Cold-adapted Β-galactosidase Research Articles

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.

Cloning, Expression and Characterization of a Novel Cold-adapted β-galactosidase from the Deep-sea Bacterium Alteromonas sp. ML52.

The bacterium Alteromonas sp. ML52, isolated from deep-sea water, was found to synthesize an intracellular cold-adapted β-galactosidase. A novel β-galactosidase gene from strain ML52, encoding 1058 amino acids residues, was cloned and expressed in Escherichia coli. The enzyme belongs to glycoside hydrolase family 2 and is active as a homotetrameric protein. The recombinant enzyme had maximum activity at 35 °C and pH 8 with a low thermal stability over 30 °C. The enzyme also exhibited a Km of 0.14 mM, a Vmax of 464.7 U/mg and a kcat of 3688.1 S−1 at 35 °C with 2-nitrophenyl-β-d-galactopyranoside as a substrate. Hydrolysis of lactose assay, performed using milk, indicated that over 90% lactose in milk was hydrolyzed after incubation for 5 h at 25 °C or 24 h at 4 °C and 10 °C, respectively. These properties suggest that recombinant Alteromonas sp. ML52 β-galactosidase is a potential biocatalyst for the lactose-reduced dairy industry.

Functional Characterization of a New Cold-Adapted β-Galactosidase from an Arctic Fjord Sediment Bacteria Enterobacter ludwigii MCC 3423

In the present study, a new cold-adapted β-galactosidase, BgalEL isolated from the fjord sediment bacteria, Enterobacter ludwigii MCC 3423, was purified and characterized. The phylogenetic analysis of partial sequence of bgalEL gene revealed 99% relatedness of the enzyme to Enterobacter cloacae β-galactosidase. BgalEL is a homotetramer with molecular weight 465 kDa composed of ~ 116.42 kDa subunits. The optimal pH range and temperature for maximum hydrolytic activity on ONPG were 7.0–8.0 and 45 °C respectively. BgalEL was stable at pH ranges 6.0–8.0. The Km and Vmax for BgalEL were recorded as 2.03 mM and 49.3 U mg−1 respectively. The product formation by BgalEL was modelled with various degradation kinetic models and zeroth was found as model was best-fitted model. The kinetic half-life period of ONPG was noted as 9.0 × 10−4. The presence of K+, Mg2+ and Mn2+ at 10 mM concentrations stimulated BgalEL activity by 31 ± 3.9, 34 ± 1.2 and 42 ± 1.9% respectively. It was interesting to see that BgalEL was least affected by the hydrolytic products, glucose and galactose. Noteworthy, effect of Ca2+ ions on BgalEL activity was negligible at concentrations of 10 mM. BgalEL hydrolyzed lactose in milk at refrigerated temperature and also displayed transglycosylation potential in the presence of substantial concentrations of lactose at 40 °C. Finally, this is the first report with such a detailed characterization of β-galactosidase enzyme from a gram-negative bacterium isolated from the Arctic region. In light of the above mentioned properties, we thus recommend the enzyme BgalEL for the large scale production lactose free milk for the safe consumption in lactose-intolerance and the prebiotic galactooligosaccharides as supplement for infant food formulas.

Purification, characterization, and gene cloning of a new cold-adapted β-galactosidase from Erwinia sp. E602 isolated in northeast China

β-Galactosidases are widely used in industry for elimination of lactose from milk products. A new β-galactosidase was obtained from bacterial strain Erwinia sp. E602, newly isolated in northeast China. The enzyme was purified with the methods of ammonium sulfate fractionation, ion exchange, and gel filtration chromatography for further study of the enzymatic characteristics. The purified enzyme had a molecular weight of near 110 kDa. The optimum reaction temperature and pH of this enzyme was determined to be 40°C and 7.0, respectively, indicating that this enzyme was a mesophilic neutral β-galactosidase. Furthermore, the enzyme retained near 10% of the activity at 0°C, which also suggested its cold-adapted property. Kinetics of the β-galactosidase was studied, and the Km (Michaelis constant) and Vmax (maximum enzymatic reaction rate) of this enzyme were 0.21 mmol/L and 263.16 µmol/mg per minute, respectively. The effects of metal ions on the enzymatic activity and the lactose hydrolysis efficiency in milk, as well as its trans-glycosylation activity, were studied in this work. The β-galactosidase coding gene was cloned to be a 3-kb length fragment, which shared at most 81% of identity with the published sequences in NCBI Blast database (https://blast.ncbi.nlm.nih.gov). Results in this work suggested it is a new β-galactosidase and it has potential to be used in dairy and food processing.

