o-nitrophenyl-β-D-galactopyranoside Research Articles

Antimicrobial Activities of α-Helix and β-Sheet Peptides against the Major Bovine Respiratory Disease Agent, Mannheimia haemolytica.

Bovine respiratory disease (BRD) is the leading cause of morbidity and mortality in cattle raised in North America. At the feedlot, cattle are subject to metaphylactic treatment with macrolides to prevent BRD, a practice that may promote antimicrobial resistance and has resulted in an urgent need for novel strategies. Mannheimia haemolytica is one of the major bacterial agents of BRD. The inhibitory effects of two amphipathic, α-helical (PRW4, WRL3) and one β-sheet (WK2) antimicrobial peptides were evaluated against multidrug-resistant (MDR) M. haemolytica isolated from Alberta feedlots. WK2 was not cytotoxic against bovine turbinate (BT) cells by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. All three peptides inhibited M. haemolytica, with WK2 being the most efficacious against multiple isolates. At 8-16 µg/mL, WK2 was bactericidal against Mh 330 in broth, and at 32 µg/mL in the presence of BT cells, it reduced the population by 3 logs CFU/mL without causing cytotoxic effects. The membrane integrity of Mh 330 was examined using NPN (1-N-phenylnaphthylamine) and ONPG (o-Nitrophenyl β-D-galactopyranoside), with both the inner and outer membranes being compromised. Thus, WK2 may be a viable alternative to the use of macrolides as part of BRD prevention and treatment strategies.

Biochemical Insights into a Novel Family 2 Glycoside Hydrolase with Both β-1,3-Galactosidase and β-1,4-Galactosidase Activity from the Arctic.

A novel GH2 (glycoside hydrolase family 2) β-galactosidase from Marinomonas sp. BSi20584 was successfully expressed in E. coli with a stable soluble form. The recombinant enzyme (rMaBGA) was purified to electrophoretic homogeneity and characterized extensively. The specific activity of purified rMaBGA was determined as 96.827 U mg-1 at 30 °C using ONPG (o-nitrophenyl-β-D-galactopyranoside) as a substrate. The optimum pH and temperature of rMaBGA was measured as 7.0 and 50 °C, respectively. The activity of rMaBGA was significantly enhanced by some divalent cations including Zn2+, Mg2+ and Ni2+, but inhibited by EDTA, suggesting that some divalent cations might play important roles in the catalytic process of rMaBGA. Although the enzyme was derived from a cold-adapted strain, it still showed considerable stability against various physical and chemical elements. Moreover, rMaBGA exhibited activity both toward Galβ-(1,3)-GlcNAc and Galβ-(1,4)-GlcNAc, which is a relatively rare occurrence in GH2 β-galactosidase. The results showed that two domains in the C-terminal region might be contributed to the β-1,3-galactosidase activity of rMaBGA. On account of its fine features, this enzyme is a promising candidate for the industrial application of β-galactosidase.

Probiotic potential of β‑galactosidase‑producing lactic acid bacteria from fermented milk and their molecular characterization.

Probiotics have attained significant interest in recent years as a result of their gut microbiome modulation and gastrointestinal health benefits. Numerous fermented foods contain lactic acid bacteria (LAB) which are considered as GRAS and probiotic bacteria. The present study aimed to investigate indigenous LAB from homemade fermented milk samples collected in remote areas of Karnataka (India), in order to isolate the most potent and well-adapted to local environmental conditions bacteria, which were then evaluated using a step-by-step approach focused on the evaluation of probiotic traits and β-galactosidase-producing ability. LAB were screened using 5-bromo-4-chloro-3-indole-D-galactopyranoside (X-Gal) and O-nitrophenyl-β-D-galactopyranoside (ONPG) as substrate, and exhibited β-galactosidase activity ranging from 728.25 to 1,203.32 Miller units. The most promising isolates were selected for 16S rRNA gene sequence analysis and identified as Lactiplantibacillus plantarum, Limosilactobacillus fermentum, Lactiplantibacillus pentosus and Lactiplantibacillus sp. Furthermore, these isolates were evaluated by in vitro, viz., survival in gastrointestinal tract, antibiotic susceptibility, antimicrobial activity, cell surface characteristics, and haemolytic activity. All eight isolates demonstrated strong adherence and prevented pathogen penetration into HT-29 cells, indicating potential of the bacteria to scale up industrial level production of milk products for lactose intolerants.

