Maximum Β-galactosidase Activity Research Articles

Modeling and Optimization of β-Galactosidase Entrapping in Polydimethylsiloxane-Modified Silica Composites.

Protein entrapment has multiple applications in enzymatic hydrolysis, drug delivery, etc. Here, we report the studies that successfully utilized the Box–Behnken design to model and optimize the parameters of β-galactosidase entrapment in sol–gel-derived silica composites. We have also demonstrated the influence of polymer–polydimethylsiloxane as a composite modifying agent on the activity of entrapped enzymes. We have determined how different sol-gel process parameters influence the activity of entrapped enzymes. The highest impact on β-galactosidase activity was exerted by the water:tetramethoxysilane ratio, followed by polydimethylsiloxane content. Optimized synthesis parameters have been utilized to obtain a composite with maximum β-galactosidase activity. Performed porosity studies have shown that the addition of polydimethylsiloxane increased the pore diameter. Microscopy studies demonstrated that polydimethylsiloxane-modified composites are softer and less rough. Studies of β-galactosidase activity using the o-NPG test showed statistically significant shifts in the enzyme temperature and pH profiles compared to the soluble form. An improvement in the reusability of the enzyme and a significant increase in the thermal stability was also observed. When lactose was used, a strong correlation was observed between the substrate concentration and the type of the catalyzed reaction. Moreover, we have demonstrated that the yields and rates of both lactose hydrolysis and galactooligosaccharides formation were correlated with reaction temperature and with the presence of polydimethylsiloxane. All these findings provide the opportunity for industrial use of optimized PDMS-modified silica composites in lactose elimination from dairy products, e.g., milk or whey.

Effect of Nutritional Factors on Growth Behaviour, Proteolytic, β-Glucosidase and β-Galactosidase Activities of Lactobacillus Cultures during Soy-Drink Fermentation

In this study, inulin, whey protein concentrate (WPC 70) and skimmed milk powder (SMP) were used as nutritional factors to improve the quality of soy-drink fermented with indigenous Lactobacillus cultures. The growth behaviour (pH, titratable acidity, viable cell count), β-glucosidase, β-galactosidase, and proteolytic activity of indigenous Lactobacillus cultures were studied with various nutritional supplements in soy-drink. Two-way analysis of variance (ANOVA) was applied and the post-hoc comparison was acquired by Tukey’s HSD test for all the data presented in the study. Due to the supplementation of nutritional factors, the pH dropped as a bit of lactic acid production was also observed due to a lack of lactose. The supplements also provided extra nutrition during their growth in different incubation hours. At 2% rate of WPC 70 supplementation, maximum proteolysis (1.17 O.D.) was found by L. fermentum K7. L. fermentum K16 showed the highest β-glucosidase enzyme activity (0.154µM/mL/min) and maximum β-galactosidase activity (0.339 µM/mL/min) was observed for L. acidipiscis K27 at 2% rate of inulin supplementation to soy-drink. In the preparation of novel fermented foods and beverages, the conjunction of nutritional supplements with indigenous Lactobacillus cultures can potentially be used.

Purification and characterization of β-galactosidase from newly isolated Aspergillus terreus (KUBCF1306) and evaluating its efficacy on breast cancer cell line (MCF-7).

β-galactosidases (EC 3.2.1.23) are able to catalyze two different types of reactions, namely hydrolysis and transgalactosylation. It is a lysosomal exoglycosidase involved in the catabolism of glycoconjugates by sequential release of β-linked terminal galactosyl residues. It has profound significance in cancer cell senescence. It can be derived from microbial sources including bacteria, yeasts, and filamentous fungi. The enzyme was purified from the crude enzyme using ammonium sulfate precipitation, dialysis, ion exchange chromatography using DEAE cellulose, fast protein liquid chromatography and high performance liquid chromatography. The enzyme was purified with 10.78 -fold with specific activity of 62U/mg of protein and yield of 28.26%. Molecular weight of β -galactosidase as estimated by using SDS-PAGE was 42kDa. Kinetic parameters Km and Vmax for purified enzyme were 0.48 and 0.96 respectively. Further the characterization and kinetic studies of purified enzyme were carried out. The optimum pH and temperature for maximum β-galactosidase activity were found to be 6, 40°C, respectively. The present study is aimed to purification, characterization and in vitro efficacy assessment in breast cancer cell line. The β-galactosidase isolated from Aspergillus terreus was found to be effective in the proliferation of MCF-7 breast cancer cells in vitro. The present study is aimed to purification and characterization of enzyme to assess in vitro efficacy of β-galactosidase on MCF-7 cell line to delineate its therapeutic efficacy.

