Factors influencing β-glucosidase production, activity and stability inNectria catalinensis

The influence of different cultivation conditions on β-glucosidase production and of some parameters on the activity and stability of this enzyme were studied inNectria catalinensis. Maximal β-glucosidase production was achieved with ammonium nitrate (0.5 g N/L) as nitrogen source. Tween 80, Tween 20 and Triton X-100 increased β-glucosidase yields, Tween 80 was the most effective for enzyme release and growth at a concentration of 3.4 mmol/L. On the other hand, Tween 20 and Triton X-100 had an inhibitory effect onN. catalinensis growth. A temperature of 23°C and an initial pH of cultures of 6.5 were optimal for biomass and β-glucosidase production. Under optimal cultural conditions (ammonium nitrate, 0.5 g N/L; Tween 80, 3.4 mmol/L; 23°C; initial pH 6.5) the β-glucosidase yield was increased more than five fold respect to the initial state. Optimal temperature for β-glucosidase activity was 45°C, the initial activity dropped 60 % after 6 h of incubation at this temperature. Optimal pH for enzyme activity was 5.3. At this pH the β-glucosidase was completely stable after 3 d of incubation. TheV andK _m values calculated from Lineweaver-Burk and Eadie-Hofstee plots were 0.23 μmol 4-nitrophenol per min per mg of protein and 0.25 mmol 4-nitrophenol β-d-glucopyranoside per L, respectively. The activation energy according to Arrhenius plot was 49.6 KJ/mol.     

Scanning assay of β-galactosidase activity

β-galactosidase, encoded by the lacZ gene in E. coli, can cleave lactose and structurally related compounds to galactose and glucose or structurally related products. Its activity can be measured using an artificial substrate, o-nitrophenyl-β-D-galactopyranoside (ONPG). Miller firstly described the standard quantitative assay of β-galactosidase activity in the cells of bacterial cultures by disrupting the cell membrane with the permeabilization solution instead of preparing cell extracts. Therefore, β-galactosidase became one of the most widely used reporters of gene expression in molecular biology to reflect intracellular gene expression difference. But the Miller assay procedure could not monitor the β-galactosidase reaction in real time and its results were greatly influenced by some operations in the Miller procedure, such as permeabilization time, reaction time and concentration of the cell suspension. A scanning method based on the Miller method to determine the intracellular β-galactosidase activity in E. coli Tuner (DE3) expressing β-galactosidase in real time was developed and the permeabilization time of cells was optimized for that. The comparison of 3 assays of β-galactosidase activity (Miller, colorimetric and scanning) was made. The results proved that scanning method for the determination of enzyme activity with using ONPG as substrate is simple, fast and reproducible.     

Inhibition of thymine synthesis inLactobacillus acidophilus R-26 by deoxyadenosine-5′-monophosphate

Growth ofLactobacillus acidophilus was inhibited in the presence of deoxyadenosine-5-monophosphate (dAMP). The other purine deoxyribonucleotides and -sides were only weak inhibitors, and pyrimidine deoxyribotides and -sides were inactive. dAMP did not act as an inhibitor if thymine, thymidine, or 5-methyldeoxycytidine-5-monophosphate was present in the medium. The inhibition by dAMP was counteracted by increasing concentrations of deoxyuridine, deoxyuridine-5-monophosphate and, to some extent, adenosine-5-monophosphate. The effect of these substances was proportional to their concentration and competitive in character. The results support the assumption that dAMP inhibits the synthesis of thymine. Mutants ofL. acidophilus resistant to inhibition by dAMP were found.     

Inducible synthesis of β-galactosidase inKluyveromyces fragilis

Lactose,d-galactose, andl-arabinose induce the synthesis of β-galactosidase inKluyveromyces fragilis. Lactose is the best inducer with a maximum effect at 1.4 mm. The induced synthesis of the enzyme in glycerol grown stationary phase cells is triggered within 30 min of inducer addition, the full induction being achieved within subsequent 30–40 min.     

Control of derepressed β-galactosidase synthesis inEscherichia coli

1. The synthesis of β-galactosidase in a constitutive mutant ofEscherichia coli (ML 308, i^-z⁺y⁺a⁺) responds to the nutritional environment. Repression can be reversed by cyclic AMP.
2. The greatest degree (%) of repression by metabolisable compounds is obtained when cells utilising glycerol (0%) are given, in addition, pyruvate (67%), serine (57%) which can be converted to pyruvate, or substrates of phosphotransferase systems (20–40%) which liberate pyruvate in their operation. Furthermore, pyruvate represses β-galactosidase synthesis in a phosphoenolpyruvate synthaseless mutant. Pyruvate, however, does not repress in a pyruvate dehydrogenaseless mutant and it follows that pyruvate itself is not the agent of repression.
3. Raffinose, a non-metabolisable galactoside, represses synthesis of β-galactosidase during growth on glycerol. Over a wide range, repression depends on raffinose concentration as does a lowered pool of ATP, rate of oxygen consumption and growth rate. All these parameters are inter-related but, in particular, β-galactosidase synthesis depends on the size of the ATP-pool presumably because this also limits synthesis of cyclic AMP under these conditions.

