Lactose/whey utilization and ethanol production by transformed Saccharomyces cerevisiae cells

Strains of Saccharomyces cerevisiae transformed with a multicopy expression vector bearing both the Escherichia coli beta-galactosidase gene under the control of the upstream activating sequence of the GAL1-10 genes and the GAL4 activator gene release part of beta-galactosidase in the growth medium. This release is due to cell lysis of the older mother cells; the enzyme maintains its activity in buffered growth media. Fermentation studies with transformed yeast strains showed that the release of beta-galactosidase allowed an efficient growth on buffered media containing lactose as carbon source as well as on whey-based media. The transformed strains utilized up to 95% of the lactose and a high growth yield was obtained in rich media. High productions of ethanol were also observed in stationary phase after growth in lactose minimal media.     

Sublethal stress in Escherichia coli: a function of salinity.

Sublethal stress in Escherichia coli was detected in various test media after exposure (in vitro) to seawater of various salinites. Stress was measured with an electrochemical detection technique and a beta-galactosidase assay. Test media included EC medium, medium A-1, and tryptic soy broth modified to contain lactose for beta-galactosidase assay experiments. Stress was defined as the difference between a predicted electrochemical response time calculated for unstarved cells from a standard curve and the observed electrochemical response time for cells starved in seawater. The higher the salinity, the greater the stress for all test media examined. Stress was most pronounced in EC and was attributed primarily to initial die-off of starved cells exposed to the test medium at the elevated temperature of 44.5 degrees C. Lag time and growth rates in test media were not significantly affected by salinity. beta-Galactosidase specific activity, assayed in starved cells after transfer to an induction medium at 44.5 degrees C for 150 min, was inversely related to the salinity of the starved cell suspension. The consequences of these observations with respect to coliform enumeration methods are discussed.     

Sublethal stress in Escherichia coli: a function of salinity.

Sublethal stress in Escherichia coli was detected in various test media after exposure (in vitro) to seawater of various salinites. Stress was measured with an electrochemical detection technique and a beta-galactosidase assay. Test media included EC medium, medium A-1, and tryptic soy broth modified to contain lactose for beta-galactosidase assay experiments. Stress was defined as the difference between a predicted electrochemical response time calculated for unstarved cells from a standard curve and the observed electrochemical response time for cells starved in seawater. The higher the salinity, the greater the stress for all test media examined. Stress was most pronounced in EC and was attributed primarily to initial die-off of starved cells exposed to the test medium at the elevated temperature of 44.5 degrees C. Lag time and growth rates in test media were not significantly affected by salinity. beta-Galactosidase specific activity, assayed in starved cells after transfer to an induction medium at 44.5 degrees C for 150 min, was inversely related to the salinity of the starved cell suspension. The consequences of these observations with respect to coliform enumeration methods are discussed.     

Physiological studies of beta-galactosidase induction in Kluyveromyces lactis

We examined the kinetics of beta-galactosidase (EC 3.2.1.23) induction in the yeast Kluyveromyces lactis. Enzyme activity began to increase 10 to 15 min, about 1/10 of a cell generation, after the addition of inducer and continued to increase linearly for from 7 to 9 cell generations before reaching a maximum, some 125- to 150-fold above the basal level of uninduced cells. Thereafter, as long as logarithmic growth was maintained, enzyme levels remained high, but enzyme levels dropped to a value only 5- to 10-fold above the basal level if cells entered stationary phase. Enzyme induction required the constant presence of inducer, since removal of inducer caused a reduction in enzyme level. Three nongratuitous inducers of beta-galactosidase activity, lactose, galactose, and lactobionic acid, were identified. Several inducers of the lac operon of Escherichia coli, including methyl-, isopropyl- and phenyl-1-thio-beta-d-galactoside, and thioallolactose did not induce beta-galactosidase in K. lactis even though they entered the cell. The maximum rate of enzyme induction was only achieved with lactose concentrations of greater than 1 to 2 mM. The initial differential rate of beta-galactosidase appearance after induction was reduced in medium containing glucose, indicating transient carbon catabolite repression. However, glucose did not exclude lactose from K. lactis, it did not cause permanent carbon catabolite repression of beta-galactosidase synthesis, and it did not prevent lactose utilization. These three results are in direct contrast to those observed for lactose utilization in E. coli. Furthermore, these results, along with our observation that K. lactis grew slightly faster on lactose than on glucose, indicate that this organism has evolved an efficient system for utilizing lactose.     

