Archives

  • 2018-07
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • 2020-08
  • 2020-09
  • 2020-10
  • 2020-11
  • 2020-12
  • 2021-01
  • 2021-02
  • 2021-03
  • 2021-04
  • 2021-05
  • 2021-06
  • 2021-07
  • 2021-08
  • 2021-09
  • 2021-10
  • 2021-11
  • 2021-12
  • 2022-01
  • 2022-02
  • 2022-03
  • 2022-04
  • 2022-05
  • 2022-06
  • 2022-07
  • 2022-08
  • 2022-09
  • 2022-10
  • 2022-11
  • 2022-12
  • 2023-01
  • 2023-02
  • 2023-03
  • 2023-04
  • 2023-05
  • 2023-06
  • 2023-08
  • 2023-09
  • 2023-10
  • 2023-11
  • 2023-12
  • 2024-01
  • 2024-02
  • 2024-03
  • br Materials and methods br Results Bgal from Alteromonas sp

    2021-10-14


    Materials and methods
    Results Bgal from Alteromonas sp. ML117 had an open reading frame of 3126 bp encoding 1042 amino L-365,260 residues with a predicted mass of 120 kDa and a theoretical pI (isoelectric point) of 5.44. The recombinant Bgal contained a His tag, as well as other additional amino acids, and had 1064 amino acid residues. The nucleotide sequence of Bgal was submitted to GenBank (Accession No. MH925305). More than 150 bacterial strains had approximately 60% similarity with Bgal from Alteromonas sp. ML117, including Alteromonas, Rickettsiakes bacterium, Pseudoalteromonas, Colwellia, Glaciecola, Vibrio, Photobacterium, and Plesiomonas (http://ekhidna2.biocenter.helsinki.fi/cgi-bin/sans/sans.cgi). Among the β-Ggalactosidases that have been characterized for enzymatic properties, β-galactosidase from Pseudoalteromonas haloplanktis [20] and Pseudoalteromonas sp. 22b were most similar to Bgal from Alteromonas sp. ML117 [21,22], with sequence similarities of 60% and 61%, respectively. Based on sequence homology analysis and hydrophobic cluster analysis [25], Bgal was classified as a member of the GH (glycosyl hydrolase) family 2. Multiple sequence alignment of the protein sequences (Fig. 1) identified the conserved amino acid residues in E. coli LacZ, Glu 461, Glu 537, Met 502, Tyr 503 and Arg 388 that were conserved in the Alteromonas sp. ML117 Bgal sequence. In the three-dimensional structure of E. coli β-galactosidase, these active-site residues were found close to each other and formed the active site pocket [[31], [32], [33]]. Bgal from Alteromonas sp. ML117 had lower arginine content (4.7% versus 6.4%) and Arg/(Arg + Lys) ratio (0.5% versus 0.77%) compared to E. coli β-galactosidase. Compared to Lys residues, Arg residues can stabilize proteins by facilitating hydrophobic interactions at the surface to adapt to high temperatures [22].
    Discussion In this study, a gene encoding a novel cold-adapted β-galactosidase from Alteromonas sp. ML117 was cloned and expressed. The recombinant β-galactosidase was purified, and its enzymatic properties were investigated. BLAST results revealed that Bgal belongs to the GH2 family. Multiple sequence alignment of Bgal with other β-galactosidases showed that the amino acid residues involved in the catalysis of substrate hydrolysis were highly conserved. Most cold-active GH2 β-galactosidases characterized to date are tetramers (Table 5). The SDS-PAGE and gel filtration of Bgal also showed that it was a tetrameric enzyme. Like other cold-adapted β-galactosidases belonging to the GH2 family, recombinant Bgal can hydrolyze ONPG and lactose. The optimum reaction temperature for the hydrolysis of ONPG and lactose by Bgal was 30 and 35 °C, respectively. Almost all the cold-adapted β-galactosidases belonging to the GH2 family are optimally active at temperatures above 10 °C, and so far only a recombinant enzyme from Arthrobacter psychrolactophilus strain F2 [15] showed the optimum hydrolysis temperature of 10 °C for lactose. At 0-30 °C, Bgal exhibited 30–100% of its optimum activity level when ONPG was the substrate and 28–83% of its optimum activity level when lactose was the substrate. Cold-adapted enzymes are often sensitive to higher temperatures and gradually lose their activity at relatively moderate temperatures. In accordance to this, Bgal did not have good thermal stability. It was inactivated after being placed at 30 °C for 1 h, and lost its activity rapidly after incubation at 35 °C for 10 min. This result was similar to the thermal stability of the recombinant β-galactosidase of Arthrobacter sp. SB [34]. The optimum pH of Bgal was similar to other cold-adapted β-galactosidases from Arthrobacter psychrolactophilus strain F2, Arthrobacter sp. 32cB, and Alkalilactibacillus ikkense [15,17,35]. Bgal retained 75% or more of its maximum activity after being incubated in pH 7.0–9.0 for 1 h. Therefore Bgal was stable at 4 °C and pH of 7.0–9.0. K+ and Mn2+ had strong activation effects on Bgal, and Na+ and Mg2+ also showed a slight promotion of its enzyme activity. These four metal ions also activated β-galactosidase from Pseudoalteromonas sp. 22b [20,21] and Arthrobacter sp. 20B [16]. However, Zn2+, Al3+, and Cu2+ completely inhibited the activity of Bgal, and these metal ions are also often reported to have an inhibitory effect on recombinant cold-adapted β-galactosidases [8,22]. EDTA had a strong inhibitory effect on recombinant Bgal, and it may have acted as a cofactor in the catalytic reaction of Bgal. One of the best known inhibitors of cold-adapted β-galactosidase enzymes is glutathione. However, the enzyme activity of Bgal is not greatly affected by glutathione, and this may be due to the fact that Cys residues do not play an important role in Bgal protein structure. Furthermore, the Cys residues are not involved in catalysis reactions facilitated by β-galactosidases of the LacZ family [36].