Production of β-galactosidase by Streptococcus salivarius subsp thermophilus 11F

The production of β-galactosidase by an autolytic strain of Streptococcus salivarius subsp thermophilus 11F was investigated in batch and fed-batch 2-L working volume stirred tank bioreactors. β-Galactosidase was released into the medium upon cell lysis within 1–2 h after the maximum biomass quantity was reached. In batch fermentations the highest β-galactosidase activity of 69 U ml⁻¹ was obtained when the temperature was increased to 42°C after a 4-h growth period at 30°C. In fed-batch experiments the highest β-galactosidase activity of 74 U ml⁻¹ was obtained at a constant 37°C. Received 18 December 1997/ Accepted in revised form 03 February 1998     

Sensitivity of Streptococcus thermophilus to Chemical Permeabilization

Streptococcus thermophilus cultures were treated with conjugated and unconjugated bile salts and tested for β-galactosidase activity. Na-deoxycholate and chenodeoxycholate were more efficient permeabilizing agents than cholate, and all three bile salts were superior to their corresponding glyco- and tauro-conjugates. Treatment with sodium dodecyl sulfate resulted in the highest measurable β-galactosidase levels in permeabilized cells, whereas response to Triton X-100 was variable and strain dependent. Na-deoxycholate, chenodeoxycholate, and sodium dodecyl sulfate caused cell injury and arrested culture growth for 4 h or longer. The nongrowing permeabilized biomass of S. thermophilus was used to hydrolyze lactose in aqueous solutions and milk.     

Heterologous expression of a gene encoding a thermostable β-galactosidase from <Emphasis Type="Italic">Alicyclobacillus acidocaldarius</Emphasis>

A genomic DNA library screen yielded the nucleotide sequence of a 12 kb fragment containing a gene (2067 bp) coding a thermostable β-galactosidase from Alicyclobacillus acidocaldarius ATCC 27009. The β-galactosidase gene was expressed in Pichia pastoris, and up to 90 mg recombinant β-galactosidase/l accumulated in shake flask cultures. Using o-nitrophenyl-β-d-galactopyranoside as a substrate, the optimum pH and temperature of the purified recombinant β-galactosidase were 5.8–6.0 and 70°C, respectively. The enzyme retained 90% of its activity when heated at 70°C for 30 min. Approximately 48% of lactose in milk was hydrolyzed following treatment with the recombinant enzyme over 60 min at 65°C.     

The structure of the α-galactosidase gene loci in Thermus brockianus ITI360 and Thermus thermophilus TH125

The Thermus thermophilus TH125 α-galactosidase gene, agaT, and flanking sequences were cloned in Escherichia coli and sequenced as well as flanking sequences of the previously cloned agaT from Thermus brockianus ITI360. Different structures of putative α-galactosidase operons in the two Thermus strains were revealed. Downstream of and overlapping with the α-galactosidase genes of both strains, a gene was identified that is similar to the galactose-1-phosphate uridylyltransferase gene (galT) of E. coli and Streptomyces lividans. Upstream of the agaT of T. brockianus ITI360, four open reading frames were observed. The deduced translation products displayed similarity to components of bacterial binding protein-dependent transport systems and a β-galactosidase. No galactoside utilization genes were identified upstream of agaT in T. thermophilus TH125. The inactivation of the α-galactosidase genes of both strains by insertional mutagenesis led to an inability to use melibiose or galactose as a single carbohydrate source. An attempt was made to isolate a gene encoding the enzyme responsible for para-nitrophenyl-(pNP-) β-galactoside hydrolyzing activity in T. thermophilus TH125. A gene designated bglT was cloned and expressed in E. coli. The inactivation of the bglT gene led to 55% reduction of the pNP-β-galactoside hydrolyzing activity in the mutant strain in comparison to the wild type. Received: April 28, 1999 / Accepted: September 9, 1999     

