o-nitrophenyl-β-D-galactopyranoside Research Articles

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.

β-Galactosidase-immobilised microreactor fabricated using a novel technique for enzyme immobilisation and its application for continuous synthesis of lactulose

Abstract In this study, several surface functionalisation techniques were used to immobilise β-galactosidase in a microreactor. β-Galactosidase was pretreated with lactose before immobilisation, and functionalised multi-walled carbon nanotubes (MWNTs), DNA-wrapped single-walled carbon nanotubes and glutaraldehyde as a linker were used to immobilise β-galactosidase on a microchannel surface. When functionalised MWNTs were used as a linker for immobilisation of pretreated β-galactosidase, the enzyme microreactor showed the highest performance. The K m(app) and V max were 2.84 mM and 0.52 mM/min, respectively, and the conversion of o -nitrophenyl-β- d -galactopyranoside (ONPG) reached 78.3% during the continuous flow reaction at a flow rate of 2.5 μL/min. In an enzyme microreactor, continuous synthesis of lactulose was performed, and the lactulose concentration was maintained at about 1.29 g/L for 48 h.

Antarctic marine bacterium Pseudoalteromonas sp. KNOUC808 as a source of cold-adapted lactose hydrolyzing enzyme

Psychrophilic bacteria, which grow on lactose as a carbon source, were isolated from Antarctic polar sea water. Among the psychrophilic bacteria isolated, strain KNOUC808 was able to grow on lactose at below 5°C, and showed 0.867 unit of o-nitrophenyl β-D-galactopyranoside(ONPG) hydrolyzing activity at 4°C. The isolate was gram-negative, rod, aerobic, catalase positive and oxidase positive. Optimum growth was done at 20°C, pH 6.8-7.2. The composition of major fatty acids in cell of KNOUC801 was C12:0 (5.48%), C12:0 3OH (9.21%), C16:0 (41.83%), C17:0 ω8 (7.24%) and C18:1 ω7 (7.04%). All these results together suggest that it is affiliated with Pseudoalteromonas genus. The 16S rDNA sequence corroborate the phenotypic tests and the novel strain was designated as Pseudoalteromonas sp. KNOUC808. The optimum temperature and pH for lactose hydrolyzing enzyme was 20°C and 7.8, respectively. The enzyme was stable at 4°C for 7 days, but its activity decreased to about 50% of initial activity at 37°C in 7 days.

Isolation and characterization of a psychrophilic bacterium producing cold active lactose hydrolyzing enzyme from soil of Mt. Himalaya in Nepal

Psychrophilic bacteria, which grew on lactose as a carbon source, were isolated from soil of Island Peak area at Mt. Himalaya in Nepal, and identified. 148 bacterial isolates were isolated via X-gal hydrolysis on plate. Among these, three isolates, referred to as KNOUC 401, 402 and 403, displayed good cold-adapted growth and activity for hydrolysis of o-nitrophenyl-β-D-galactopyranoside (ONPG) and lactose in cell free extracts. Strain KNOUC403 showing negative activity in β-hemolysis was selected and identified. The strain was gram-positive bacilli, non-motile, catalase-positive and aerobic. Optimum growth was done at 20°C and pH 6.5~6.8. Anteiso-C15:0 (68.14%) was predominant in the fatty acid composition of cell. The physiological and biochemical properties, cellular fatty acid composition and sequence of 16S rDNA, suggest that strain KNOUC403 indicate a taxonomic affiliation to Arthrobacter psychrolactophilus. The optimal conditions for ONPG hydrolysis of cell free extracts from the strain were 15°C and pH 6.0, and the enzyme was stable at 4°C for 42 h. Zymogram assay of cell free extract from the strain in nondenaturing polyacrylamide gel showed a distinct band of X-gal hydrolysis. Key words: Psychrophilic bacteria, Arthrobacter psychrolactophilus, cold-active lactose hydrolyzing enzyme, Mt. Himalaya.