Utilization of Cheese Whey Using Synergistic Immobilization of β-Galactosidase and Saccharomyces cerevisiae Cells in Dual Matrices.

Whey is a byproduct of the dairy industry, which has prospects of using as a source for production of various valuable compounds. The lactose present in whey is considered as an environmental pollutant and its utilization for enzyme and fuel production, may be effective for whey bioremediation. The dairy yeast Kluyveromyces marxianus have the ability to utilize lactose sharply as the major carbon source for the production of the enzyme. Five strains were tested for the production of the β-galactosidase using whey. The maximum β-galactosidase activity of 1.74IU/mg dry weight was achieved in whey using K. marxianus MTCC 1389. The biocatalyst was further immobilized on chitosan macroparticles and exhibited excellent functional activity at 35°C. Almost 89% lactose hydrolysis was attained for concentrated whey (100g/L) and retained 89% catalytic activity after 15cycles of reuse. Finally, β-galactosidase was immobilized on chitosan and Saccharomyces cerevisiae on calcium alginate, and both were used together for the production of ethanol from concentrated whey. Maximal ethanol titer of 28.9g/L was achieved during fermentation at 35°C. The conclusions generated by employing two different matrices will be beneficial for the future modeling using engineered S. cerevisiae in scale-up studies.

Evaluation of probiotic potential of lactic acid bacteria isolated from traditional Malaysian fermented Bambangan (Mangifera pajang)

This study aims to evaluate the probiotic and technological properties of lactic acid bacteria (LAB) isolated from an indigenous fermentation of Bambangan (Mangifera pajang). The LAB were evaluated for their tolerance to low pH, bile salt, antimicrobial potential, auto-aggregation ability, microbial adhesion to solvents, tolerance to high temperature and osmotic pressure. Approximately 36% of the isolated LAB strains displayed excellent survival at pH 3.0 with at least 4 log CFU/ml after 24 hours at 2.0% bile salt. A high aggregation activity (>20%) was found in most of the LAB strains. Five Lactobacillus plantarum strains showed at least 70% viability at 60°C for 10 minutes while one L. brevis and three L. plantarum strains were tolerant to 6% sodium chloride. Furthermore, the maximum β-galactosidase activity was found in four L. plantarum strains. In conclusion, these LAB strains could serve as promising probiotic candidates for the preparation of functional food products.

Transgalactosylating β-galactosidase from probiotic Lactobacillus plantarum MCC2156: production and permeabilization for use as whole cell biocatalyst.

Key nutritional factors were optimized for the maximum production of transgalactosylating β-galactosidase from Lactobacillus plantarum MCC2156. Galactose, yeast extract, sodium acetate and manganese sulphate were the most important nutrients affecting β-galactosidase production. Maximum β-galactosidase production (3015 miller units) was obtained by culturing L. plantarum in the optimized fermentation medium containing (w/v) galactose (4%), yeast extract (2%), sodium acetate (3%) and manganese sulphate (0.075%) with an optimum medium pH of 7.0, after 14h of incubation at 35°C. Further, permeabilization of L. plantarum cells using various chemical/ solvents for maximum β-galactosidase activity was performed for use as whole cell biocatalyst. Mixture of ethanol: n-butanol was found to effectively permeabilize the cells with maximum β-galactosidase activity under the following optimum conditions; 1: 1 mixture of ethanol (10%, v/v): n-butanol (30%, v/v) with a contact time of 10min at 28 ± 2°C.