Kinetic and modeling analyses of lactose‑hydrolyzing β‑galactosidase from Lactiplantibacillus plantarum GV54

The enzyme β‑galactosidase plays a very essential role in food and in the pharmaceutical industry. The aim of the present study was to identify the most effective lactic acid bacteria isolated from homemade fermented milk samples and screened for β‑galactosidase production using the substrate, 5‑bromo‑4‑chloro‑3‑indolyl β‑D‑galactopyranoside (X‑gal). For the optimization of the enzyme production with carbon and nitrogen sources, four lactic acid bacterial isolates were used. Of these, only the <em>Lactiplantibacillus plantarum</em> (<em>L. plantarum</em>) GV54 and <em>L. plantarum</em> GV64 isolates significantly increased the production of β‑galactosidase with lactose as the carbon source. Similar results were obtained when nitrogen sources, such as ammonium sulfate and beef extract were used. The extraction of β‑galactosidase using the toluene/acetone method and SDS‑PAGE were performed to determine the molecular weight. The <em>L. plantarum</em> GV54 isolate exhibited the maximum β‑galactosidase activity (162.960±0.36 Miller units) compared with the other isolates with O‑nitrophenyl‑β‑D‑galactopyranoside (ONPG) as the substrate. The β‑galactosidase activity was high at pH 7.0 and a temperature of 37˚C. In addition, Mg²⁺, Mn²⁺ and Fe²⁺ had an indirect effect on the activity of β‑galactosidase. The kinetic constant was found to be 27.37574 mM, and its maximum velocity was 0.259289 U/min for pure β‑galactosidase in relation to ONPG. Comparative modeling and structural annotation analysis were performed, and the structural comparison of β‑galactosidase enzyme from <em>L. plantarum</em> and the crystal structure of <em>Bifidobactarum bifidium</em> was also performed. It was observed that the isolated enzyme shared the highest degree of similarity. Considering these characteristics of β‑galactosidase isolated from <em>L. plantarum</em> GV54, this isolate may be a promising commercial enzyme for various industrial and other biotechnological applications.

Self-supply of H2O2 and O2 by a composite nanogenerator for chemodynamic therapy/hypoxia improvement and rapid therapy of biofilm-infected wounds

Bacterial wound infection is a major challenge to human health worldwide. In this study, a composite nanogenerator, CaO2@ZIF-67-PLL, was fabricated via a bottom-up approach with chemodynamic therapy (CDT)/hypoxia improvement for rapid therapy of biofilm-infected wounds. Under a pathologically acidic microenvironment, the out layer ZIF-67 can be decomposed to rapidly release Co2+ and CaO2; Subsequently, the unprotected CaO2 reacts with H2O to produce O2 and H2O2. The self-supply of O2 relieves hypoxia and inflammatory response induced by infections, while the generated H2O2 and poly-l-lysine (PLL) exhibited synergistic strong anti-biofilm capability against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) as assessed by reactive oxygen species (ROS) generation, membrane permeability change, ATP reduction, o-nitrophenyl-β-d-galactopyranoside (ONPG) hydrolysis and intracellular component leakage. Meanwhile, CaO2@ZIF-67-PLL exhibited good cytocompatibility and promoted the adhesion, proliferation, and migration of NIH-3T3 cells by enhancing the expression of wound healing-related genes (TGF-β1, VEGF, α-SMA, Col I, Col III, and Wnt-5a). Notably, CaO2@ZIF-67-PLL could significantly improve the healing process of S. aureus-infected wounds via suppressed inflammatory responses, enhanced collagen disposition and angiogenesis, and stimulated HIF-1α, CD31, and Wnt-5a/β-Catenin signaling pathways. These results indicated the potential of CaO2@ZIF-67-PLL nanogenerator for clinical application in infected wound treatment.