Detection of Liposome Lysis Utilizing an Enzyme–Substrate System

A novel optical reporter system was developed to verify encapsulation and subsequent release of a foreign molecule in liposomes. The protocol utilizes a single enzyme and substrate. We encapsulate o-nitrophenyl-β,D: -galactopyranoside (ONPG) and measure its release by detecting the levels of o-nitrophenol created when the encapsulated ONPG is released and hydrolyzed by β-galactosidase. Using this method, liposome formation and subsequent lysis with Triton X-100 were verified. This new protocol eliminates the complications of multiple reaction enzyme detection methods, along with the chance for false negatives and unreliable data seen when using fluorescent particles as reporters.

Antarctic marine bacterium Pseudoalteromonas sp. KNOUC808 as a source of cold-adapted lactose hydrolyzing enzyme

Psychrophilic bacteria, which grow on lactose as a carbon source, were isolated from Antarctic polar sea water. Among the psychrophilic bacteria isolated, strain KNOUC808 was able to grow on lactose at below 5°C, and showed 0.867 unit of o-nitrophenyl β-D-galactopyranoside(ONPG) hydrolyzing activity at 4°C. The isolate was gram-negative, rod, aerobic, catalase positive and oxidase positive. Optimum growth was done at 20°C, pH 6.8–7.2. The composition of major fatty acids in cell of KNOUC801 was C12:0 (5.48%), C12:0 3OH (9.21%), C16:0 (41.83%), C17:0 ω8 (7.24%) and C18:1 ω7 (7.04%). All these results together suggest that it is affiliated with Pseudoalteromonas genus. The 16S rDNA sequence corroborate the phenotypic tests and the novel strain was designated as Pseudoalteromonas sp. KNOUC808. The optimum temperature and pH for lactose hydrolyzing enzyme was 20°C and 7.8, respectively. The enzyme was stable at 4°C for 7 days, but its activity decreased to about 50% of initial activity at 37°C in 7 days.

Effect of methyl methanesulfonate on hsp70 expression and tissue damage in the third instar larvae of transgenic Drosophila melanogaster (hsp70-lacZ) Bg.

Methyl methanesulfonate (MMS) is an anti-carcinogenic drug and its toxicity has been reported in various experimental models. The hsp70s are a family of ubiquitously expressed heat shock proteins. In the recent years, hsp70 has been considered to be one of the candidate genes for predicting cytotoxicity against environmental chemicals. Nowadays emphasis is given to the use of alternatives to mammals in testing, research and education. The European Centre for the Validation of Alternative Methods (EVCAM) has recommended the use of Drosophila as an alternative model for scientific studies. Almost all living organisms possess proteins with a similar structure to that of hsp70s. In the present study, the toxicity of MMS was evaluated by quantifying hsp70 expression and tissue damage in the third instar larvae of transgenic Drosophila melanogaster (hsp70-lacZ) Bg 9, at different doses and hours of exposure. We studied the effect of 0.25, 0.50, 0.75 and 1.0 µl/ml of MMS at 2, 4, 24 and 48 hours of exposure on hsp70 expression by using the soluble O-nitrophenyl-β-D-galactopyranoside (ONPG) assay and on establishing the tissue damage by the Trypan blue exclusion assay in the third instar larvae of transgenic Drosophila melanogaster (hsp70-lacZ) Bg 9. A dose-dependent increase in the expression of hsp70 was observed at 0.25, 0.50, and 0.75 µl/ml of MMS compared to the control. At the highest dose, i.e. 1.0 µl/ml of MMS, the activity of hsp70 was decreased due to tissue damage.

Selective testing of β-galactosidase activity in the laboratory identification of Salmonella and Shigella species

BackgroundSalmonella and Shigella species are pathogens of great medical and public health importance. However, laboratory identification of these organisms is time consuming. Using current standard laboratory algorithms, the vast majority...

ChemInform Abstract: Enzymatic Synthesis of ω-Hydroxyalkyl and n-Alkyl β-D- Galactopyranosides by the Transglycosylation Reaction of β- Galactosidase.

ω-Hydroxyalkyl and n-alkyl β-D-galactopyranosides were directly prepared in high yield from 1,8-octanediol, 1,10-decanediol, 1-octanol and 1-decanol by the transglycosylation reaction of o-nitrophenyl β-D-galactopyranoside (ONPG) or lactose using β-galactosidase.