     

Effect of compressed fluids treatment on β-galactosidase activity and stability

This study aimed at assessing the influence of different pressurized fluids treatment on the enzymatic activity and stability of a lyophilized β-galactosidase. The effects of system pressure, exposure time and depressurization rate, using propane, n-butane, carbon dioxide and liquefied petroleum gas on the enzymatic activity were evaluated. The β-galactosidase activity changed significantly depending on the experimental conditions investigated, allowing the selection of the proper compressed fluid for advantageous application of this biocatalyst in enzymatic reactions. The residual activity ranged from 32.1 to 93.8?% after treatment. The storage stability of the enzyme after high-pressure pre-treatment was also monitored, and results showed that the biocatalyst activity presents strong dependence of the fluid used in the pretreatment. The activity gradually decreases over the time for the enzyme treated with LGP and propane, while the enzyme treated with n-butane maintained 96?% of its initial activity until 120?days. For CO₂, there was a reduction of around 40?% in the initial activity 90?days of storage. The enzyme treated with n-butane also showed a better thermostability in terms of enzymatic half-life.     

Mycelia-associated β-galactosidase activity in microbial pellets of Aspergillus and Penicillium strains

Pellet formation and production of mycelia-associated -galactosidase were investigated in 15 Aspergillus and Penicillium strains. Mycelia-associated enzyme activity was measured in sonicated homogenates. The properties of the mycelia-associated -galactosidase of A. phoenicis QM 329 was investigated. The pH optimum of the mycelia-associated enzyme was 4.0. The optimum temperature under assay conditions was 70°C and the optimum temperature for repeated lactose hydrolysis was 60°C. Repeated batch hydrolysis of lactose was made with pellets from five Aspergillus strains. A. phoenicis QM 329 showed the least enzyme leakage from the pellets during hydrolysis. From repeated lactose hydrolysis experiments it was estimated that 50% of the mycelia-associated -galactosidase activity remained after 1300 h. Correspondence to: F. Tjerneld     

Growth and β-galactosidase synthesis in aerobic chemostat cultures of Kluyveromyces lactis

Growth and β-galactosidase (β-gal) expression were characterized in the yeast Kluyveromyces lactis strain NRRL Y-1118 growing in aerobic chemostat cultures under carbon, nitrogen or phosphate limitation. In lactose or galactose-limited cultures, β-gal accumulated in amounts equivalent to 10–12% of the total cell protein. The induced β-gal expression was repressed when cells were grown under N- or P-limitation. In lactose medium, enzyme levels were 4–8 times lower than those expressed in C-limited cultures. A similar response was observed when galactose was the carbon source. These results suggest that a galactose-dependent signal (in addition to glucose) may have limited induction when cells were grown in carbon-sufficient cultures. Constitutive β-gal expression was highest in lactate-limited and lowest in glucose-limited media and was also repressed in glucose-sufficient cultures. Other K. lactis strains (NRRL Y-1140 and CBS 2360) also showed glucose repression (although with different sensitivity) under non-inducing conditions. We infer that these strains share a common mechanism of glucose repression independent of the induction pathway. The kinetics of β-gal induction observed in C-limited cultures confirms that β-gal induction is a short-term enzyme adaptation process. Applying a lactose pulse to a lactose-limited chemostat culture resulted in ‘substrate-accelerated death’. Immediately after the pulse, growth was arrested and β-gal was progressively inactivated. Yeast metabolism in C-limited cultures was typically oxidative with the substrate being metabolized solely to biomass and CO₂. Cells grown under P- or N-limitation, either with glucose or lactose, exhibited higher rates of sugar consumption than C-limited cells, accumulated intracellular reserve carbohydrates and secreted metabolic products derived from the glycolytic pathway, mainly glycerol and ethanol. Received 16 October 1997/ Accepted in revised form 17 April 1998     