Escherichia coli growth on lactose requires cycling of beta-galactosidase products into the medium

Growth on lactose was found to be restricted in an Escherichia coli strain deficient in its ability to transport glucose and galactose. If the latter sugars were removed from the medium as they were being produced, a wild-type strain grew only poorly, while the transport-deficient strain did not grow at all. These results suggested that all of the products of beta-galactosidase action on lactose are released into the medium before being metabolized. This contention was strongly supported by the finding that the appearance of products in the medium was equal to lactose disappearance at three limiting lactose concentrations and by an experiment which showed that essentially all of the label from added lactose ( [1-14C]glucose) was found in the medium as glucose when chased with unlabelled lactose.     

Immunocytological investigation of protein synthesis in Escherichia coli.

Ferritin-conjugated specific antibodies have been used to localize beta-galactosidase and both the monomer and active dimer of alkaline phosphatase in frozen thin sections of cells of Escherichia coli O8 strain F515. The even distribution of the ferritin marker throughout cells that had been induced for beta-galactosidase synthesis, frozen, sectioned, and exposed to ferritin-anti-beta-galactosidase conjugate showed that this enzyme was present throughout the cytoplasm of these cells. Frozen thin sections of cells that had been derepressed for the synthesis of alkaline phosphatase were exposed to both ferritin-anti-alkaline phosphatase monomer and ferritin-anti-alkaline phosphatase dimer conjugates, and the ferritin markers showed a peripheral distribution of both the monomer and the dimer of this enzyme. This indicates that alkaline phosphatase is present only in the peripheral regions of the cell and argues against the existence of a cytoplasmic pool of inactive monomers of this enzyme. This peripheral location of both the monomers and dimers of alkaline phosphatase supports the developing concensus that this enzyme is, like other wall-associated enzymes, synthesized in association with the cytoplasmic membrane and vectorially transported to the periplasmic area, where it assumes its tertiary and quaternary structure and acquires its enzymatic activity.     

Regulation of the formation of protease byBacillus megaterium

The formation of protease takes place in washed cells ofBacillus megaterium incubated in a nitrogen-free medium. The rate of enzyme synthesis is decreased much less than that of cell proteins as compared with growing cells. The synthesis of protease in a nitrogen-free medium requires the presence of glucose. The omission of glucose results in stopping of the enzyme formation and substantial decrease of the rate of protein synthesis. Protease is not synthesized when the washed cells are incubated in a phosphate, free medium. The incubation of the cells in a nitrogen-free medium results in a decrease of the concentration of amino acids in the pool. In a phosphate-free medium the content of free amino acids increases temporarily and decreases again later. When the culture grown in the medium containing threonine or threonine and isoleucine in addition to NH₄ ions is transferred into the medium without amino acids, no protease formation is found during derepression of enzymes synthesizing both amino acids. The cells grown in a medium containing casamino acids begin to form the enzyme after a short lag period when transferred into the medium containing NH₄ as a sole nitrogen source or into a nitrogen-free medium.     