β-Galactosidase Assay

When a transient or stable transfection assay is developed for a promoter, a primary objective is to quantify promoter strength. Because transfection efficiency in such assays can be low, promoters are commonly fused to heterologous reporter genes that encode enzymes that can be quantified using highly sensitive assays. The reporter protein's activity or fluorescence within a transfected cell population is approximately proportional to the steady-state mRNA level. Although the Escherichia coli lacZ gene, encoding beta-galactosidase (beta-gal), can be used as a standard reporter for monitoring the strength of a promoter or enhancer in a transient or stable transfection assay, it is predominantly used as an internal control during transient transfection experiments. When used in this manner, cells are usually transfected with the control plasmid (containing a ubiquitously active viral promoter fused to the E. coli lacZ gene) and an experimental plasmid containing another reporter gene (e.g., luciferase or chloramphenicol acetyltransferase [CAT]) under the control of the promoter or enhancer of interest. The basic colorimetric assay described here is the simplest and least expensive assay for quantifying beta-gal activity. The cells are lysed and, after determining the total protein concentration in the extracts, an aliquot of the extract is mixed with the reaction substrate, O-nitrophenyl-beta-D-galactopyranoside (ONPG), in a buffer containing sodium phosphate and magnesium chloride. When the yellow product becomes visible, the optical densities of the samples are determined spectrophotometrically.

Neisseria lactamica Arthritis and Septicemia Complicating Myeloma

Neisseria lactamica is a common and normally harmless commensal of the upper respiratory tract found especially in young children. In the July 2006 edition of this journal, Zavascki and colleagues reported the first case of N. lactamica causing cavitatory lung disease in an adult organ transplant recipient (3). The same year, Wang et al. reported a case of N. lactamica bacteremic pneumonia in an adult with liver cirrhosis (2). Apart from a 1991 report of N. lactamica meningitis following skull trauma (1), there are no other published reports of invasive N. lactamica infection in adults or cases listed in selected bacteremia databases from New Zealand, Australia, or North America. We present a case of N. lactamica arthritis and septicemia in a patient immune suppressed by myeloma and corticosteroids. This 60-year-old male from a small mining city in the West Australian outback presented with 1 day of rigors, hypotension, and acute knee pain and swelling. He had been prescribed prednisolone at 50 mg daily, reduced to 15 mg daily, for an infective exacerbation of chronic obstructive airways disease during the 17 days before admission. The myeloma was associated with an IgG kappa-type paraprotein, mild suppression of serum IgA and IgM, and lytic bony lesions. HIV serology was negative. The cloudy knee fluid aspirate had neutrophilic pleocytosis (100 white cells/mm3) and Gram-negative cocci by microscopy. Culture of both joint fluid and blood yielded a Neisseria species, provisionally identified as Neisseria gonorrhoeae. This caused distress for the patient and his wife, who denied sexual activity for 5 years. The isolate was oxidase and catalase positive but Superoxol and Phadebact Monoclonal GC test (Bactus AB, Huddinge, Sweden) negative. API NH (bioMerieux, La Balme-les-Grottes, France) identified N. lactamica (99.9%, profile 5041). BLAST analysis (http://www.ncbi.nlm.nih.gov/blast/) of an amplified 2,098-nucleotide 16S rRNA sequence (GenBank accession no. {type:entrez-nucleotide,attrs:{text:GU980596,term_id:294874967,term_text:GU980596}}GU980596) showed 98% homology with both N. lactamica (GenBank accession no. {type:entrez-nucleotide,attrs:{text:AJ239305.1,term_id:5327203,term_text:AJ239305.1}}AJ239305.1) and Neisseria polysaccharea (GenBank accession no. {type:entrez-nucleotide,attrs:{text:AJ239314.1,term_id:5327212,term_text:AJ239314.1}}AJ239314.1). The isolate was o-nitrophenyl-β-d-galactopyranoside (ONPG) positive and generated acid from sucrose, excluding N. polysaccharea. Our isolate was thus identified biochemically and genetically as N. lactamica but may represent a new virulent strain. The penicillin MIC was 3 mg/liter, and the ceftriaxone MIC was 0.006 mg/liter, both determined by Etest. An echocardiogram showed no endocarditis, and the neisserial infection was cured with 11 days of ceftriaxone therapy. It should not be a surprise that N. lactamica causes septic arthritis, as there are case reports of septic arthritis, discitis, or bursitis caused by other commensal Neisseria spp., including N. sicca, N. subflava, and N. mucosa. Neisseria meningitidis (especially serogroup W-135) and N. gonorrhoeae also cause septic arthritis. Commensal neisserial infections have often been reported in compromised patients, including those who are neutropenic, have invasive devices, or have undergone procedures. Meningococcal infections (especially nonpathogenic strains) have also been reported in compromised patients, including those with complement deficiency, prior corticosteroid use, myeloma, Waldenstrom's disease, and hypogammaglobulinemia. Like the case presented by Zavascki et al. (3), our patient represents a rare example of invasive adult N. lactamica infection and highlights the propensity for commensal neisserial species to cause bloodstream and joint infections and to be of special concern to patients with immune compromises. We encourage others isolating clinically significant N. lactamica strains to deposit the sequences in GenBank to enable further study of this emerging pathogen in compromised patients.

