Activation of LacZ gene in Escherichia coli DH5α via α-complementation mechanism for β-galactosidase production and its biochemical characterizations

Hamed, Ahmed A.; Khedr, Mohamed; Abdelraof, Mohamed

doi:10.1186/s43141-020-00096-w

Cited by 9 publications

(6 citation statements)

References 45 publications

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“…Inhibition% = 100 − Viability% a good enzymatic recovery of 73.96% being valuable for the industry in terms of economic point of view [23]. A. fumigatus L-methioninase was subjected under only two purification steps; which was better than those obtained for L-methioninase purification by Trichoderma harzianum [12], A. flavipes [24], Candida tropicalis [14], requires to three purification steps.…”

Section: Purification Of a Fumigatus L-methioninasementioning

confidence: 99%

Purification, Characterization and anticancer activity of L-methionine γ-lyase from thermo-tolerant Aspergillus fumigatus

et al. 2023

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Purification of L-methionine γ-lyase (MGL) from A. fumigatus was sequentially conducted using heat treatment and gel filtration, resulting in 3.04 of purification fold and 73.9% of enzymatic recovery. The molecular mass of the purified MGL was approximately apparent at 46 KDa based on SDS-PAGE analysis. The enzymatic biochemical properties showed a maximum activity at pH 7 and exhibited plausible stability within pH range 5.0–7.5; meanwhile the highest catalytic activity of MGL was observed at 30–40 °C and the enzymatic stability was noted up to 40 °C. The enzyme molecule was significantly inhibited in the presence of Cu2+, Cd2+, Li2+, Mn2+, Hg2+, sodium azide, iodoacetate, and mercaptoethanol. Moreover, MGL displayed a maximum activity toward the following substrates, L-methionine < DL-methionine < Ethionine < Cysteine. Kinetic studies of MGL for L-methioninase showed catalytic activity at 20.608 mM and 12.34568 µM.min−1. Furthermore, MGL exhibited anticancer activity against cancerous cell lines, where IC50 were 243 ± 4.87 µg/ml (0.486 U/ml), and 726 ± 29.31 µg/ml (1.452 U/ml) against Hep-G2, and HCT116 respectively. In conclusion, A. fumigatus MGL had good catalytic properties along with significantly anticancer activity at low concentration which makes it a probably candidate to apply in the enzymotherapy field.

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Section: Purification Of a Fumigatus L-methioninasementioning

confidence: 99%

Purification, Characterization and anticancer activity of L-methionine γ-lyase from thermo-tolerant Aspergillus fumigatus

et al. 2023

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“…Mutagenesis using UV as a physical method for enhancement enzymes production from different fungal strains was studied by many workers in scientific field [27][28][29]. Fig ( 2) showed that UVmutagenesis play a critical role in alkaline cellulase production enhancement.…”

Section: Uv-mutagenesis For a Fumigatus Nrcf-122 Alkaline Cellulase Enhancemenmentioning

confidence: 99%

Cellulases and animal feed production by solid-state fermentation by Aspergillus fumigatus NRCF-122 mutant

et al. 2021

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A high cellulase-producing fungus was isolated from agricultural waste compost, purified, identified genetically as Aspergillus fumigatus NRCF-122 (accession no. MT318341), and improved by UV-irradiation for cellulase production (named as A. fumigatus mutant-17). Optimal cellulase production conditions were studied using sugar beet pulp "SBP" as the substrate of solid-state fermentation "SSF" technique. The optimal conditions were: initial moisture content ratio (1:2 w/v), incubation temperature (30°C), initial pH of solid culture (10), incubation period (4 days), aeration area (1:100 ratio ((1%)), SBP substrate weight : flask volume of moistened SBP in a 250 mL Erlenmeyer flask, fungal inoculum (seeding) culture age (3 days) and inoculum size (20%, v/w). Studies were also involved the effect of different nitrogen sources where urea is the best nitrogen source for cellulase production. Concerning the application of bioethanol production from yeast, using rice straw cellulose as a source of fermentable reducing sugars, the lingo-cellulosic waste rice straw was first pretreated (with NaOH 1.5%) to reduce crystallinity of cellulose & removing lignin and other inhibitors, enhancing the subsequent process (saccharification) of rice straw cellulose. Then, its cellulose was saccharified enzymatically by cellulases (produced from SBP as substrate (under SSF condition) as substrate under the optimum conditions) resulting in reducing sugars. one of the largest livestock production operating costs is the feed bill. In our study we evaluated the resulted crude protein content of the fermented SBP and the results declared that, the total digest nutrient (TDN) and gross energy performance were increased, where as crude fiber, acid detergent fiber ADF & neutral detergent fiber NDF were decreased, thereby upgrading the feed quality of biologically treated SBP.

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“…β-Galactosidase is a vital enzyme. The most extensively researched β-galactosidase (coded by the lacZ gene) is derived from Escherichia coli, which is widely used in the laboratory for DNA cloning, protein expression, and synthetic biology research (Hamed et al, 2020;Gao et al, 2021). The α-complementation phenomenon is a well-known feature of E. coli β-galactosidase (Ullmann et al, 1967).…”

Section: Introductionmentioning

confidence: 99%

A food-grade vector for Streptococcus thermophilus based on the α-complementation of β-galactosidase

Liang

Kong

et al. 2022

Journal of Dairy Science

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Streptococcus thermophilus is a common yogurt starter that consumes lactose as its primary carbon source. The enzyme β-galactosidase is essential for the lactose metabolism and the growth of this species. Streptococcus thermophilus appears to be a promising cell factory. Food-grade vectors have advantages in heterologous protein expression. This study aimed to determine whether the β-galactosidase of S. thermophilus has the α-complementary characteristic and to develop a novel food-grade vector based on this phenomenon. The Nterminal 7 to 36 AA residues of the β-galactosidase in S. thermophilus were deleted. The obtained mutant S. thermophilus Δα lost β-galactosidase activity and growth ability in the lactose medium. Subsequently, plasmids expressing α-fragments with different lengths of 1 to 36 (Sα1), 1 to 53 (Sα2), and 1 to 88 (Sα3) AA were constructed and transformed into S. thermophilus Δα. Recombinant S. thermophilus Δα expressing Sα2 or Sα3 recovered the ability to grow in the lactose medium, and their β-galactosidase activity accounted for 24.5% or 11.5% of the wild strain, respectively. These results indicated that the α-complementation system of β-galactosidase existed in S. thermophilus. Based on the characteristic, a food-grade vector pSEα was constructed. Except for Sα2, vector pSEα expressed the α-donor derived from E. coli β-galactosidase. This facilitated the construction of recombinant plasmids in E. coli DH5α and thus improved the transformation efficiency of S. thermophilus. Green fluorescent protein as a reporter protein could be highly expressed in S. thermophilus using this vector. As a result, pSEα is an efficient and safe vector for S. thermophilus with potential food applications.

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Activation of LacZ gene in Escherichia coli DH5α via α-complementation mechanism for β-galactosidase production and its biochemical characterizations

Cited by 9 publications

References 45 publications

Purification, Characterization and anticancer activity of L-methionine γ-lyase from thermo-tolerant Aspergillus fumigatus

Purification, Characterization and anticancer activity of L-methionine γ-lyase from thermo-tolerant Aspergillus fumigatus

Cellulases and animal feed production by solid-state fermentation by Aspergillus fumigatus NRCF-122 mutant

A food-grade vector for Streptococcus thermophilus based on the α-complementation of β-galactosidase

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