Influence of amino acids on low-density Escherichia coli responses to nutrient downshifts

A vast bibliography on nutrient effects on high-density cultures exists, while it has been overlooked that low densities of starving cells are often the rule in natural environments. By means of a novel sensitive beta-galactosidase assay, we examined Escherichia coli transitions to minimal media when the cell concentration was 100 to 10,000 cells per ml. As in high-density cultures, the enzyme activity depended on amino acid availability and was subject to catabolite repression and stringent control. In all conditions tested, despite the presence of other nutrient sources, the relationship between beta-galactosidase activity and the l-amino acid pool was hyperbolic. The affinity constant when the amino acid pool was the only nutrient source averaged 14 muM after 90 min and increased up to 222 muM after 4.5 h. While investigating the transition from lag phase to exponential phase, we observed that the cells did not enter into starvation mode in the presence of amino acids, even when the nutrient amount was insufficient to support full survival. Based on these premises, the switch from starvation to hunger was investigated in relation to the amino acid pools. A critical range of concentrations at which E. coli linearly synthesized beta-galactosidase despite, at the same time, suffering a large decrease in cell viability was then recognized. Since both beta-galactosidase production and the dilution rate were reduced by more than half in the absence of leucine, we examined the contribution of leucine to cell recovery capabilities.     

Differentiation between binding and transport of dansylgalactosides in Escherichia coli.

The results presented in this paper confirm and extend previous observations which indicate that fluorescent dansylgalactsodes bind to the beta-galactoside carrier protein but do not penetrate the cytoplasmic membrane. The conclusion is supported by the following observations. (a) Although 2'-(N-dansyl)aminoethyl-beta-D-thiogalactopyranoside and 2'-(N-dansyl)aminoethyl-beta-D-galactopyranoside are competitive inhibitors of lactose transport in intact cells of Escherichia coli and induce the in vitro synthesis of beta-galactosidase, they do not induce beta-galactosidase in vivo. (b) p-Chloromercuribenzenesulfonate does not cause efflux of lactose from the intravesicular pool, but causes rapid reversal of D-lactate-induced dansylgalactoside fluorescence. (c) Dansylgalactosides inhibit dilution-induced, carrier-mediated lactose efflux.     

Alcoholysis and reverse hydrolysis reactions in organic one-phase system with a hyperthermophilic beta-glycosidase

Alcoholysis and reverse hydrolysis reactions were performed enzymatically in one-phase water-saturated 1-heptanol systems. Lactose or glucose was used as substrate to produce heptyl-beta-galactoside and/or heptyl-beta-glucoside, respectively. When alcoholysis of lactose was performed at 37 degrees C with beta-galactosidase from Escherichia coli, the initial rate was 14 nmol/mL min, and the limiting factors were the poor solubility of the substrate in 1-heptanol and low thermal stability of the enzyme. When a hyperthermophilic beta-glycosidase was used at 90 degrees C, the rate was 3.14-fold higher; in this case a higher concentration of soluble lactose in the water-saturated heptanol was available to the enzyme due to the higher temperature. The hyperthermophilic beta-glycosidase was also able to use glucose and galactose as substrates to achieve the reverse hydrolysis reaction. As a consequence, when lactose was used as substrate, heptyl-beta-galactoside was formed by alcoholysis, while the released glucose moiety was used in a secondary reverse hydrolysis reaction to produce heptyl-beta-glucoside. Both reactions followed Michaelis-Menten kinetics behavior. Neither lactose nor heptyl glycosides were hydrolyzed by this enzyme in water-saturated heptanol. However, the conversion was limited by a strong product inhibition and the formation of oligosaccharides, especially at high substrate concentrations, reducing the final glycoside yield.     

Cloning of chemically synthesized lactose operators.

Recombinant DNA molecules, constructed from the ColE1-Mk5 hybrid plasmid PMB9 and a chemically synthesized wild-type lactose operator segment, have been used to transform Escherichia coli. Up to 10% of the transformants (selected for the tetracycline-resistance property of PMB9) are partially constitutive for the lactose operon enzyme beta-galactosidase. In vitro studies demonstrate that these partially constitutive transformants contain plasmid DNA molecules which carry one or more lactose operators, and which will bind purified lactose repressor. Preliminary results with some modified operator sequences are also presented.     

