Molecular Cloning and Nucleotide Sequence of the<i>β</i>-Galactosidase Gene from<i>Entevobacter cloacae</i>GAO

Nagano, Hiroko; Kawaguchi, Takashi; Omori, Masashi; Shoji, Zenya; Arai, Motoo

doi:10.1271/bbb.58.1866

Cited by 8 publications

(5 citation statements)

References 1 publication

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“…cloacae GAO (isolated from a spontaneous apple leachate fermentation), which consists of 3,087 nucleotides encoding a 1,028 amino-acid protein [17]. The identity between the two proteins was 88%.…”

Section: Discussionmentioning

confidence: 99%

“…To clone the bgaB5 gene, two forward primers (F1: 5'-GGGCATGTTAAC-CAAAATTGTGAGC-3'; F2: 5'-CGCTTCGCAAAGAGGTTAACA-3') and three reverse primers (R1: 5'-TTAAGGGTTCTGCTGCCA-3'; R2: 5'-CCGCATCTTCTGGTCAGTTA-3'; R3: 5'-ACGCTCTGTGGCCGCATCTT-3') were designed based on the sequence of bgalactosidase gene from E. cloacae GAO [17] (Fig. 1).…”

Section: Gene Cloning and Heterogenous Expressionmentioning

confidence: 99%

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A novel transglycosylating β-galactosidase from Enterobacter cloacae B5

Xiao

et al. 2009

Process Biochemistry

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Section: Discussionmentioning

confidence: 99%

Section: Gene Cloning and Heterogenous Expressionmentioning

confidence: 99%

A novel transglycosylating β-galactosidase from Enterobacter cloacae B5

Xiao

et al. 2009

Process Biochemistry

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“…The Trp at position 999 of the β-galactosidase of E. coli is conserved in many related β-galactosidases (3,(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26).…”

mentioning

confidence: 99%

“…The Trp at position 999 of the β-galactosidase of E. coli is conserved in many related β-galactosidases ( 3 , − . Structural studies indicate that the aromatic side chain of Trp-999 (part of the fifth domain of the subunit) is one component of the binding site ( , ).…”

mentioning

confidence: 99%

Trp-999 of β-Galactosidase (Escherichia coli) Is a Key Residue for Binding, Catalysis, and Synthesis of Allolactose, the Natural Lac Operon Inducer

et al. 2003

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Trp-999 is a key residue for the action of beta-galactosidases (Escherichia coli). Several site specific substitutions (Phe, Gly, Tyr, Leu) for Trp-999 were made. Each substitution caused greatly decreased affinities for substrates and inhibitors that bind in the "shallow" mode, while the affinities of inhibitors that bind in the "deep" mode were not decreased nearly as much. This shows that Trp-999 is important for binding in the shallow mode. The residue is also very important for binding glucose to galactosyl-beta-galactosidase (as a transgalactosidic acceptor). Substitution greatly diminished the affinity for glucose. Substitutions also changed the activation thermodynamics and, subsequently, the rates of the catalytic reactions. The enthalpies of activation of the glycolytic bond cleavage step (galactosylation, k(2)) became less favorable while the entropies of activation of that step became more favorable as a result of the substitutions. Differing magnitudes of these enthalpic and entropic effects with ONPG as compared to PNPG caused the k(2) values for ONPG to decrease but to increase for PNPG. The enthalpies of activation for the common hydrolytic step (degalactosylation, k(3)) increased while the entropies of activation for this step did not change much. As a result, k(3) became small and rate determining for each substituted enzyme. The substitutions caused the rate constant (k(4)) of the transgalactosidic acceptor reactions with glucose (for the formation of allolactose) to become much larger and of the same order of magnitude as the normally large rate constants for transgalactosidic acceptor reactions with small alcohols. This is probably because glucose can approach with less restriction in the absence of Trp-999. However, since glucose binds very poorly to the galactosyl-beta-galactosidases with substitutions for Trp-999, the proportion of lactose molecules converted to allolactose is small. Thus, Trp-999 is also important for ensuring that an appropriate proportion of lactose is converted to allolactose.

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“…Members of GH-2 contain more AA residues than those of GH-42, including β-galactosidase from Saccharopolyspora rectivirgula [39] (1251 AA residues), β-galactosidase from Alteromonas sp. ML52 [40] (1059 AA residues), and β-galactosidase from Enterobacter cloacae GAO [41] (1028 AA residues). β-galactosidases from GH-35 and GH-1 contain a smaller number of AA residues.…”

Section: Heterologous Expression and Purification Of Laczbamentioning

confidence: 99%

A Novel Thermal-Activated β-Galactosidase from Bacillus aryabhattai GEL-09 for Lactose Hydrolysis in Milk

Luan

Duan

2022

Foods

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β-Galactosidase has been greatly used in the dairy industry. This study investigated a novel thermostable β-galactosidase (lacZBa) from Bacillus aryabhattai GEL-09 and evaluated the hydrolytic performance of this enzyme. Firstly, the lacZBa-encoding gene was cloned and overexpressed in Escherichia coli BL21(DE3). Phylogenetic analyses revealed that lacZBa belonged to the glycoside hydrolase family 42. Using SDS-PAGE, we determined that the molecular weight of lacZBa was ~75 kDa. Purified lacZBa exhibited a maximum activity at 45 °C, pH 6.0, and could be activated following incubation at 45 °C for several minutes. The half-life of lacZBa at 45 °C and 50 °C was 264 h and 36 h, respectively. While Co2+, Mn2+, Zn2+, Fe2+, Mg2+, and Ca2+ enhanced enzymatic activity, Cu2+ and ethylenediaminetetraacetic acid inhibited enzymatic activity. Moreover, lacZBa could hydrolyze lactose and oNPG with Km values of 85.09 and 14.38 mM. Molecular docking results revealed that lacZBa efficiently recognized and catalyzed lactose. Additionally, the hydrolysis of lactose by lacZBa was studied in lactose solution and commercial milk. Lactose was completely hydrolyzed within 4 h with 8 U/mL of lacZBa at 45 °C. These results suggested that lacZBa identified in this study has potential applications in the dairy industry.

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Molecular Cloning and Nucleotide Sequence of theβ-Galactosidase Gene fromEntevobacter cloacaeGAO

Cited by 8 publications

References 1 publication

A novel transglycosylating β-galactosidase from Enterobacter cloacae B5

A novel transglycosylating β-galactosidase from Enterobacter cloacae B5

Trp-999 of β-Galactosidase (Escherichia coli) Is a Key Residue for Binding, Catalysis, and Synthesis of Allolactose, the Natural Lac Operon Inducer

A Novel Thermal-Activated β-Galactosidase from Bacillus aryabhattai GEL-09 for Lactose Hydrolysis in Milk

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