Permeabilization of Kluyveromyces marxianus with Mild Detergent for Whey Lactose Hydrolysis and Augmentation of Mixed Culture

Cheese whey is a by-product of cheese-manufacturing industries, and the utilization of whey is a challenging problem either to use it or dispose it, because only few microorganisms can metabolize the whey lactose. Enzymatic hydrolysis of whey lactose to glucose and galactose by β-galactosidase is the approach for biotechnological application. Kluyveromyces marxianus cells were permeabilized with non-toxic, biodegradable, anionic detergent N-lauroyl sarcosine (N-LS) for the enzyme activity. The permeabilization process parameters (N-LS concentration, solvent volume, temperature and incubation time) were optimized. The maximum β-galactosidase activity of 1,220 IU/g dry weight was obtained using permeabilized cells under optimized conditions. Moreover, viability of the permeabilized cells was also evaluated, which showed that cells were alive; however, viability was reduced by two log cycles. The permeabilized cells were evaluated for whey lactose hydrolysis. The maximum lactose hydrolysis of 91% was observed with 600 mg (dry cell weight/100 mL) in whey powder (5% w/v) solution at 180-min incubation, pH 6.5 and 30 °C. Further, the hydrolyzed whey was evaluated for amelioration of growth of non-lactose-consuming yeast Saccharomyces cerevisiae. S. cerevisiae was able to grow in hydrolyzed whey simultaneously with K. marxianus. The study confirmed that N-LS could be used to permeabilize K. marxianus cells to make available the enzyme activity.

Study on β-galactosidase enzymatic activity of herbal yogurt

Different types of herbal yogurts were developed by mixing standardized milk with pretreated herbs, namely tulsi leaf (Ocimum sanctum), pudina leaf (Mentha arvensis) and coriander leaf (Coriandrum sativum), with leaves separately and a 1:1(v/v) mixture of the strains of lactic starter cultures—Lactobacillus acidophilus (NCIM 2903) and Lactobacillus plantarum (NCIM 2083)—followed by incubation at 40°C for 6h. The β-galactosidase enzymatic activity of the abovementioned herbal yogurts was determined and interestingly noted to exhibit higher enzymatic activity compared with the control yogurt (without any herbs). Among all herbal yogurts, tulsi yogurt had the maximum β-galactosidase activity.

The activity of β-galactosidase and lactose metabolism in Kluyveromyces lactis cultured in cheese whey as a function of growth rate

Kluyveromyces lactis was cultured in cheese whey permeate on both batch and continuous mode to investigate the effect of time course and growth rate on beta-galactosidase activity, lactose consumption, ethanol production and protein profiles of the cells. Cheese whey was the substrate to grow K. lactis as a batch or continuous culture. In order to precise the specific growth rate for maximum beta-galactosidase activity a continuous culture was performed at five dilution (growth) rates ranging from 0.06, 0.09, 0.12, 0.18 to 0.24 h(-1). The kinetics of lactose consumption and ethanol production were also evaluated. On both batch and continuous culture a respirofermentative metabolism was detected. The growth stage for maximum beta-gal activity was found to be at the transition between late exponential and entrance of stationary growth phase of batch cultures. Fractionating that transition stage in several growth rates at continuous culture a maximum beta-galactosidase activity at 0.24 h(-1) was observed. Following that stage beta-gal activity undergoes a decline which does not correlate to the density of its corresponding protein band on the gel prepared from the same samples. The maximum beta-galactosidase activity per unit of cell mass was found to be 341.18 mmol ONP min(-1) g(-1) at a dilution rate of 0.24 h(-1). The physiology of K. lactis growing in cheese whey permeate can proven useful to optimize the conversion of that substrate in biomass rich in beta-gal or in ethanol fuel. In addition to increasing the native enzyme the conditions established here can be set to increase yields of recombinant protein production based on the LAC4 promoter in K. lactis host.

Cultural condition affecting the growth and production of β-galactosidase by Bifidobacterium longum CCRC 15708 in a jar fermenter

In the present study, the growth and production of β-galactosidase by Bifidobacterium longum CCRC 15708 in a 5-L jar fermenter as influenced by cultivation temperature (27–42 °C), medium pH (4.5–7.5) and agitation speed (5–200 rpm) were evaluated. In general, it was found that a cultivation temperature of 37 °C proved optimal for both growth and β-galactosidase production by the test organism. Although the growth of the test organism was the highest in the culture with pH controlled at 4.5–6.5, the culture with pH controlled at 6.5 resulted in the highest production of β-galactosidase. Further, agitation at 100 rpm or more was found to enhance both the growth and production of β-galactosidase. Fermentation conducted in a jar fermenter having the pH of the culture medium, the cultivation temperature, and the agitation speed controlled at 6.5, 37 °C, and 100 rpm, respectively, a maximum β-galactosidase activity of 36.7 U/ml and a maximum transgalactosylation activity of 0.49 U/ml was achieved in 10 h of fermentation. There are ca 2.0 and 12.3 fold greater than the reported maximum β-galactosidase and transgalactosylation activity, respectively, produced by B. longum CCRC 15708 in a flask culture system.