A novel cold-adapted β-galactosidase from Alteromonas sp. ML117 cleaves milk lactose effectively at low temperature

A novel β-galactosidase gene (Bgal) was cloned from the marine bacterium Alteromonas sp. ML117. Bgal from Alteromonas sp. ML117 was heterologously expressed in Escherichia coli BL21 (DE3) cells to study the enzymatic properties of the recombinant β-galactosidase. Using gel-filtration chromatography and SDS-PAGE, the recombinant enzyme was identified to be a tetrameric enzyme that belonged to the glycoside hydrolase family GH2. The purified recombinant Bgal was able to hydrolyze lactose and o-nitrophenyl-β-d-galactopyranoside (ONPG), and showed maximum activity at pH 8.0 and 30 °C to hydrolyze ONPG and at 35 °C to hydrolyze lactose. The enzyme’s activity level quickly diminished when incubated at 35 °C, suggesting that it was a cold-adapted variant. At 10 °C, the purified enzyme had Km values of 1.6 mM for ONPG and 3.8 mM for lactose. The enzyme tolerated NaCl, and retained 80% of its activity in a 30% ethanol reaction system. In addition, the recombinant Bgal hydrolyzed 86% of the lactose from milk over 24 h at 10 °C. Taken together, this study identified a recombinant Alteromonas sp. ML117 β-galactosidase that may be used for industrial applications.

Evaluation of lactase activity in new isolated Lactobacillus strains

The aim of this study was to isolate and characterize new Lactobacillus strains in order to check their β-galactosidase activity. A total of 28 strains were isolated from camel and goat milks collected from the Algerian Sahara. These strains were identified with the method of Polymerase Chain Reaction (PCR) of the 16S rRNA gene. Their ability to hydrolyze O-nitrophenyl-β-D ga-lactopyranoside (ONPG) was determined. Cell lysis method involving soni-cation and lysozyme treatment was used for β-galactosidase release from the isolated strains. Analysis of their 16S rRNA gene sequences revealed that the strains clustered in the Lactobacillus genus, 17 isolates were identified as L. plantarum, 10 isolates as L. herbarum and one isolate as L. brevis. The best intracellular enzymatic β-galactosidase activities obtained in descending order are 14.84 (L. plantarum P4), 14.66 (L. plantarum P17), 14.33 (L. planta-rum P12), 14.10 (L. plantarum P10), 13.49 (L. plantarum P6), 12.13 (L. herba-rum H10) and 11.27 (L. plantarum P7) U/mL; while a very low level of this activity was found for Lactobacillus brevis B1. These findings are an argu-ment for the use of these isolated Lactobacilli in the dairy industry in order to alleviate lactose intolerance.

In Vitro Screening of Indigenous Lactobacillus Isolates for Selecting Organisms with Better Health-Promoting Attributes.

Lactobacilli have several attributes that provide health benefits to the host. The aim of this study was to screen indigenous lactobacilli from human gut and fermented foods for such attributes as production of β- and α-galactosidase and also their ability to reduce serum cholesterol. Lactobacilli were cultured on MRS broth and β-galactosidase activity was determined using o-nitrophenyl-β-D-galactopyranoside (ONPG) as a substrate. Three isolates Lactobacillus fermentum GPI-3 and L. fermentum GPI-6 and Lactobacillus salivarius GPI-1(S) showed better β-galactosidase activity than the standard strains Lactobacillus rhamnosus GG (LGG) and Lactobacillus plantarum ATCC 8014. The isolates showed variability in assimilating cholesterol during growth. Several isolates showed excellent cholesterol-lowering ability compared to standard strains LGG and L. plantarum ATCC 8014. Isolate L. rhamnosus SCB being the highest acid producer (pH4.38) also showed the highest cholesterol reduction compared to other strains including standard strains. The ability of these isolates to produce α-galactosidase was also studied and the maximum α-galactosidase activity was found in isolate L. salivarius GPI-1(S) followed by L. fermentum FA-5 and Lactobacillus helveticus FA-7. This study therefore reports Lactobacillus isolates that have superior probiotic properties when compared to the standard strains; hence, they could be considered as potential probiotic strains, which can provide health benefits to the Indian population.

Continuous Packed Bed Reactor with Immobilized β-Galactosidase for Production of Galactooligosaccharides (GOS)