大肠杆菌O121侵袭性菌株的发现与研究

本文报告从一急性腹泻患儿的脓血便中分离的具有侵袭性的大肠杆菌。该菌株发酵葡萄糖,产酸产气,不发酵乳糖,β-半乳糖苷酶试验阳性,在醋酸钠培养基上生长,赖氨酸脱羧酶阴性,无动力,能引起豚鼠角膜结膜炎,侵入上皮细胞,具有140Md的质粒带,经血清学试验证实其血清型为O121:H-,是国内外首次报道的新的侵袭性大肠杆菌血清型。     

The production of protease by dense suspensions ofBacillus megaterium KM Sp−

The synthesis and secretion of extracellular protease was demonstrated during the incubation of dense susponsions of the asporogenicBacillus megaterium KM. The overall production of the enzyme by cells incubated with glucose in a nitrogen-free medium was found to be only slightly lower than that in the presence of an inorganic nitrogen source. The capacity to form protease decreased exponentially with increasing density of the bacterial suspension. The synthesis of the enzyme was interrupted after the exhaustion of glucose. A repeated exchange of the medium made it possible to reach relatively high and continuous production of protease for several hours. The total amount of extracellular proteins synthesized during incubation of the dense suspension in media with or without a nitrogen source was less than 2% of a total of newly formed proteins. The amount of these extracellular proteins was slightly lower in the absence of Ca²⁺ being considerably decreased when the dense suspension was incubated with chloramphenicol.     

The hydrophobic domain of cytochrome b5 is capable of anchoring beta-galactosidase in Escherichia coli membranes.

Cytochrome b5 is inserted posttranslationally into membranes in vivo and spontaneously into liposomes in vitro by a short carboxyl-terminal hydrophobic membrane-anchoring sequence. DNA corresponding to this hydrophobic sequence has been synthesized, and two gene fusions with the Escherichia coli enzyme beta-galactosidase have been constructed by locating the hydrophobic domain in one case at the EcoRI site near the C terminus and in the other at the normal C terminus of the enzyme. The latter fusion protein was enzymatically active, having approximately 50% of the specific activity of beta-galactosidase, and cells expressing this protein grew normally with lactose as the sole carbon source. Both fusion proteins were localized to the E. coli inner membrane, converting beta-galactosidase from a cytoplasmic enzyme to a membrane-associated enzyme. The hydrophobic domain of cytochrome b5 therefore contains the information required to target polypeptides containing this domain to the membrane. Use of the cytochrome b5 hydrophobic peptide, either alone or in conjunction with other localizing sequences such as signal sequences, provides a general procedure for associating proteins with membranes. Polypeptides bearing this hydrophobic peptide may have considerable use as pharmaceuticals when associated with liposomes or cellular membranes.     

Hydrolysis of lactose by immobilized microorganisms.

Cells of Lactobacillus bulgaricus, Escherichia coli, and Kluyveromyces (Saccharomyces) lactis immobilized in polyacrylamide gel beads retained 27 to 61% of the beta-galactosidase activity of intact cells. Optimum temperature and pH and thermostability of these microbial beta-galactosidases were negligibly affected by the immobilization. Km values of beta-galactosidase in immobilized cells of L. bulgaricus, E. coli, and K. lactis toward lactose were 4.2, 5.4, and 30 mM, respectively. Neither inhibition nor activation of beta-galactosidase in immobilized L. bulgaricus and E. coli appeared in the presence of galactose, but remarkable inhibition by galactose was detected in the case of the enzyme of immobilized K. lactis. Glucose inhibited noncompetitively the activity of three species of immobilized microbial cells. These kinetic properties were almost the same as those of free beta-galactosidase extracted from individual microorganisms. The activity of immobilized K. lactis was fairly stable during repeated runs, but those of E. coli and L. bulgaricus decreased gradually. These immobilized microbial cells, when introduced into skim milk, demonstrated high activity for converting lactose to monosaccharides. The flavor of skim milk was hardly affected by treatment with these immobilized cells, although the degree of sweetness was raised considerably.     

Hydrolysis of lactose by immobilized microorganisms.