Production of β-galactosidase by Bifidobacteria as influenced by various culture conditions

β-Galactosidase production by Bifidobacterium longum CCRC 15708, Bifidobacterium longum B6 and Bifidobacterium infantis CCRC 14633 was first examined with B. longum CCRC 15708 showing the highest production of β-galactosidase and the highest specific activity. Further study with B. longum CCRC 15708 revealed that the highest level of β-galactosidase was produced with lactose and yeast extract as carbon and nitrogen sources, respectively. Optimal enzyme production occurred at an initial pH of 6.5 and at 37 °C. Under these optimum culture conditions, a maximumβ-galactosidase activity of 18.6 U/ml could be obtained after 16 h of fermentation in a medium contain 4% lactose, 3.5% yeast extract, 0.3% K 2HPO 4, 0.1% KH 2PO 4, 0.05% MgSO 4·7H 2O and 0.03% l-cysteine. The highest transgalactosylation activity was also detected in this culture after 14–16 h of fermentation.

Effect of Growth Rate on the Production of β-Galactosidase from Escherichia Coli in Bacillus Subtilis Using Glucose-Limited Exponentially Fedbatch Cultures

The effect of specific growth rate (μ) on the production of β-galactosidase from Escherichia coli in Bacillus subtilis was assessed using glucose-limited exponentially fedbatch cultures (EFBC). A B subtilis strain carrying a chromosomal copy of the lacZ gene under the control of the subtilisin (aprE) regulatory region was used in this study. This strain also carries the hpr2 and degU32(Hy) mutations which are known to overexpress the subtilisin (aprE) gene. Constant μ in the range of 0.058–0.35 h −1 were maintained using mineral media and computer-controlled predetermined glucose feeding profiles. At all growth rates tested, a pseudo-steady state in μ was obtained; however, a sudden decrease in the slope of log of biomass concentration against time and accumulation of glucose and acetic acid indicated that control on μ was lost at all growth rates tested. Cultures maintained at a μ value close to the maximum obtained in batch cultures had a specific enzymatic activity relatively constant during the fedbatch phase. In contrast, cultures maintained at a μ value lower than 0.2 h −1 had specific and volumetric β-galactosidase activities that decreased during the fedbatch phase, reaching a value close to zero before glucose feeding was stopped. The maximum β-galactosidase activity after the EFBC phase was obtained at the maximum μ tested whereas it strongly decreased at lower μ values. This suggests that glucose limitation and μ reduction during exponential growth phase represses β-galactosidase production during the exponential and stationary phases.

Modulation of the function of the signal receptor domain of XylR, a member of a family of prokaryotic enhancer-like positive regulators.

The XylR protein controls expression from the Pseudomonas putida TOL plasmid upper pathway operon promoter (Pu) in response to aromatic effectors. XylR-dependent stimulation of transcription from a Pu::lacZ fusion shows different induction kinetics with different effectors. With toluene, activation followed a hyperbolic curve with an apparent K of 0.95 mM and a maximum beta-galactosidase activity of 2,550 Miller units. With o-nitrotoluene, in contrast, activation followed a sigmoidal curve with an apparent K of 0.55 mM and a Hill coefficient of 2.65. m-Nitrotoluene kept the XylR regulator in an inactive transcriptional form. Therefore, upon binding of an effector, the substituent on the aromatic ring leads to productive or unproductive XylR forms. The different transcriptional states of the XylR regulator are substantiated by XylR mutants. XylRE172K is a mutant regulator that is able to stimulate transcription from the Pu promoter in the presence of m-nitrotoluene; however, its response to m-aminotoluene was negligible, in contrast with the wild-type regulator. These results illustrate the importance of the electrostatic interactions in effector recognition and in the stabilization of productive and unproductive forms by the regulator upon aromatic binding. XylRD135N and XylRD135Q are mutant regulators that are able to stimulate transcription from Pu in the absence of effectors, whereas substitution of Glu for Asp135 in XylRD135E resulted in a mutant whose ability to recognize effectors was severely impaired. Therefore, the conformation of mutant XylRD135Q as well as XylRD135N seemed to mimic that of the wild-type regulator when effector binding occurred, whereas mutant XylRD135E seemed to be blocked in a conformation similar to that of wild-type XylR and XylRE172K upon binding to an inhibitor molecule such as m-nitrotoluene or m-aminotoluene.