The β-galactosidase from Bacillus circulans was covalently attached to aldehyde-activated (glyoxal) agarose beads and assayed for the continuous production of galactooligosaccharides (GOS) in a packed-bed reactor (PBR). The immobilization was fast (1 h) and the activity of the resulting biocatalyst was 97.4 U/g measured with o-nitrophenyl-β-d-galactopyranoside (ONPG). The biocatalyst showed excellent operational stability in 14 successive 20 min reaction cycles at 45 °C in a batch reactor. A continuous process for GOS synthesis was operated for 213 h at 0.2 mL/min and 45 °C using 100 g/L of lactose as a feed solution. The efficiency of the PBR slightly decreased with time; however, the maximum GOS concentration (24.2 g/L) was obtained after 48 h of operation, which corresponded to 48.6% lactose conversion and thus to maximum transgalactosylation activity. HPAEC-PAD analysis showed that the two major GOS were the trisaccharide Gal-β(1→4)-Gal-β(1→4)-Glc and the tetrasaccharide Gal-β(1→4)-Gal-β(1→4)-Gal-β(1→4)-Glc. The PBR was also assessed in the production of GOS from milk as a feed solution. The stability of the bioreactor was satisfactory during the first 8 h of operation; after that, a decrease in the flow rate was observed, probably due to partial clogging of the column. This work represents a step forward in the continuous production of GOS employing fixed-bed reactors with immobilized β-galactosidases.

Characterization and Effects of β–Galactosidase in the Crude Extracts of Cuminum cyminum and Curcuma longa in Preparation of Delactosed Milk and Whey

&lt;p&gt;β-galactosidase (EC 3.2.1.23) was extracted from &lt;em&gt;Cuminum cyminum &lt;/em&gt;and&lt;em&gt; Curcuma longa&lt;/em&gt;. The crude extracts of these plants were then characterized in term of pH, temperature, and enzyme kinetic. The crude extracts were also used in hydrolysis of lactose in milk and whey. The enzyme activity was measured by its ability to hydrolyze the substrate o-nitrophenyl β -D-galactopyranoside (ONPG).&lt;/p&gt;&lt;p&gt;It was found that β-galactosidase in the crude extracts of &lt;em&gt;Cuminum cyminum &lt;/em&gt;exhibited maximum activity at pH 8.0 and optimum temperature at 60 °C. While, β-galactosidase in the crude extracts of &lt;em&gt;Curcuma longa&lt;/em&gt; have optimum pH at 5.0 and 7.0 and optimum temperature at 50 °C.The K&lt;sub&gt;m&lt;/sub&gt; and V&lt;sub&gt;max&lt;/sub&gt; values of the β-galactosidase in the crude extracts of &lt;em&gt;Cuminum cyminum&lt;/em&gt; and &lt;em&gt;Curcuma longa &lt;/em&gt;were 4.16 mM and 0.087 μmol/min, and 2.63 mM and 0.333μmol/min, respectively.&lt;/p&gt;&lt;p&gt;The results showed that 96.84-97.08% of lactose was hydrolyzed in cow’s milk and whey when treated with crude extracts of &lt;em&gt;Cuminum cyminum&lt;/em&gt; and 90-98.6% when treated with crude extracts of &lt;em&gt;Curcuma longa&lt;/em&gt;.&lt;/p&gt;

Genotoxicity studies of tetrahydro-naphthalenebenzimidazole/ thiazolidinedione as retinoid derivatives / Tetrahidro-naftalen-benzimidazol/tiyazolidindion retinoid türevlerinin genotoksisitesi

Abstract Objective: Mutagenicity is an undesirable side effect of clinically prescribed drugs, thus raises the question of their potential carcinogenicity. Taking into account that nitro compounds are known for their genotoxicity, it will be considerable interest to assess the genotoxic activities of the benzimidazole/thiazolidinedione retinoid derivatives. For this reason, the present study reports the genotoxicity of previously synthesized benzimidazole/ thiazolidinedione-retinoid derivatives (Ates-Alagoz and Buyukbingol, 2001; Ates-Alagoz et. al., 2009) by the umu-microplate test system. Methods: In this study, since this derivates involve nitro groups, makes advantageous the use of the umu-test, using NM2009 and NM1011 strains especially designed for detecting the mutagenicity of nitro compounds. Two bacterial strains Salmonella typhimurium NM1011 and S. typhimurium NM2009, expressing high levels of nitroreductase (NR) and O-acetyltransferase (O-AT) respectively, were used in the tests. Chlorophenol red-β-D-galactopyranoside (CPRG), O-nitrophenyl- β-D-galactopyranoside (ONPG) were used as substrate in the enzyme assays along with a well-known genotoxic nitro compound, 4-nitroquinoline 1-oxide (4NQO), as the positive control in the test. Results: Benzimidazole/ thiazolidinedione-retinoids that contain NO2 group with different position on benzene ring showed no genotoxicity in both strains with both substrates. However, using CPRG as the colorimetric substrate resulted in stronger activity than using ONPG in test conducted in both strains with all compounds. Although the β-galactosidase activities with using CPRG were three fold higher than ONPG, parallel results were obtained for both substrates and strains with all compounds tested. For all compounds, the induction of umuC gene expression was found to be almost the same for the strains that overexpress nitroreductase and O-acetyltransferase. Conclusion: None of the derivatives showed genotoxic effects, a very important data, making them a potential drug candidate.