Lactose and galactose uptake by genetically engineered Pediococcus species

The ability to utilize lactose is requisite for lactic acid bacteria used as starters in the dairy industry. Modern genetic recombination techniques have facilitated the introduction of the lactose-positive phenotype into bacteria such as Pediococcus species, which traditionally have not been used as dairy starters. This study investigated lactose and galactose uptake along with phospho-β-galactosidase activity in pediococci that had been transformed with a Latococcus lactis lactose plasmid. Lactose-positive transformants, Pediococcus acidilactici SAL and Pediococcus pentosaceus SPL-2, demonstrated an ability to accumulate [¹⁴C]lactose at a rate greater than the Lactococcus lactis control. Phospho-β-galactosidase activity was also higher in transformants versus Lactococcus lactis. Studies of [³H]galactose uptake suggested that a wild-type galactose transport system and the introduced lactose phosphotransferase system both functioned in galactose uptake by Pediococcus spp. transformants. Significantly lower levels of free galactose were detected in milk fermented with Lactobacillus helveticus LH100 and SAL or SPL-2 than in milk fermented with a LH100 plus Streptococcus thermophilus TA061 control starter blend. Received: 16 September 1997 /  Received revision: 11 November 1997 / Accepted: 21 November 1997     

Selenium-mediated differential response of β-glucosidase and β-galactosidase of germinatingTrigonella foenum-graecum

β-Glucosidase and β-galactosidase activity profile tested in different seeds during 24 h germination revealed reasonably high levels of activity inVigna radiata, Cicer arietinum, andTrigonella foenum-graecum. In all seeds tested, β-galactosidase activity was, in general, higher than that of β-glucosidase.T. foenum-graecum seedlings exhibited maximal total and specific activities for both the enzymes during 72 h germination. Se supplementation as Na₂SeO₃ up to 0.75 ppm was found to be beneficial to growth and revealed selective enhancement of β-galactosidase activity by 40% at 0.5 ppm Se. The activities of both the enzymes drastically decreased at 1.0 ppm level of Se supplementation. On the contrary, addition of Na₂SeO₃ in vitro up to 1 ppm to the enzyme extracts did not influence these activities. Hydrolytic rates of β-glucosidase in both control and Se-supplemented groups were enhanced by 20% with 0.05M glycerol in the medium and 30% at 0.1M glycerol. The rates were marginally higher in Se-supplemented seedlings than the controls, irrespective of added glycerol in the medium. In contrast, hydrolysis by β-galactosidase showed a trend of decrease in Se-supplemented seedlings compared to the control, when glycerol was present in the medium. Addition of Se in vitro in the assay medium showed no difference in the hydrolytic rate by β-galactosidase when compared to control, while the activity of β-glucosidase declined by 50%. Se-grown seedlings showed an enhancement of transglucosidation rate by 40% in the presence of 0.1M glycerol. The study reveals a differential response to Se among the β-galactosidase and β-glucosidase ofT. foenumgraecum with increase in the levels of β-galactosidase activity.     

The β-Galactosidase Activity in <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> CBS6556 Decreases by High Concentrations of Galactose

In this paper we report on the effect of different concentrations of lactose and galactose in the production of β-galactosidase by Kluyveromyces marxianus CBS6556. The results clearly demonstrate a decrease in enzyme specific activity during cultivation at high concentrations of L-lactose or D-galactose, despite the fact that these carbohydrates are normally used for induction of the β-galactosidase activity. Therefore, maximum induction of β-galactosidase in K. marxianus batch cultures was obtained at low concentrations of the inducer carbohydrates, in the range between 0.5 to 15 mM. Those informations can help to design low cost medium with higher β-galactosidase productivity by K. marxianus cells. Received: 8 August 2001 / Accepted: 15 October 2001     

Simultaneous on line monitoring of intracellular β-galactosidase activity and biomass using flow injection analysis inEscherichia coli batch fermentations

Summary A novel method to monitor on-line intracellular β-galactosidase activity and biomass simultaneously, using flow injection analysis (FIA), has been developed. The automatic ultrasonic cell disruption and FIA analysis allow the processing of 10 samples/hour with a wide and variable linear working range of β-galactosidase activity and biomass and a maximum relative standard deviation (RSD) of 1.5%. The system has been optimized by monitoring biomass and intracellular β-galactosidase activity inEscherichia coli batch fermentation.     