Cells of Lactobacillus bulgaricus, Escherichia coli, and Kluyveromyces (Saccharomyces) lactis immobilized in polyacrylamide gel beads retained 27 to 61% of the beta-galactosidase activity of intact cells. Optimum temperature and pH and thermostability of these microbial beta-galactosidases were negligibly affected by the immobilization. Km values of beta-galactosidase in immobilized cells of L. bulgaricus, E. coli, and K. lactis toward lactose were 4.2, 5.4, and 30 mM, respectively. Neither inhibition nor activation of beta-galactosidase in immobilized L. bulgaricus and E. coli appeared in the presence of galactose, but remarkable inhibition by galactose was detected in the case of the enzyme of immobilized K. lactis. Glucose inhibited noncompetitively the activity of three species of immobilized microbial cells. These kinetic properties were almost the same as those of free beta-galactosidase extracted from individual microorganisms. The activity of immobilized K. lactis was fairly stable during repeated runs, but those of E. coli and L. bulgaricus decreased gradually. These immobilized microbial cells, when introduced into skim milk, demonstrated high activity for converting lactose to monosaccharides. The flavor of skim milk was hardly affected by treatment with these immobilized cells, although the degree of sweetness was raised considerably.     

Construction of lactose-utilizing Xanthomonas campestris and production of xanthan gum from whey.

Xanthomonas campestris pv. campestris possesses a low level of beta-galactosidase and therefore is not able to grow and produce significant amounts of xanthan gum in a medium containing lactose as the sole carbon source. In this study, a beta-galactosidase expression plasmid was constructed by ligating an X. campestris phage phi LO promoter with pKM005, a ColE1 replicon containing Escherichia coli lacZY genes and the lpp ribosome-binding site. It was then inserted into an IncP1 broad-host-range plasmid, pLT, and subsequently transferred by conjugation to X. campestris 17, where it was stably maintained. The lacZ gene under the control of the phage promoter was expressed at a high level, enabling the cells to grow in a medium containing lactose. Production of xanthan gum in lactose or diluted whey by the engineered strain was evaluated, and it was found to produce as much xanthan gum in these substrates as the cells did in a medium containing glucose.     

Immobilization of beta-galactosidase for application in organic chemistry using a chelating peptide

The strong interaction of hexa-histidine fusion proteins with metal chelate adsorbents was utilized to immobilize beta-galactosidase with a hexa-histidine peptide at the N-terminus to the Ni(2+)-nitrilotriacetic acid adsorbent. The fusion protein was cloned and expressed in Escherichia coli. The purified soluble fusion protein showed the same specific activity as the purified beta-galactosidase and retained 64 percent of its beta-galactosidase activity when bound to the adsorbent. To demonstrate the potential of the immobilized beta-galactosidase in organic chemistry, allyl-beta-D-galactosidase was synthesized from lactose and allyl alcohol on a gram scale. The same enzyme preparation was reused in three subsequent batches to prepare the model compound with high yield. (c) 1993 John Wiley & Sons, Inc.     

Construction of lactose-utilizing Xanthomonas campestris and production of xanthan gum from whey

Xanthomonas campestris pv. campestris possesses a low level of beta-galactosidase and therefore is not able to grow and produce significant amounts of xanthan gum in a medium containing lactose as the sole carbon source. In this study, a beta-galactosidase expression plasmid was constructed by ligating an X. campestris phage phi LO promoter with pKM005, a ColE1 replicon containing Escherichia coli lacZY genes and the lpp ribosome-binding site. It was then inserted into an IncP1 broad-host-range plasmid, pLT, and subsequently transferred by conjugation to X. campestris 17, where it was stably maintained. The lacZ gene under the control of the phage promoter was expressed at a high level, enabling the cells to grow in a medium containing lactose. Production of xanthan gum in lactose or diluted whey by the engineered strain was evaluated, and it was found to produce as much xanthan gum in these substrates as the cells did in a medium containing glucose.