Effect of the pH in the formation of β-galactosidase microparticles produced by a spray-drying process

The objective of this work was to investigate the influence of pH in the microencapsulation process, using a modified chitosan to microencapsulate the enzyme β-galactosidase, by a spray-drying technique. Structural analysis of the surface of the particles was performed by scanning electron microscopy (SEM), showing that the obtained microparticles have an average diameter smaller than 3.5μm and in general a regular shape. The activity of the enzyme was studied by spectrophotometric methods using the substrate O-nitrophenyl-β,d-galactopyranoside (ONPG). The parameters of Michaelis–Menten were calculated. The value of Km decreases with the decrease of the pH, which can be associated to an increase of the affinity between the enzyme and substrate to smaller pH's. The highest value of the parameter Vmax, representing the maximum reaction rate at a given enzyme concentration, was obtained at pH 6.

Kinetic studies on exploring lactose hydrolysis potential of β galactosidase extracted from Lactobacillus plantarum HF571129.

A novel intracellular β-galactosidases produced by Lactobacillus plantarum HF571129, isolated from an Indian traditional fermented milk product curd was purified and characterized. The β-galactosidases is a hetrodimer with a molecular weight of 60kDa (larger subunit) and 42kDa (smaller subunit), as estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was purified 7.23 fold by ultrasonication, ultrafiltration and gel filtration chromatography with an overall recovery of 30.41%. The optimum temperature for hydrolysis of its preferred substrates, o-nitrophenyl- β-D-galactopyranoside (ONPG) and lactose, are 50°C (both), and optimum pH for these reactions is 6.5 and 7.5, respectively. The β-galactosidases showed higher affinity for ONPG (Km, 6.644mM) as compared to lactose (Km, 23.28mM). Galactose, the end product of lactose hydrolysis was found to be inhibited (47%). The enzyme activity was drastically altered by the metal ion chelators EDTA, representing that this enzyme is a metalloenzyme. The enzyme was activated to a larger extent by Mg(2+) (73% at 1mM), while inhibited at higher concentrations of Na(+) (54% at 100mM), K(+) (16% at 100mM) and urea (16% at 100mM). The thermal stability study indicated an inactivation energy of Ed = 171.37kJmol(-1). Thermodynamic parameters such as ∆H, ∆S and ∆G, were determined as a function of temperature. About 88% of lactose was hydrolyzed at room temperature within 1h. The study suggested that this enzyme showed its obvious superiority in the industrial lactose conversion process.

Microencapsulation of β-galactosidase with different biopolymers by a spray-drying process

The aim of this work was to investigate the possibility of producing microparticles containing β-galactosidase, using different biopolymers (arabic gum, chitosan, modified chitosan, calcium alginate and sodium alginate) as encapsulating agents by a spray-drying process. This study focused on the enzyme β-galactosidase, due to its importance in health and in food processing. Encapsulation of β-galactosidase can increase the applicability of this enzyme in different processes and applications. A series of β-galactosidase microparticles were prepared, and their physicochemical structures were analyzed by laser granulometry analysis, zeta potential analysis, and by scanning electron microscopy (SEM). Microparticles with a mean diameter around 3μm have been observed, for all the biopolymers tested. The microparticles formed with chitosan or arabic gum presented a very rough surface; on the other hand, the particles formed with calcium or sodium alginate or modified chitosan presented a very smooth surface. The activity of the enzyme was studied by spectrophotometric methods using the substrate ONPG (O-nitrophenyl-β,d-galactopyranoside). The microencapsulated β-galactosidase activity decreases with all the biopolymers. The relative enzyme activity is 37, 20, 20 and 13%, for arabic gum, modified chitosan, calcium alginate and sodium alginate, respectively, when compared with the free enzyme activity. The enzyme microparticles formed with arabic gum shows the smallest decrease of Vmax, followed by the calcium alginate, sodium alginate, and modified chitosan.

