A novel cold-active β-D-galactosidase from the Paracoccus sp. 32d - gene cloning, purification and characterization

<p>Abstract</p> <p>Background</p> <p>β-<smcaps>D</smcaps>-Galactosidases (EC 3.2.1.23) catalyze the hydrolysis of terminal non-reducing β-<smcaps>D</smcaps>-galactose residues in β-<smcaps>D</smcaps>-galactosides. Cold-active β-<smcaps>D</smcaps>-galactosidases have recently become a focus of attention of researchers and dairy product manufactures owing to theirs ability to: (i) eliminate of lactose from refrigerated milk for people afflicted with lactose intolerance, (ii) convert lactose to glucose and galactose which increase the sweetness of milk and decreases its hydroscopicity, and (iii) eliminate lactose from dairy industry pollutants associated with environmental problems. Moreover, in contrast to commercially available mesophilic β-<smcaps>D</smcaps>-galactosidase from <it>Kluyveromyces lactis </it>the cold-active counterparts could make it possible both to reduce the risk of mesophiles contamination and save energy during the industrial process connected with lactose hydrolysis.</p> <p>Results</p> <p>A genomic DNA library was constructed from soil bacterium <it>Paracoccus </it>sp. 32d. Through screening of the genomic DNA library on LB agar plates supplemented with X-Gal, a novel gene encoding a cold-active β-<smcaps>D</smcaps>-galactosidase was isolated. The <it>in silico </it>analysis of the enzyme amino acid sequence revealed that the β-<smcaps>D</smcaps>-galactosidase <it>Paracoccus </it>sp. 32d is a novel member of Glycoside Hydrolase Family 2. However, owing to the lack of a BGal_small_N domain, the domain characteristic for the LacZ enzymes of the GH2 family, it was decided to call the enzyme under study 'BgaL'. The <it>bgaL </it>gene was cloned and expressed in <it>Escherichia coli </it>using the pBAD Expression System. The purified recombinant BgaL consists of two identical subunits with a combined molecular weight of about 160 kDa. The BgaL was optimally active at 40°C and pH 7.5. Moreover, BgaL was able to hydrolyze both lactose and <it>o</it>-nitrophenyl-β-<smcaps>D</smcaps>-galactopyranoside at 10°C with <it>K</it>_mvalues of 2.94 and 1.17 mM and <it>k</it>_catvalues 43.23 and 71.81 s^-1, respectively. One U of the recombinant BgaL would thus be capable hydrolyzing about 97% of the lactose in 1 ml of milk in 24 h at 10°C.</p> <p>Conclusions</p> <p>A novel <it>bgaL </it>gene was isolated from <it>Paracoccus </it>sp. 32d encoded a novel cold-active β-<smcaps>D</smcaps>-galactosidase. An <it>E. coli </it>expression system has enabled efficient production of soluble form of BgaL <it>Paracoccus </it>sp. 32d. The amino acid sequence analysis of the BgaL enzyme revealed notable differences in comparison to the result of the amino acid sequences analysis of well-characterized cold-active β-<smcaps>D</smcaps>-galactosidases belonging to Glycoside Hydrolase Family 2. Finally, the enzymatic properties of <it>Paracoccus </it>sp. 32d β-<smcaps>D</smcaps>-galactosidase shows its potential for being applied to development of a new industrial biocatalyst for efficient lactose hydrolysis in milk.</p

Biochemical characterization of a novel monospecific endo-β-1,4-glucanase belonging to GH Family 5 from a rhizosphere metagenomic library

Cellulases have a broad range of different industrial applications, ranging from food and beverages to pulp and paper and the biofuels area. Here a metagenomics based strategy was used to identify the cellulolytic enzyme CelRH5 from the rhizosphere. CelRH5 is a novel monospecific endo-β-1,4-glucanase belonging to the glycosyl hydrolase family 5 (GH5). Structural based modelling analysis indicated that CelRH5 is related to endo-β-1,4-glucanases derived from thermophilic microorganisms such as Thermotoga maritima, Fervidobacterium nodosum and Ruminiclostridium thermocellum sharing 30-40% amino acid sequence identity. The molecular weight of the enzyme was determined as 40.5 kDa. Biochemical analyses revealed that the enzyme displayed good activity with soluble forms of cellulose as a substrate such as ostazin brilliant red hydroxyethyl cellulose (OBR-HEC), carboxymethylcellulose (CMC), hydroxyethyl cellulose (HEC) and insoluble azurine cross-linked hydroxyethylcellulose (AZCL-HEC). The enzyme shows highest enzymatic activity at pH 6.5 with high pH tolerance, remaining stable in the pH range 4.5 – 8.5. Highest activity was observed at 40 ˚C, but CelRH5 is psychrotolerant being active and stable at temperatures below 30 ˚C. The presence of final products of cellulose hydrolysis (glucose and cellobiose) or metal ions such as Na+, K+, Li+ and Mg2+, as well as ethylenediaminetetraacetic acid (EDTA), urea, dithiothreitol (DTT), dimethyl sulfoxide (DMSO), 2-mercaptoethanol (2-ME) or glycerol, did not have a marked effect on CelRH5 activity. However, the enzyme is quite sensitive in presence of 10 mM ions Zn2+, Ni2+, Co2+, Fe3+ and reagents such as 1 M guanidine HCl, 0.1% sodium dodecyl sulphate (SDS) and 20% ethanol. Given that it is psychrotolerant and retains activity in the presence of final cellulose degradation products, metal ions and various reagents, which are common in many technological processes; CelRH5 may be potential suitability for a variety of different biotechnological applications