Heterogeneous Properties of Individual Molecules of β-Galactosidase from the Thermophilic Bacteria <Emphasis Type="Italic">Geobacillus stearothermophilus</Emphasis>

Single enzyme molecule assays were performed on β-galactosidase from the thermophilic bacteria Geobacillus stearothermophilus using a capillary electrophoresis-based continuous flow assay and the substrate DDAO-β-d-galactoside. The enzyme was found to be heterogeneous with respect to catalytic rate, electrophoretic mobility and activation energy of catalysis. Catalytic rate was also found to vary over time for individual molecules at elevated temperature. Comparison with β-galactosidase from the mesophilic bacteria Escherichia coli showed that the variation in activity over time was less pronounced and the average activation energy of catalysis was lower for the Geobacillus stearothermophilus enzyme. Attempts to measure the properties of individual β-galactosidase molecules from the thermophilic bacteria Thermus thermophilus and the cold-adapted bacteria Pseudoalteromas haloplanktis using this assay were unsuccessful.     

Improvement of culture conditions to overproduce β-galactosidase from Escherichia coli in Bacillus subtilis

The effect of some culture variables in the production of β-galactosidase from Escherichia coli in Bacillus subtilis was evaluated. The lacZ gene was expressed in B. subtilis using the regulatory region of the subtilisin gene aprE. The host contained also the hpr2 and degU32 mutations, which are known to overexpress the aprE gene. We found that, when this overproducing B. subtilis strain was grown in mineral medium supplemented with glucose (MMG), β-galactosidase production was partially growth-associated, as 40%–60% of the maximum enzyme activity was produced before the onset of the stationary phase. In contrast, when a complex medium was used, β-galactosidase was produced only at low levels during vegetative growth, whereas it accumulated to high levels during early stationary phase. Compared with the results obtained in complex media, a 20% increase in specific β-galactosidase activity in MMG supplemented with 11.6 g/l glucose was obtained. On the 1-l fermenter scale, a threefold increase in volumetric β-galactosidase activity was obtained when the glucose concentration was varied from 11 g/l to 26 g/l. In addition, glucose feeding during the stationary phase resulted in a twofold increase in volumetric enzyme activity as cellular lysis was prevented. Finally, we showed that oxygen uptake and carbon dioxide evolution rates can be used for on-line determination of the onset of stationary phase, glucose depletion and biomass concentration. Received: 18 April 1996 / Received revision: 27 August 1996 / Accepted: 6 September 1996     

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     

Production of β-galactosidase by Kluyveromyces marxianus MTCC 1388 using whey and effect of four different methods of enzyme extraction on β-galactosidase activity

Whey containing 4.4% (w/v) lactose was inoculated with Kluyveromyces marxianus MTCC 1389 for carrying out studies related to β-galactosidase production. β-galactosidase activity was found to be maximum after 30 h and further incubation resulted in decline in activity. The maximum cell biomass of 2.54 mg mL⁻¹ was observed after 36 h of incubation. Lactose concentration dropped drastically to 0.04 % from 4.40% after 36 h of incubation. Out of the four methods tested for extraction of enzyme, SDS — Chlorofom method was found to be best followed by Toluene — Acetone, sonication and homogenization with glass beads in that order. It could be concluded through this study that SDS — Chloroform is cheap and simple method for enzyme extraction from Kluyveromyces cells, which resulted in higher enzyme activity as compared to the activity observed using the remaining extraction methods. The study could also establish that whey could effectively be utilized for β-galactosidase production thus alleviating water pollution problems caused due to its disposal into the water streams.     

Cloning,production and secretion of β-galactosidase inAcetobacter pasteurianus

The gene coding for β-galactosidase fromEscherichia coli was cloned into plasmid pACT71 containing the replicon from plasmid pAC1 fromAcetobacter pasteurianus. E. coli MC4100,E. coli JM105,E. coli LE392.23 andA. pasteurianus 3614 were transformed with the recombinant plasmid pACB815. Cells were cultivated in LB, YPG and M media supplemented with glucose, glycerol, lactose or ethanol and β-galactosidase activity was detected in the cells and in the cultivation medium. The best substrate for production of β-galactosidase was lactose. To release β-galactosidase fromA. pasteurianus cells amino acids were added to the cultivation medium. The highest secretory activity was achieved using 1.5% glycine after 36 h of cultivation in the M medium.     