Identification and characterization of an unusual glycosyltransferase-like enzyme with β-galactosidase activity from a soil metagenomic library

Glycosyltransferases and glycoside hydrolases are two diversified groups of carbohydrate-active enzymes (CAZymes) in existence, they serve to build and break down the glycosidic bonds, respectively, and both categories have formed many sequence-based families. In this study, a novel gene (glyt110) conferring β-galactosidase activity was obtained from a metagenomic library of Turpan Basin soil. Sequence analysis revealed that glyt110 encoded a protein of 369 amino acids that, rather than belonging to a family typically known for β-galactosidase activity, belonged to glycosyltransferase family 4. Because of this unusual sequence information, the novel gene glyt110 was subsequently expressed in Escherichia coli BL21(DE3), and the recombinant enzyme (Glyt110) was purified and characterized. Biochemical characterization revealed that the β-galactosidase activity of Glyt110 toward o-nitrophenyl-β-d-galactopyranoside (ONPG) and lactose were identified to be 314±18.3 and 32±2.7U/mg, correspondingly. In addition, Glyt110 can synthesize galacto-oligosaccharides (GOS) using lactose as substrate. A GOS yield of 47.2% (w/w) was achieved from 30% lactose solution at 50°С, pH 8.0 after 10h reaction. However, Glyt110 was unable to glycosylate either N-acetylated saccharides or lactose and galactose using UDP-gal as sugar donor, and its glycosyltransferase activity needs further investigation. These results indicated that Glyt110 is an unusual enzyme with β-galactosidase activity but phylogenetically related to glycosyltransferase. Our findings may provide opportunities to improve the insight into the relationship between glycosyltransferases and glycoside hydrolases and the sequence-based classification.

Purification, recovery, and characterization of chick pea (Cicer arietinum) β-galactosidase in single step by three phase partitioning as a rapid and easy technique

In this study chick pea β-galactosidase was first time purified and recovered in single step by three phase partitioning (TPP). Optimal purification parameters for TPP were 60% ammonium sulfate saturation (w/v) with 1:0.5 (v/v) ratio of crude extract:t-butanol at pH 6.8, which gave 10.1 purification fold with 133% recovery of β-galactosidase. SDS–PAGE analysis showed that protein has two subunits with molecular masses of 48 and 38kDa, respectively. Characterization of enzyme showed that optimal pH of purified enzyme was 2.8 and optimal temperature was 50°C. Enzyme was further characterized by the Arrhenius activation energy and Michael–Menten kinetic constants. Activation energy (Ea) was calculated by using Arrhenius equation and determined to be 15.52kcalmol−1. Km value of purified enzyme was estimated for the o-nitrophenyl β-d galactopyranoside (ONPG) substrate as 1.09mM, while its maximum velocity, Vm was 0.90U/mL/min at 37°C. TPP improved substrate affinity of enzyme by the increased flexibility during the partitioning. TPP is simple, easy and economic technique for purification and recovery of β-galactosidase from chick pea, and has a big potential use for industrial applications.

Effect of Ce3+ on membrane permeability of Escherichia coli cell

This study aimed to delineate the antibacterial mechanism of rare-earth ion Ce3+ to the target organism Escherichia coli cell, and the most important purpose was to identify its biological effect of increasing the E. coli cell membrane permeability. The antibacterial activities of Ce3+ to E. coli cells were tested, and then the permeability of outer membrane (OM) and inner membrane (IM) were studied by N-phenyl-1-naphthylamine (NPN) and o-nitrophenyl-β-D-galactopyranoside (ONPG) methods separately. Through these experiments we concluded that the E. coli cells grown to log phage were more sensitive to Ce3+ than the ones not at this stage; the structure of membrane was destroyed and the permeability of both OM and IM was obviously increased by Ce3+; there should be certain interactions between Ce3+ and some proteins inside the cell, which impeded the physiological activities of bacteria.