The novel sterilization device: the prototype testing

Abstract Currently, there are numerous methods that can be used to neutralize pathogens (i.e., devices, tools, or protective clothing), but the sterilizing agent must be selected so that it does not damage or change the properties of the material to which it is applied. Dry sterilization with hydrogen peroxide gas (VHP) in combination with UV-C radiation is well described and effective method of sterilization. This paper presents the design, construction, and analysis of a novel model of sterilization device. Verification of the sterilization process was performed, using classical microbiological methods and flow cytometry, on samples containing Geobacillus stearothermophilus spores, Bacillus subtilis spores, Escherichia coli, and Candida albicans. Flow cytometry results were in line with the standardized microbiological tests and confirmed the effectiveness of the sterilization process. It was also determined that mobile sterilization stations represent a valuable solution when dedicated to public institutions and businesses in the tourism sector, sports & fitness industry, or other types of services, e.g., cosmetic services. A key feature of this solution is the ability to adapt the device within specific constraints to the user’s needs

The emission spectra of rhodamine B (RB; 1.225×10⁻³ g L⁻¹) and RSFP protein (0.081 g L⁻¹).

<p>The emission spectra of rhodamine B (RB; 1.225×10⁻³ g L⁻¹) and RSFP protein (0.081 g L⁻¹).</p

The UV-VIS absorption spectra of rhodamine B (brown and orange lines) in the presence of the different concentration of <i>E. coli</i> proteins, respectively, and the UV-VIS absorption spectrum of rhodamine B in the absence of <i>E. coli</i> proteins (pink line).

<p>The concentration of <i>E. coli</i> proteins in the first analyzed sample (brown line) was twice higher than the concentration of <i>E. coli</i> proteins (0.023 g L⁻¹) in the second one (orange line), respectively. The concentration of RB (3.5×10⁻⁴ g L⁻¹) was the same in the all assayed samples.</p

X-ray data collection and crystal structure refinement statistics.

1<p>Values in parentheses correspond to the last resolution shell.</p>2<p>R_int = ∑_h∑_j | I_hj−h> |/∑_h∑_j I_hj, where I_hj is the intensity of observation j of reflection h.</p>3<p>R = ∑_h | | F_o|−| F_c| |/∑_h | F_o| for all reflections, where F_o and F_c are observed and calculated structure factors, respectively.</p><p>R_free is calculated analogously for the test reflections, randomly selected and excluded from the refinement.</p

Interactions of RB with RSFP: (A) trimmer of RSFP with visible RB molecules on the interface between monomers; (B) binding site of RB1 interacting with neighbouring monomer (deep pink and violet); (C) binding site of RB2.

<p>Interactions of RB with RSFP: (A) trimmer of RSFP with visible RB molecules on the interface between monomers; (B) binding site of RB1 interacting with neighbouring monomer (deep pink and violet); (C) binding site of RB2.</p

SDS-PAGE analysis of the cell extract with RSFP protein and the purified enzyme.

<p>Protein weight marker (lane M), cell extract of <i>E. coli</i> LMG194/pBADRSFP (lane A), pooled fraction after Fractogel EMD DEAE chromatography (lane B), pooled fraction after Resource Q chromatography (lane C).</p

The UV-VIS absorption spectra of rhodamine B (green and blue lines) in the presence of the different concentration of RSFP protein, respectively, and the UV-VIS absorption spectrum of rhodamine B in the absence of RSFP protein (pink line).

<p>The concentration of RSFP protein in the first analyzed sample (green line) was twice higher than the concentration of RSFP protein (0.023 g L⁻¹) in the second one (blue line), respectively. The concentration of RB (3.5×10⁻⁴ g L⁻¹) was the same in the all assayed samples.</p

The pink fluorescence assay: RB in PBS buffer (A), PBS buffer (B), RB+RSFP in PBS buffer (C), RSFP in PBS buffer (D), RB+<i>E. coli</i> LMG194/pBADRSFP cell lysate in PBS buffer (E), <i>E. coli</i> LMG194/pBADRSFP cell lysate in PBS buffer (F), RB+<i>E. coli</i> LMG194/pBADMycHisA cell lysate in PBS buffer (G), and <i>E. coli</i> LMG194/pBADMycHisA cell lysate in PBS buffer (H).

<p>The pink fluorescence assay: RB in PBS buffer (A), PBS buffer (B), RB+RSFP in PBS buffer (C), RSFP in PBS buffer (D), RB+<i>E. coli</i> LMG194/pBADRSFP cell lysate in PBS buffer (E), <i>E. coli</i> LMG194/pBADRSFP cell lysate in PBS buffer (F), RB+<i>E. coli</i> LMG194/pBADMycHisA cell lysate in PBS buffer (G), and <i>E. coli</i> LMG194/pBADMycHisA cell lysate in PBS buffer (H).</p