Construction of the industrial ethanol-producing strain of Saccharomyces cerevisiae able to ferment cellobiose and melibiose

The gene mel1, encoding α-galactosidase in Schizosaccharomyces pombe, and the gene bgl2, encoding and α-glucosidase in Trichoderma reesei, were isolated and co-expressed in the industrial ethanolproducing strain of Saccharomyces cerevisiae. The resulting strains were able to grow on cellobiose and melibiose through simultaneous production of sufficient extracellular α-galactosidase and β-glucosidase activity. Under aerobic conditions, the growth rate of the recombinant strain GC1 co-expressing 2 genes could achieve 0.29 OD600 h⁻¹ and a biomass yield up to 7.8 g l⁻¹ dry cell weight on medium containing 10.0 g l⁻¹ cellobiose and 10.0 g l⁻¹ melibiose as sole carbohydrate source. Meanwhile, the new strain of S. cerevisiae CG1 demonstrated the ability to directly produce ethanol from microcrystalline cellulose during simultaneous saccharification and fermentation process. Approximately 36.5 g l⁻¹ ethanol was produced from 100 g of cellulose supplied with 5 g l⁻¹ melibose within 60 h. The yield (g of ethanol produced/g of carbohydrate consumed) was 0.44 g/g, which corresponds to 88.0% of the theoretical yield.     

Utilization of protein-hydrolyzed cheese whey for production of β-galactosidase by Kluyveromyces marxianus

We studied the utilization of protein-hydrolyzed sweet cheese whey as a medium for the production of β-galactosidase by the yeasts Kluyveromyces marxianus CBS 712 and CBS 6556. The conditions for growth were determined in shake cultures. The best growth occurred at pH 5.5 and 37°C. Strain CBS 6556 grew in cheese whey in natura, while strain CBS 712 needed cheese whey supplemented with yeast extract. Each yeast was grown in a bioreactor under these conditions. The strains produced equivalent amounts of β-galactosidase. To optimize the process, strain CBS 6556 was grown in concentrated cheese whey, resulting in a higher β-galactosidase production. The β-galactosidase produced by strain CBS 6556 produced maximum activity at 37°C, and had low stability at room temperature (30°C) as well as at a storage temperature of 4°C. At −4°C and −18°C, the enzyme maintained its activity for over 9 weeks. Received 20 January 1999/ Accepted in revised form 30 April 1999     

Immobilization and properties of β-D-galactosidase fromLactobacillus bulgaricus

β-D-galactosidase (EC 3.2.1.23) fromLactobacillus bulgaricus (1373) was immobilized by entrapment in a Polyacrylamide gel lattice. The enzymatic properties of the immobilized β-galactosidase were compared with those of the native enzyme. The temperature and pH optima were not affected by the immobilization. After entrapment of the enzyme no significant change was observed in its thermostability. The pH stability of the immobilized enzyme was higher than that of the native enzyme on the acidic side. TheK _m values for the immobilized and native β-galactosidase with both lactose ando-nitrophenyl-β-D-galactoside as substrates were comparable. The immobilized enzyme could be repeatedly used 12 times without any loss of activity. No loss in the activity of the immobilized β-galactosidase was found after its storage for 30 days at 4°C and for 20 days at 25°C.     

Effects of medium composition and nutrient limitation on loss of the recombinant plasmid pLG669-z and β -galactosidase expression by Saccharomyces cerevisiae

The effects of medium composition, nutrient limitation and dilution rate on the loss of the recombinant plasmid pLG669-z and plasmid-borne β -galactosidase expression were studied in batch and chemostat cultures of Saccharomyces cerevisiae strain CGpLG. The difference in growth rates between plasmid-free and plasmid-containing cells (Δμ) and the rate of segregation (R) were determined and some common factors resulting from the effect of medium composition on plasmid loss were identified. Glucose-limited chemostat cultures of CGpLG grown on defined medium were more stable at higher dilution rates and exhibited Δμ -dominated plasmid loss kinetics. Similar cultures grown on complex medium were more stable at lower dilution rates and exhibited R-dominated plasmid loss kinetics. Overall plasmid stability was greatest in phosphate-limited chemostat cultures grown on defined medium and was least stable in magnesium-limited cultures grown on defined medium. Δμ decreased and R increased with increased dilution rate, irrespective of medium composition. Increased plasmid loss rates at high or low dilution rates would appear to be characteristic of loss kinetics dominated by R or Δμ, respectively. Growth of glucose-limited chemostat cultures on complex medium decreased Δμ values but increased R values, in comparison to those cultures grown on defined medium. Any increased stability that a complex medium-induced reduction of Δμ may have conferred was counteracted by an increased R value. Increased β-galactosidase productivity was correlated with increased plasmid stability only in glucose-limited chemostat cultures grown on defined medium and not in those grown on complex medium. Previous studies have yielded contrasting responses with regard to the effect of dilution rate on recombinant plasmid loss from S. cerevisiae. Our findings can account for these differences and may be generally valid for the stability of similar yeast plasmid constructs. This information would facilitate the design of bioprocesses, where recombinant plasmid instability results in reduced culture productivity. Received 08 July 1996/ Accepted in revised form 14 January 1997     