Single step method of preparation of detergent-permeabilized Kluyveromyces fragilis for lactose hydrolysis

β-Galactosidase activity of CTAB-permeabilized K. fragilis depends upon the cell to detergent ratio and the pH of the permeabilizing medium. Maximum β-galactosidase activity was observed when the cell to detergent ratio was 1:0·01 at neutral pH. A simple and single step method for the preparation of whole cell β-galactosidase is described which involves adjusting the pH of the growth medium at the beginning of the stationary phase to 7·0, addition of CTAB directly to the growth medium at 1% concentration of biomass (wet weight), before harvesting cells by centrifugation. This whole cell enzyme activity readily hydrolysed high concentrations of lactose in buffer and whey.

Induction of umu gene expression in Salmonella typhimurium TA1535/pSK1002 by dimethyl sulfoxide (DMSO)

The genotoxicity of dimethyl sulfoxide (DMSO) was demonstrated by the umu test using Salmonella typhimurium TA1535/pSK1002 carrying the umuC-lacZ fusion gene. The level of β-galactosidase activity which shows umu gene expression in the test system was dependent on the concentration of DMSO in the culture medium. The maximum β-galactosidase activity was approximately 3.5 times as high as the background level with 10% of DMSO in the culture medium. The lowest concentration of DMSO required for a response of over twice the background level was approximately 5%. Four structurally related chemicals (acetone, di- n-butylsulfoxide, dimethylsulfide, methylphenylsulfoxide) did not show umu gene expression at their non-toxic doses.

Affinity Immobilization of Escherichia coli : Catalysis by Intact and Permeable Cells Bound to Starch

The binding of viable Escherichia coli cells to an immobilized ligand of a surface receptor for maltodextrins has recently been demonstrated (T. Ferenci and K. S. Lee, J. Mol. Biol. 160:431-444, 1982). The interaction of bacteria and ligand immobilized in a chromatographic column was investigated over a wide range of applied cell densities, temperatures, eluant pH values, osmotic concentrations, and flow rates. Over 95% retention of bacteria applied to starch-Sepharose was found at cell densities up to 10 per ml of matrix, between pH 5.5 and 8.0, between 8 and 55 degrees C, in the presence of 0 to 0.5 M NaCl, and at elution flow rates up to 37 column volumes per h. The catalytic capability and stability of affinity-immobilized cells was demonstrated with the cytoplasmic beta-galactosidase activity of starch-bound cells. Intact immobilized bacteria exhibited slowly increasing beta-galactosidase activity over several days with a plateau after 6 days. Bacteria made permeable by treatment with toluene were also bound to starch-Sepharose but showed maximum beta-galactosidase activity within 1 day and exhibited no loss of enzyme activity in 8 days of continuous elution at ambient temperatures.

Fractionation of j-methylumbelliferyl-beta-galactosidase activities in liver in gargoylism.

ockerman, P. A. & Hultberg, B. Fractionation of 4-methylumbelliferyl-β-galactosidase Activities in Liver in Gargoylism. Scand. J. clin. Lab. Invest. 22, 199-202, 1968.The pH-dependency of the 4-methyl-umbelliferyl-β-galactosidase activity was studied in homogenates of liver biopsy specimens from controls and patients with gargoylism. In the controls maximum β-galacto-sidase activity was seen at around pH 4.5 with very little variation, whereas in the gargoylism patients maximum activity was noted near a pH of 5.0-5.5.By gel filtration on Sephadex G-150 the 4-methyl-umbelliferyl-β-galactosidase activity could be separated into three distinct peaks. The maximum β-galactosidase activity was at around pH 4.0-4.2 for peak I, pH 4.4-4.7 for peak II and pH 5.0-5.5 for peak III. In the controls, peak II was larger than peak I and III. In the patients with gargoylism a different pattern was seen, involving a very marked diminution of peak II, a slight decrease of peak I and no significant change of peak III. These...