Catabolite repression of β-galactosidase synthesis in Escherichia coli

1. Repression by glucose of β-galactosidase synthesis is spontaneously reversible in all strains of Escherichia coli examined long before the glucose has all been consumed. The extent of recovery and the time necessary for reversal differ among various strains. Other inducible enzymes show similar effects. 2. This transient effect of glucose repression is observed in constitutive (i⁻) and permease-less (y⁻) cells as well as in the corresponding i⁺ and y⁺ strains. 3. Repression is exerted by several rapidly metabolizable substrates (galactose, ribose and ribonucleosides) but not by non-metabolized or poorly metabolized compounds (2-deoxyglucose, 2-deoxyribose, phenyl thio-β-galactoside and 2-deoxyribonucleosides). 4. The transient repression with glucose is observed in inducible cells supplied with a powerful inducer of β-galactosidase synthesis (e.g. isopropyl thio-β-galactoside) but not with a weak inducer (lactose); in the latter instance glucose repression is permanent. Diauxic growth on glucose plus lactose can be abolished by including isopropyl thio-β-galactoside in the medium. 5. In some strains phosphate starvation increases catabolite repression; in others it relieves it. Adenine starvation in an adenine-requiring mutant also relieves catabolite repression by glycerol but not that by glucose. Restoration of phosphate or adenine to cells starved of these nutrients causes a pronounced temporary repression. Alkaline-phosphatase synthesis is not affected by the availability of adenine. 6. During periods of transient repression of induced enzyme synthesis the differential rate of RNA synthesis, measured by labelled uracil incorporation in 2min. pulses, shows a temporary rise. 7. The differential rate of uracil incorporation into RNA falls during exponential growth of batch cultures of E. coli. This is equally true for uracil-requiring and non-requiring strains. The fall in the rate of incorporation has been shown to be due to a real fall in the rate of RNA synthesis. The significance of the changes in the rate of RNA synthesis is discussed. 8. A partial model of catabolite repression is presented with suggestions for determining the chemical identification of the catabolite co-repressor itself.     

Enzymatic synthesis of fucose-containing galacto-oligosaccharides using β-galactosidase and identification of novel disaccharide structures

Fucosylated oligosaccharides have an important role in maintaining a healthy immune system and homeostatic gut microflora. This study employed a commercial β-galactosidase in the production of fucose-containing galacto-oligosaccharides (fGOS) from lactose and fucose. The production was optimized using experiment design and optimal conditions for a batch production in 3-liter scale. The reaction product was analyzed and the produced galactose-fucose disaccharides were purified. The structures of these disaccharides were determined using NMR and it was verified that one major product with the structure Galβ1–3Fuc and two minor products with the structures Galβ1–4Fuc and Galβ1–2Fuc were formed. Additionally, the product composition was defined in more detail using several different analytical methods. It was concluded that the final product contained 42% total monosaccharides, 40% disaccharides and 18% of larger oligosaccharides. 290 μmol of fGOS was produced per gram of reaction mixture and 37% of the added fucose was bound to fGOS. The fraction of fGOS from total oligosaccharides was determined as 44%. This fGOS product could be used as a new putative route to deliver fucose to the intestine.     

Influence of lactose and lactate on growth and β-galactosidase activity of potential probiotic Propionibacterium acidipropionici

Dairy propionibacteria are microorganisms of interest for their role as starters in cheese technology and as well as their functions as probiotics. Previous studies have demonstrated that Propionibacterium acidipropionici metabolize lactose by a β-galactosidase that resists the gastrointestinal transit and the manufacture of a Swiss-type cheese, so that could be considered for their inclusion in a probiotic product assigned to intolerant individuals. In the present work we studied the effect of the sequential addition of lactose and lactate as first or second energy sources on the growth and β-galactosidase activity of P. acidipropionici Q4. The highest β-galactosidase activity was observed in a medium containing only lactate whereas higher final biomass was obtained in a medium with lactose. When lactate was used by this strain as a second energy source, a marked increase of the intracellular pyruvate level was observed, followed by lactate consumption and increase of specific β-galactosidase activity whereas lactose consumption became negligible. On the contrary, when lactose was provided as second energy source, lactic acid stopped to be metabolized, a decrease of the intracellular pyruvate concentration was observed and β-galactosidase activity sharply returned to a value that resembled the observed during the growth on lactose alone. Results suggest that the relative concentration of each substrate in the culture medium and the intracellular pyruvate level were decisive for both the choice of the energetic substrate and the β-galactosidase activity in propionibacteria. This information should be useful to decide the most appropriate vehicle to deliver propionibacteria to the host in order to obtain the highest β-galactosidase activity.     

Glucose inhibition of galactose-induced synthesis of β-galactosidase in Streptomyces violaceus

Various carbon compounds inhibited galactose induced synthesis of a -galactosidase activity in Streptomyces violaceus. Glucose and 2-deoxyglucose, but not methyl--d-glucose, caused inhibition of galactose uptake activity. In addition, glucose, or one of its metabolites, inhibited the synthesis of the glactose uptake system. Therefore it is concluded that the main inhibitory activity of glucose on galactose induced enzyme synthesis is exerted through inducer exclusion. Other carbon sources, such as d-ribose, d-gluconate, cellobiose or dl--glycerophosphate, did not inhibit uptake of the inducer galactose and may exert their effect through catabolite repression, inactivation or direct enzyme inhibition.     