Characterization of Lactose Utilization and β-Galactosidase in Lactobacillus brevis KB290, the Hetero-Fermentative Lactic Acid Bacterium

Unlike dairy lactic acid bacteria, Lactobacillus brevis cannot ferment milk. We characterized the lactose utilization by L. brevis KB290. In a carbohydrate fermentation assay using API 50 CHL, we showed during 7 days L. brevis did not ferment lactose. L. brevis grew to the stationary phase in 2 weeks in MRS broth containing lactose as the carbon source. L. brevis slowly consumed the lactose in the medium. L. brevis hydrolyzed lactose and a lactose analog, o-nitrophenyl-β-D-galactopyranoside (ONPGal). This β-galactosidase activity for ONPGal was not repressed by glucose, galactose, fructose, xylose, or maltose showing the microorganism may not have carbon catabolite repression. We purified the L. brevis β-galactosidase using ammonium sulfate precipitation and several chromatographies. The enzyme's molecular weight is estimated at 72 and 37 kDa using SDS-PAGE analysis. The N-terminal amino acid sequence of the larger protein was 90 % similar to the sequence of the putative β-galactosidase (YP_796339) and the smaller protein was identical to the sequence of the putative β-galactosidase (YP_796338) in L. brevis ATCC367. This suggests the enzyme is a heterodimeric β-galactosidase. The specific activity of the purified enzyme for lactose is 55 U/mg. We speculate inhibition of lactose transport delays the lactose utilization in L. brevis KB290.

Wide range of interacting partners of pea Gβ subunit of G-proteins suggests its multiple functions in cell signalling.

Climate change is a major concern especially in view of the increasing global population and food security. Plant scientists need to look for genetic tools whose appropriate usage can contribute to sustainable food availability. G-proteins have been identified as some of the potential genetic tools that could be useful for protecting plants from various stresses. Heterotrimeric G-proteins consisting of three subunits Gα, Gβ and Gγ are important components of a number of signalling pathways. Their structure and functions are already well studied in animals but their potential in plants is now gaining attention for their role in stress tolerance. Earlier we have reported that over expressing pea Gβ conferred heat tolerance in tobacco plants. Here we report the interacting partners (proteins) of Gβ subunit of Pisum sativum and their putative role in stress and development. Out of 90 transformants isolated from the yeast-two-hybrid (Y2H) screening, seven were chosen for further investigation due to their recurrence in multiple experiments. These interacting partners were confirmed using β-galactosidase colony filter lift and ONPG (O-nitrophenyl-β-D-galactopyranoside) assays. These partners include thioredoxin H, histidine-containing phosphotransfer protein 5-like, pathogenesis-related protein, glucan endo-beta-1, 3-glucosidase (acidic isoform), glycine rich RNA binding protein, cold and drought-regulated protein (corA gene) and soluble inorganic pyrophosphatase 1. This study suggests the role of pea Gβ subunit in stress signal transduction and development pathways owing to its capability to interact with a wide range of proteins of multiple functions.

A β-galactosidase from chick pea (Cicer arietinum) seeds: Its purification, biochemical properties and industrial applications

A β-galactosidase from Cicer arietinum seeds has been purified to apparent electrophoretic homogeneity using a combination of various fractionation and chromatographic techniques, giving a final specific activity of 220 units mg(-1), with approximately 1840 fold purification. Analysis of the protein by SDS-PAGE revealed two subunits with molecular masses of 48 and 38 kDa, respectively. These bands were further confirmed with LC-MS/MS, indicating that Chick pea β-galactosidase (CpGAL) is a heterodimer. Molecular mass was determined to be 85 kDa by Superose-12 FPLC column, which is in agreement with the molecular mass suggested by mass spectroscopy to be 83 kDa. The optimum pH of the enzyme was 2.8 and it hydrolysed o-nitrophenyl β-d galactopyranoside (ONPG) with a K(m) value of 1.73 mM at 37°C. The energy of activation (E(a)) calculated in the range of 35 to 60°C, using Arrhenius equation, was determined to be 11.32 kcal mol(-1). The enzyme could also hydrolyse lactose, with an optimum pH of 4.0 at 40°C. K(m) and E(a) for lactose hydrolysis was found to be 10mM and 10.57 kcal mol(-1), respectively. The enzyme was found to be comparatively thermostable showing maximum activity at 60°C for both ONPG and lactose. Galactose was found to be the competitive inhibitor. β-Galactosidase also exhibited glycoproteineous properties when applied on Con-A Sepharose column. The enzyme was localised in germinated seeds with X-gal activity staining and shown to be expressed prominently at grown radical tip and seed coat. Sequence alignment of CpGAL with other known plant β-galactosidase showed high amino acid sequence homology.