Preparation and characterization of gellan gum microspheres containing a cold-adapted β-galactosidase from Rahnella sp. R3

R-β-Gal is a cold-adapted β-galactosidase that is able to hydrolyze lactose and has the potential to produce low-lactose or lactose-free dairy products at low temperatures (4°C). Cold-adapted enzymes unfold at moderate temperatures due to the lower intramolecular stabilizing interactions necessary for flexibility at low temperatures. To increase stability and usage-performance, R-β-Gal was encapsulated in gellan gum by injecting an aqueous solution into two different hardening solutions (10mM CaCl2 or 10mM MgCl2). Enzyme characteristics of both free and encapsulated R-β-Gal were carried out, and the different effects of two cations were investigated. R-β-Gal showed better thermal and pH stability after encapsulation. Ca2+ gels had higher encapsulation efficiency (71.4%) than Mg2+ (66.7%) gels, and Ca2+ formed larger inner and surface pores. R-β-Gal was released from the Ca2+ hydrogel beads more rapidly than the Mg2+ hydrogels during storage in aqueous solution due to the larger inner/surface pores of the matrix.

Construction of recombinant Pichia pastoris strains producing cold-adapted β-galactosidase from Paracoccus sp. 32d

Construction of recombinant Pichia pastoris strains producing cold-adapted β-galactosidase from Paracoccus sp. 32d

Structure analysis of a glycosides hydrolase family 42 cold-adapted β-galactosidase from Rahnella sp. R3

The β-galactosidase isolated from a psychrotrophic bacterium, Rahnella sp. R3 (R-β-Gal), exhibits high activity at low temperature and has potential in the dairy industry.

Cloning, expression and structural stability of a cold-adapted β-galactosidase from Rahnella sp. R3

A novel gene was isolated for the first time from a psychrophilic gram-negative bacterium Rahnella sp. R3. The gene encoded a cold-adapted β-galactosidase (R-β-Gal). Recombinant R-β-Gal was expressed in Escherichia coli BL21 (DE3), purified and characterized. R-β-gal belongs to the glycosyl hydrolase family 42. Circular dichroism spectrometry of the structural stability of R-β-Gal with respect to temperature indicated that the secondary structures of the enzyme were stable to 45°C. In solution, the enzyme was a homo-trimer and was active at temperatures as low as 4°C. The enzyme did not require the presence of metal ions to be active, but Mg2+, Mn2+, and Ca2+ enhanced its activity slightly, whereas Fe3+, Zn2+ and Al3+ appeared to inactive it. The purified enzyme displayed Km values of 6.5mM for ONPG and 2.2mM for lactose at 4°C. These values were lower than the corresponding Kms reported for other cold-adapted β-Gals.

Stepwise optimization of a low-temperature Bacillus subtilis expression system for "difficult to express" proteins.

In order to improve the overproduction of "difficult to express" proteins, a low-temperature expression system for Bacillus subtilis based on the cold-inducible promoter of the desaturase-encoding des gene was constructed. Selected regulatory DNA sequence elements from B. subtilis genes known to be cold-inducible were fused to different model genes. It could be demonstrated that these regulatory elements are able to mediate increased heterologous gene expression, either by improved translation efficiency or by higher messenger RNA (mRNA) stability. In case of a cold-adapted β-galactosidase from Pseudoalteromonas haloplanktis TAE79A serving as the model, significantly higher expression was achieved by fusing its coding sequence to the so-called "downstream box" sequence of cspB encoding the major B. subtilis cold-shock protein. The combination of this fusion with a cspB 5'-UTR stem-loop structure resulted in further enhancement of the β-galactosidase expression. In addition, integration of the transcription terminator of the B. subtilis cold-inducible bkd operon downstream of the target genes caused a higher mRNA stability and enabled thus a further significant increase in expression. Finally, the fully optimized expression system was validated by overproducing a B. subtilis xylanase as well as an α-glucosidase from Saccharomyces cerevisiae, the latter known for tending to form inclusion bodies. These analyses verified the applicability of the engineered expression system for extracellular and intracellular protein synthesis in B. subtilis, thereby confirming the suitability of this host organism for the overproduction of critical, poorly soluble proteins.

A novel cold-adapted β-galactosidase isolated from Halomonas sp. S62: gene cloning, purification and enzymatic characterization

A novel 1,170 bp β-galactosidase gene sequence from Halomonas sp. S62 (BGalH) was identified through whole genome sequencing and was submitted to GenBank (Accession No. JQ337961). The BGalH gene was heterologously expressed in Escherichia coli BL21(DE3) cells, and the enzymatic properties of recombinant BGalH were studied. According to the polyacrylamide gel electrophoresis results and the sequence alignment analysis, BGalH is a dimeric protein and cannot be classified into one of the known β-galactosidase families (GH1, GH2, GH35, GH42). The optimal pH and temperature were determined to be 7.0 and 45 °C, respectively; the K m and K cat were 2.9 mM and 390.3 s(-1), respectively, for the reaction with the substrate ortho-nitrophenyl-β-D-galactopyranoside. At 0-20 °C, BGalH exhibited 50-70 % activity relative to its activity under the optimal conditions. BGalH was stable over a wide range of pHs (6.0-8.5) after a 1 h incubation (>93 % relative activity) and was thermostable at 50 °C and below (>60 % relative activity). The enzyme hydrolyzes lactose completely in milk over 24 h at 7 °C. The characteristics of this novel β-galactosidase suggest that BGalH may be a good candidate for medical researches and food industry applications.