Activity of plasma membrane β-galactosidase and β-glucosidase

Human fibroblasts produce ceramide from sialyllactosylceramide on the plasma membranes. Sialidase Neu3 is known to be plasma membrane associated, while only indirect data suggest the plasma membrane association of β-galactosidase and β-glucosidase. To determine the presence of β-galactosidase and β-glucosidase on plasma membrane, cells were submitted to cell surface biotinylation. Biotinylated proteins were purified by affinity column and analyzed for enzymatic activities on artificial substrates. Both enzyme activities were found associated with the cell surface and were up-regulated in Neu3 overexpressing cells. These enzymes were capable to act on both artificial and natural substrates without any addition of activator proteins or detergents and displayed a trans activity in living cells.     

Effect of ethionine on the synthesis of β-galactosidase inEscherichia coli

Ethionine at concentrations of 10⁻³M, 5×10⁻³M and 10⁻²M inhibits growth, both of β-galactosidase inducible ML-30 and constitutive ML-308Escherichia coli strains. The protein synthesis (measured by the incorporation of l-leucine-¹⁴C and l-aspartic-¹⁴C acid into proteins) of these strains is inhibited to the same extent as their growth. The synthesis of inducible and constitutive β-galactosidase produced by the strains ML-30 and ML-308, respectively, is considerably inhibited by ethionine.     

Simultaneous and gradual induction of β-galactosidase and tryptophanase synthesis in anEscherichia coli batch culture

β-Galactosidase and tryptophanase can be induced inEscherichia coli simultaneously or gradually during a batch cultivation. In the strainEscherichia coli K 12 and ML 30, in which the synthesis of the two enzymes was induced simultaneously, only the synthesis of tryptophanase partially decreased, whereas the synthesis of β-galactosidase was not influenced. In the strains B 28 and ATCC 9637 the synthesis of both enzymes was partially decreased. On a gradual induction of these enzymes in the strainEscherichia coli E 12 only the synthesis of tryptophanase decreased. Thus, the results obtained here resemble those observed during the simultaneous induction. In addition, it was found that it is not important which of the two enzymes is induced as the first one.     

Enzymatic synthesis of D-tagatose from lactose with the combination of β-galactosidase and L-arabinose isomerase

将大肠杆菌K-12中的β-半乳糖苷酶基因lacZ和L-阿拉伯糖异构酶基因araA以串联方式克隆到载体pET-28a(+)上,并转入大肠杆菌BL21( DE3)中进行表达.通过SDS-PAGE分析发现,重组菌株能表达出大量可溶性β-半乳糖苷酶蛋白和L-阿拉伯糖异构酶蛋白.以重悬菌液为酶源,可将乳糖降解为D-半乳糖,并将D-半乳糖转化为D-塔格糖.在温度为50℃,pH 7.0的缓冲液中,经一段时间反应后,D-塔格糖的转化率可达21％以上.加入Mn2+、Co2+和Fe2+均能够使D-塔格糖的转化率提高.     

Transgalactosylation and hydrolytic activities of commercial preparations of β-galactosidase for the synthesis of prebiotic carbohydrates

β-Galactosidases exhibit both hydrolytic and transgalactosylation activities; the former has been used traditionally for the production of delactosed milk and dairies, while the latter is being increasingly used for the synthesis of lactose-derived oligosaccharides: balance between both activities was highly dependent on the enzyme origin: β-galactosidases from Aspegillus oryzae and Bacillus circulans exhibited high transgalactosylation activity, while those from one from Kluyveromyces exhibited high hydrolytic activity but quite low transgalactosylation activity. Also the affinity for the donors (lactose or lactulose) and the acceptors (lactose, lactulose or fructose) of transgalactosylated galactose was dependent on the enzyme origin, as reflected by the Michaelis constants obtained in the synthesis of galacto-oligosaccharides, fructosyl-galacto-oligosaccharides and lactulose. Finally, the balance between transgalactosylation and hydrolytic activities of β-galactosidases could be tuned by changing the concentration of galactose donor.     

Design,synthesis and antioxidant activity evaluation of novel β-elemene derivatives

Forty β-elemene derivatives were prepared and their antioxidant activity in H₂O₂-treated human umbilical vein endothelial cells (HUVECs) was first investigated. Among which, the dimer compounds 5r and 5s exhibited the most potent antioxidant activity against reactive oxygen species production. Meanwhile, 5r and 5s led to a significant increase in superoxide dismutase and nitric oxide levels and decrease in malonyldialdehyde and lactate dehydrogenase contents. Furthermore, MTT assay showed that 5r and 5s did not produce obvious cytotoxicity and had significantly cytoprotective effects against oxidative damage on HUVECs.