Gene transfer into mouse embryonic stem cell-derived cardiac myocytes mediated by recombinant adenovirus

Summary The main purpose of this study was to examine, for the first time, the ability of recombinant adenovirus to mediate gene transfer into cardiac myocytes derived from mouse embryonic stem (ES) cells differentiating in vitro. In addition, observations were made on the effect of adenovirus infection on cardiac myocyte differentiation and contractility in this in vitro system of cardiogenesis. ES cell cultures were infected at various times of differentiation with a recombinant adenovirus vector (AdCMVlacZ) containing the bacterial lacZ gene under the control of the cytomegalovirus (CMV) promoter. Expression of the lacZ reporter gene was determined by histochemical staining for β-galactosidase activity. LacZ expression was not detected in undifferentiated ES cells infected with AdCMVlacZ. In contrast, infection of differentiating ES cell cultures showed increasing transgene expression with continued time in culture. Expression in ES-cell-derived cardiac myocytes was demonstrated by codetection of β-galactosidase activity and troponin T with indirect immunofluorescence. At 24 h postinfection, approximately 27% of the cardiac myocytes were β-galactosidase positive, and lacZ gene expression appeared to be stable for up to 21 postinfection. Adenovirus infection had no apparent effect on the onset, extent, or duration of spontaneously contracting ES-cell-derived cardiomyocytes, indicating that cardiac differentiation and contractile function were not significantly altered in the infected cultures. The demonstration of adenovirus-mediated gene transfer into ES-cell-derived cardiac myocytes will aid studies of gene expression with this in vitro model of cardiogenesis and may facilitate future studies involving the use of these myocytes for grafting experiments in vivo.     

Purification and characterization of a sodium-stimulated β-galactosidase from Bifidobacterium longum CCRC 15708

Summary β-galactosidase from Bifidobacterium longum CCRC 15708 was first extracted by ultrasonication then purified by Q Fast-Flow chromatography and gel chromatography on a Superose 6 HR column. These steps resulted in a purification of 15.7-fold, a yield of 29.3%, and a specific activity of 168.6 U mg⁻¹ protein. The molecular weight was 357 kDa as determined from Native-PAGE. Using o-nitrophenyl-β-d-galactopyranoside (ONPG) as a substrate, the pH and temperature optima of the purified β-galactosidase were 7.0 and 50 °C, respectively. The enzyme was stable at a temperature up to 40 °C and at pH values of 6.5–7.0. K _m and V _max for this purified enzyme were noted to be 0.85 mM and 70.67 U/mg, respectively. Na⁺ and K⁺ stimulated the enzyme up to 10-fold, while Fe³⁺, Fe²⁺, Co²⁺, Cu²⁺, Ca²⁺, Zn²⁺, Mn²⁺ and Mg²⁺ inhibited the activity of β-galactosidase. Furthermore, although glucose, galactose, maltose, or raffinose exerted little or no effect on the β-galactosidase activity, lactose and fructose inhibited the enzyme activity. The effect of lactose on the enzyme activity for ONPG is probably a case of competitive inhibition. A relatively high specific activity of β-galactosidase from B. longum CCRC 15708 could be obtained by Q Fast-Flow chromatography and gel chromatography on a Superose 6 HR column. In some aspects, particularly the activation by monovalent cations, the properties of β-galactosidase of B. longum CCRC 15708 are different from those obtained from other sources. Data collected in the present study are of value and indispensable when β-galactosidase from B. longum CCRC 15708 is employed in practical application.