Study on b-galactosidase enzyme produced by isolated lactobacilli from milk and cheese

β-galactosidases enzyme have been used in the dairy industry for the improvement of lactose intolerance. The aim of this study was to detect b-galactosidase enzyme produced by isolated lactobacilli from milk and cheese. Isolated lactobacilli were cultured on MRS agar. Lactobacilli were identified by Gram stain and standard bacteriological and biochemical methods. Their ability to hydrolyze 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) and O-nitrophenyl-β-D-galactopyranoside (ONPG) was determined. A protein band of indicated b-galactosidase enzyme was also detected by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) method. The colonies that produced green color on X-Gal plates were lactobacilli with b-galactosidase enzyme which had ONPG positive results. The highest enzymatic value (1,966 U/ml) was observed in one strain of Lactobacillus delbrueckii. A 116 kDa protein band was detected in some strains (37%) with highest enzyme value and in others (63%), protein band was weak by SDS-PAGE method. By adding Lactobacilli producing b-galactosidase enzyme as probiotic to dairy products, could help lactose intolerant people. Key words: Lactobacillus, β-galactosidase, X-Gal, ONPG, SDS-PAGE.

Encapsulation of β-galactosidase from Aspergillus oryzae based on “fish-in-net” approach with molecular imprinting technique

By using tetraethylorthosilicate as a silica resource and triblock copolymer P123 as a template, the encapsulations of β-galactosidase with three different models of without protection, protection of protective agent and molecular imprinting technique pretreatment were accomplished through modified “fish-in-net” route at pH 5.0. The highest enzymatic activity of β-galactosidase was gained by using pretreatment of molecular imprinting technique. Scanning electron microscopy (SEM) images showed that the matrix of encapsulated β-galactosidase was made of an aggregation of uniform microspheres of 200–300 nm, and N 2 adsorption/desorption isotherms demonstrated that the matrix of encapsulated β-galactosidase possessed average Brunauer–Emmett–Teller (BET) pore size of 27 Å and narrow pore size distribution. More importantly, compared with encapsulated β-galactosidase without protection, the hydrolytic activity of encapsulated β-galactosidase pretreated by molecular imprinting technique was about 3 times and 1.8 times, while the enzymatic activity of encapsulated β-galactosidase with the protection of protective agent increased only 1.3-fold when lactose and o-nitrophenyl-β- d-galactopyranoside (ONPG) were used as substrates, respectively. The protective effect of molecular imprinting technique pretreatment on the enzymatic activity after encapsulation was better than that by protective agent.

Immobilization of β-galactosidase onto Sepharose and stabilization in room temperature ionic liquids

The hydrolysis of o-nitrophenyl-β- d-galactopyranoside (ONPG) by β-galactosidase immobilized on Sepharose CL-4B ® was investigated in five different ionic liquids (ILs), 1-butyl-3-methylimidazolium X −; [ X = CF 3SO 3 −, BF 4 −, PF 6 −, CH 3SO 4 −and N(CN) 2 −]. Michaelis–Menten kinetic studies were conducted in phosphate buffer and in the five ionic liquids. For the immobilized enzyme in the ILs, the K m values were lower (0.36–1.2 mmol ONPG) while the V max values were higher (0.04–0.008 min − 1 ) compared to those in aqueous phosphate buffer suggesting a marked increase in the efficiency of the immobilized enzyme in the ionic liquid. For the free enzyme in the ionic liquids, the K m values, in general, were larger (0.45–4.96 mmol ONPG) than those of the immobilized enzyme in the ionic liquid. A postulated mechanism for the hydrolysis is suggested, involving interception of the intermediate oxonium ion species by the counter ion of the ionic liquid, thereby enabling the hydrolysis to occur at a faster rate.