Antarctic, cold-adapted β-galactosidase of Pseudoalteromonas sp. 22b as an effective tool for alkyl galactopyranosides synthesis

The antarctic Pseudoalteromonas sp. 22b β-galactosidase was found to catalyze synthesis of alkyl galactopyranosides. The number of carbon atoms in C3–C6 alcohol molecules only slightly affected the yield of products. Reactions of transgalactosylation were conducted for 80 h and the maximum accumulation of their products was observed between 40 and 50 h. The highest and almost the same yields of alkyl galactosides were achieved at pH 6–9 and 10–30% water concentration. Like known mesophilic β-galactosidases, the antarctic enzyme more efficiently synthesized alkyl galactosides when reactions were carried out in mixtures of buffers and organic solvents (below 50%, v/v).

Molecular modelling of a psychrophilic β-galactosidase

An Antarctic strain of bacteria was isolated from the digestive tract of the crustacean Thysanoessa macrura and classified as Pseudoalteromonas sp. 22b based on 16SrRNA gene sequence and physiological as well as biochemical properties. This bacterium turned out to be a good producer of a cold-adapted β-galactosidase. The enzyme displays high catalytic and molecular adaptation to low temperatures. Here we present a homology model of the psychrophilic β-galactosidase based on the structural template of the mesophilic β-galactosidase from Escherichia coli (PDB code: 1JZ7, resolution 1.5 Å). Our aim was to identify and characterize potential cold-adaptational features of the target psychrophilic β-galactosidase at the level of the three-dimensional structure rather than solely from the analysis of the amino acid sequence. We report the results of comparisons between the psychrophilic and mesophilic β-galactosidases and point out similarities and differences in the catalytic site and in other parts of the structure. The model allowed us to pinpoint a number of characteristics that are frequently observed in psychrophilic enzymes and allowed interpretation of the results of immunochemical and biochemical analyses.

Cloning, expression, and purification of a recombinant cold-adapted β-galactosidase from antarctic bacterium Pseudoalteromonas sp. 22b

The gram-negative antarctic bacterium Pseudoalteromonas sp. 22b, isolated from the alimentary tract of krill Thyssanoessa macrura, synthesizes an intracellular cold-adapted β-galactosidase. The gene encoding this β-galactosidase has been PCR amplified, cloned, expressed in Escherichia coli, purified, and characterized. The enzyme is active as a homotetrameric protein, and each monomer consists of 1028 amino acid residues. The enzyme was purified to homogeneity (50% recovery of activity) by using the fast, two-step procedure, including affinity chromatography on PABTG–Sepharose. Enzymatic properties of the recombinant protein are identical to those of native Pseudoalteromonas sp. 22b β-galactosidase. The enzyme is cold-adapted and at 10 °C retains 20% of maximum activity. The purified enzyme displayed maximum activity close to 40 °C and at pH of 6.0–8.0. PNPG was its preferred substrate (58% higher activity than against ONPG). The enzyme was particularly thermolabile, losing all activities within 10 min at 50 °C. The hydrolysis of lactose in a milk assay revealed that 90% of milk lactose was hydrolyzed during 6 h at 30 °C and during 28 h at 15 °C. Because of its attributes, the recombinant Pseudoalteromonas sp. 22b β-galactosidase could be applied at refrigeration temperatures for production of lactose-reduced dairy products.

Antarctic marine bacterium Pseudoalteromonas sp. 22b as a source of cold-adapted β-galactosidase

The marine, psychrotolerant, rod-shaped and Gram-negative bacterium 22b (the best of 41 β-galactosidase producers out of 107 Antarctic strains subjected to screening), classified as Pseudoalteromonas sp. based on 16S rRNA gene sequence, isolated from the alimentary tract of Antarctic krill Thyssanoessa macrura, synthesizes an intracellular cold-adapted β-galactosidase, which efficiently hydrolyzes lactose at 0–20 °C, as indicated by its specific activity of 21–67 U mg −1 of protein (11–35% of maximum activity) in this temperature range, as well as k cat of 157 s −1, and k cat/K m of 47.5 mM −1 s −1 at 20 °C. The maximum enzyme synthesis (lactose as a sufficient inducer) was observed at 6 °C, thus below the optimum growth temperature of the bacterium (15 °C). The enzyme extracted from cells was purified to homogeneity (25% recovery) by using the fast, three-step procedure, including affinity chromatography on PABTG-Sepharose. The enzyme is a tetramer composed of roughly 115 kDa subunits. It is maximally active at 40 °C (190 U mg −1 of protein) and pH 6.0–8.0. PNPG is its preferred substrate (50% higher activity than against ONPG). The Pseudoalteromonas sp. 22b β-galactosidase is activated by thiol compounds (70% rise in activity in the presence of 10 mM dithiotreitol), some metal ions (K +, Na +, Mn 2+—40% increase, Mg 2+—15% enhancement), and markedly inactivated by pCMB and heavy metal ions, particularly Cu 2+. Noteworthy, Ca 2+ ions do not affect the enzyme activity, and the homogeneous protein is stable at 4 °C for at least 30 days without any stabilizers.