of the fermentation were estimated from experimental data. In the FOS mixture fermentations, approximately 12% of the total reducing sugars (mainly fructose) in the feed were not metabolized by the bacterium. In fermentations of fructose and the FOS mixture, biomass concentration increased as the dilution rate increased and, once maximum values were reached [3.90 (D=0.20 h^–1) and 2.54 g l^–1 (D=0.15 h^–1), respectively], decreased rapidly as the culture was washed out. Formic acid was detected at low dilution rates in glucose and fructose fermentations. The main products in fermentations of the three carbon sources were lactic and acetic acids. Average values of the molar ratio between acetic and lactic acids of 1.18, 1.21 and 0.83 mol mol^–1 were obtained in glucose, fructose and FOS mixture fermentations, respectively. In batch fermentations carried out without pH control this molar ratio was lower than 1.5 only when fructose was used as the carbon source.     

Mutants of Rhizobium meliloti defective in succinate metabolism.

We characterized mutants of Rhizobium meliloti SU47 that were unable to grow on succinate as the carbon source. The mutants fell into five groups based on complementation of the succinate mutations by individual recombinant plasmids isolated from a R. meliloti clone bank. Enzyme analysis showed that mutants in the following groups lacked the indicated common enzyme activities: group II, enolase (Eno); group III, phosphoenolpyruvate carboxykinase (Pck); group IV, glyceraldehyde-3-phosphate dehydrogenase (Gap), and 3-phosphoglycerate kinase (Pgk). Mutants in groups I and V lacked C4-dicarboxylate transport (Dct-) activity. Wild-type cells grown on succinate as the carbon source had high Pck activity, whereas no Pck activity was detected in cells that were grown on glucose as the carbon source. It was found that in free-living cells, Pck is required for the synthesis of phosphoenolpyruvate during gluconeogenesis. In addition, the enzymes of the lower half of the Embden-Meyerhoff-Parnas pathway were absolutely required for gluconeogenesis. Eno, Gap, Pck, and one of the Dct loci (ntrA) mapped to different regions of the chromosome; the other Dct locus was tightly linked to a previously mapped thi locus, which was located on the megaplasmid pRmeSU47b.     

Substrate utilization by recombinant Yarrowia lipolytica growing on sucrose

We report the study of the dynamics of substrate utilization by the genetic modified strain Yarrowia lipolytica H222-S4(p67ICL1) T5. In contrast to its wild-type equivalent, this recombinant strain is able to excrete the sucrose cleaving enzyme invertase. Both the sucrose degradation rate and the glucose and fructose consumption rate have been investigated. In all experiments, satisfied amounts of invertase were produced so that all sucrose was cleaved into its monomers. While glucose and fructose as sole carbon sources were consumed with the same uptake rate, a clear preference for glucose uptake was detected in cultivations with sucrose as sole carbon source or mixed substrates when compared with fructose. Nevertheless, no real diauxie could be observed because of partly simultaneous consumption of both monosaccharides. Fructose being present in the cultivation medium at the beginning of the fermentation led to the retardation of glucose uptake. This effect was observed for various fructose starting concentrations in the range of 5–85 g/l.     

Carbon and nitrogen requirements of Fusarium oxysporum causing cotton wilt

Summary The present work was carried out to study the nutritional requirements of the cotton wilt-inducing fungus, i.e.Fusarium oxysporum on a synthetic liquid medium with regard to the carbon and nitrogen sources at varying concentrations in terms of the average mycelial dry weights.The optimum carbon requirements of the fungus ranged from 7000–8000 p.p.m. irrespective of the carbon source used in experiment. Carbon utilization was best on sucrose followed by maltose, starch, glucose, fructose and cellulose successively.The optimum nitrogen requirements of the fungus were 300 p.p.m. of nitrogen in the medium; nitrogen utilization was best on using nitrate-nitrogen followed by glycine, glutamic acid, ammonium nitrate, asparagine and ammonium sulphate.Maximum growth of the fungus took place on media containing a C/N ratio ranging between 22.8 and 25.7.Colour formation is correlated with varying either source or concentration of nitrogen and not carbon.     

Carbon Source Control of Cellobiohydrolase I and II Formation by Trichoderma reesei

Regulation of the formation and secretion of two cellulase components from Trichoderma reesei QM 9414, cellobiohydrolases I and II (CBH I and CBH II, respectively), by the carbon source was investigated. With monoclonal antibodies against CBH I and CBH II it was found that during cultivation on carbon sources which enable fast growth (glucose, glycerol, and fructose), no formation of CBH I occurred, whereas low levels of CBH II were formed. Lactose and cellulose, which allow comparably slower growth, promoted the formation of both CBH I and CBH II. However, noncarbohydrate carbon sources as citrate or acetate, which also enable only slow growth, did not promote the formation of CBH I or CBH II. The addition of glucose or glycerol to lactose- or cellulose-pregrown mycelia, on the other hand, only partially reduced the formation of CBH I. This reduction was also achieved by several other metabolizable and nonmetabolizable carbon compounds, e.g., fructose, galactose, β-methylglucoside, 2-deoxyglucose, and rhamnose, as well as by transfer to no carbon source at all. This result indicates that the control of CBH I synthesis by the carbon source is due to induction and not to repression. The use of cycloheximide and 5-fluorouracil as inhibitors at and before translation, respectively, revealed a half-life for CBH I mRNA of at least several hours, which may, at least in part, account for the prolonged synthesis of some CBH I under these conditions. Northern (RNA) hybridization with full copies of cbh1 and cbh2 genes indicated that the control of CBH I and CBH II biosyntheses by the carbon source operates mainly at the pretranslational level. We conclude that the low rate of cellulase synthesis on glucose and some other carbon sources is due to the lack of an inducer and not to carbon source repression.