Synthesis and biological evaluation of 11C-labeled β-galactosyl triazoles as potential PET tracers for in vivo LacZ reporter gene imaging

In our aim to develop LacZ reporter probes with a good retention in LacZ expressing cells, we report the synthesis and preliminary evaluation of two carbon-11 labeled β-galactosyl triazoles 1-(β- d-galactopyranosyl)-4-( p-[ 11C]methoxyphenyl)-1,2,3-triazole ([ 11C]- 6) and 1-(β- d-galactopyranosyl)-4-(6-[ 11C]methoxynaphthyl)-1,2,3-triazole ([ 11C]- 13). The precursors for the radiolabeling and the non-radioactive analogues ( 6 and 13) were synthesized using straightforward ‘click’ chemistry. In vitro incubation experiments of 6 with β-galactosidase in the presence of o-nitrophenyl β- d-galactopyranoside (ONPG) showed that the triazolic compound was an inhibitor of β-galactosidase activity. Radiolabeling of both precursors was performed using [ 11C]methyl iodide as alkylating agent at 70 °C in DMF in the presence of a small amount of base. The log P values were −0.1 and 1.4, respectively, for [ 11C]- 6 and [ 11C]- 13, the latter therefore being a good candidate for increased cellular uptake via passive diffusion. Biodistribution studies in normal mice showed a good clearance from blood for both tracers. [ 11C]- 6 was mainly cleared via the renal pathway, while the more lipophilic [ 11C]- 13 was excreted almost exclusively via the hepatobiliary system. Despite the lipophilicity of [ 11C]- 13, no brain uptake was observed. Reversed phase HPLC analysis of murine plasma and urine revealed high in vivo stability for both tracers. In vitro evaluation in HEK-293T cells showed an increased cell uptake for the more lipophilic [ 11C]- 13, however, there was no statistically higher uptake in LacZ expressing cells compared to control cells.

β-Galactosidase activity assay using far-red-shifted fluorescent substrate DDAOG

β-Galactosidase (β-gal) is commonly used as a reporter gene in biological research, and a wide variety of substrates have been developed to assay its activity. One substrate, 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) β- d-galactopyranoside (DDAOG), can be cleaved by β-gal to produce 7-hydroxy-9H(I,3-dichloro-9,9-dimethylacridin-2-one) (DDAO). On excitation, DDAO generates a far-red-shifted fluorescent signal. Using this substrate, we developed a β-gal activity assay method. The DDAO signal was stable for at least 18 h. The signal intensity was linearly related to both the enzyme amount and substrate concentration. An optimized buffer for the β-gal/DDAOG assay was also formulated. When compared with the colorimetric substrate o-nitrophenyl-β- d-galactopyranoside (ONPG), the signal-to-background ratio of the DDAOG method was approximately 12-fold higher. The β-gal/DDAOG assay method was also tested in transiently transfected cells employing both pharmacologically and genetically inducible gene expression systems. The ability to detect signal induction is comparable to a similar assay using luciferase as the signal generating moiety. The β-gal/DDAOG assay method should provide a fluorescent reporter assay system for the wide variety of β-gal systems currently in use.

DETECTION OF MICROCYSTINS BASED ON YEAST TWO-HYBRID SYSTEM

Microcystins(MCs) are secondary metabolites produced by toxic cyanobacteria,which belong to the family of monocyclic heptapeptides with molecular weight about 1000Da.MCs are claimed to possess a variety of toxic effects and considered as a strong tumor inducer.Up to now,the simpler and easier MCs detection method has not been fully established.The present study aims to establish a novel MCs detection methodology by using the principle of yeast two-hybrid system.In previous study,several polypeptides interacting with MCs had been identified from the random 12-mer peptide phage displayed library and 7-mer peptide phage displayed library.We chose three polypeptides with high affinity for MCs and cloned them into the yeast two-hybrid system.Firstly,DNA fragments encoding for those polypeptides were constructed into the yeast two hybrid expression vectors harboring Active Domain or Biding Domain,then the recombinant plasmids were co-transfected into yeast strain AH109.Theβ-Gal reporter's activity could be easily measured by using ONPG(O-nitrophenyl-β-D-galactopyranoside) as substrate and the change in β-Gal reporter's activity represented the presence of MCs in culture medium,thus this paper provided a more convenient approach to detect MCs.

Synthesis and Evaluation of 18F- and 11C-Labeled Phenyl-Galactopyranosides as Potential Probes for in Vivo Visualization of LacZ Gene Expression using Positron Emission Tomography

3-Hydroxy-2-nitrophenyl 2,3,4,6-tetra-O-acetyl-beta-D-galactopyranoside, a derivative of the chromogenic beta-galactosidase (beta-gal) substrate o-nitrophenyl beta-D-galactopyranoside (ONPG) was synthesized using a Koenigs-Knorr glycosylation reaction. It was alkylated with 2-[(18)F]fluoroethyl triflate or [(11)C]methyl triflate, followed by deacetylation of the sugar hydroxyl groups to obtain radiolabeled 3-(2'-[(18)F]fluoroethoxy)-2-nitrophenyl beta-D-galactopyranoside ([(18)F]-2c) and 3-[(11)C]methoxy-2-nitrophenyl beta- d-galactopyranoside ([(11)C]-3c), which were evaluated as potential reporter probes for in vivo visualization of LacZ gene expression with positron emission tomography (PET). In vitro, [(18)F]- 2c and [(11)C]-3c were good substrates of beta-gal and showed, respectively, a 7.5- and 2.5-fold higher uptake into beta-gal expressing cells (LacZ cells) compared to control cells. However, reversed-phase HPLC analysis of the LacZ cell lysate and supernatant showed that labeled 3-(2'-[(18)F]fluoroethoxy)-2-nitrophenol, the hydrolysis product formed by beta-gal-mediated cleavage of [(18)F]-2c, substantially leaked out of the cells, which would lead to loss of PET signal. In a microPET study of [(18)F]-2c in a mouse with a beta-gal expressing tumor, high retention was observed in liver and kidneys, but only negligible accumulation was seen in the tumor. As a general conclusion, it can be stated that the synthesized PET tracers [ (18)F]-2c and [(11)C]-3c are not suitable for use as LacZ reporter probes. Further structural modifications to improve the diffusion over the tumor cell membrane and to increase retention in beta-gal expressing cells may lead to more favorable in vivo imaging probes.

Molecular Cloning and Characterization of the Gene Encoding Cold-Active β-Galactosidase from a Psychrotrophic and Halotolerant Planococcus sp. L4

The gene bgaP encoding cold-active beta-galactosidase from a psychrotrophic and halotolerant Planococcus sp. L4 was cloned into Escherichia coli and sequenced. A sequence analysis of the DNA responsible for the BgaP gene revealed an open reading frame of 2031 bp encoding for a protein of 677 amino acid residues. The BgaP was heterologously expressed in E. coli and purified followed by Ni2+ affinity chromatography. The molecular mass of the native enzyme was approximately 156 kDa as determined by gel filtration. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the deduced amino acid sequence of the BgaP indicated molecular masses of 78 and 77.311 kDa, respectively, suggesting that the BgaP is a dimer. The purified BgaP had an isoelectric point of 4.8 and exhibited maximal activity at 20 degrees C and pH 6.8 under the assay conditions used. The enzyme is particularly thermolabile, losing all activity in only 10 min at 45 degrees C. It was able to hydrolyze lactose as a substrate, as well as o-nitrophenyl-beta-D-galactopyranoside (ONPG); the Km values with ONPG and lactose were calculated to be 5.4 and 20.4 mM at 5 degrees C, respectively. The catalytic efficiencies of BagP for lactose at 5 and 20 degrees C had 14 and 47 times more than that of E. coli beta-galactosidase at 20 degrees C, respectively. Therefore, cold-active beta-galactosidase from the psychrotrophic and halotolerant Planococcus sp. L4 could conceivably be developed to fulfill the practical requirements to enable its use for lactose removal in milk and dairy products at low temperature or a reporter enzyme for psychrophilic genetic systems.

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

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. Km and Vmax 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.