Y93F Substitution of Cyclodextrin Glucanotransferase from Bacillus sp. A2-5a and its Enzyme Characterization

Rostinawat, Tina; Riani, Catur; Elfahmi, Elfahmi; Sumirtapur, Yeyet Cahyati; Retnoningr, Debbie Sofie

doi:10.3923/biotech.2015.181.187

Cited by 2 publications

(2 citation statements)

References 28 publications

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“…Gene expression using this vector had previously been able to produce sufficient recombinant proteins. 17 This vector use lac operon system which can produce high amounts of recombinant protein. 18 The recombinant protein to be expressed too as intracellular protein to supplement this anti-HER2 scFv which had previously been designed as an extracellular protein.…”

Section: Discussionmentioning

confidence: 99%

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Characterization of Anti-HER2 scFv Gene Expression as Intracellular Protein in Escherichia coli BL21 (DE3)

Rostinawati

Paramita

Wicaksono

et al. 2020

IJPI

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Objectives: In patients with breast cancer, Human Epidermal Growth Factor is over expressed until 30%. Monoclonal antibodies was an alternative detection cancer in molecular level. The aim of the experiment was protein recombinant of anti-HER2 scFv was constructed from the gene encoding single chain variable fragment of anti-HER2 antibody wich was fused with Histag and can be expressed in the Eschericia coli BL21(DE3) to be used as a diagnostic protein for breast cancer cells. Methods: The recombinant pJ401express_anti-HER2 scFv fused with histaq was transformed into E. coli BL21 (DE3) and expressed as recombinant anti-HER2 scFv protein with various inducer concentration. Then, those protein was purified with the nickel polyhistidine tag (Ni-NTA) affinity chromatography using imidazole concentration i.e 100 and 150 mM. Finally, the existence of this recombinant protein was determined with anti histaq antibody in western blot assay. Results: Plasmid isolation from E. coli BL21 (DE3) cells revelaed the existence of the recombinant pJ401express_anti-HER2 scFv. The optimum condition for using IPTG as inducer for the intracellular expressed anti-HER2 scFv gene was 1 mM IPTG which was entered into broth medium at the 3.5 th hr of growth time of E. coli BL21(DE3). Then, the higher amout of more purified anti-HER2 scFv was obtained using imidazole at 150 mM. The recombinant protein was also bound to anti histaq antibody in western blot assay. Conclusion: the recombinant pJ401express_anti-HER2 scFv was successfully expressed as anti-HER2 scFv protein.

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Section: Discussionmentioning

confidence: 99%

“…25,26 The use of a gradual imidazole concentration elution solution can remove impurity proteins. 17 Ultimately, this recombinant protein was detected as an histaq protein using anti-histag antibody. The Histaq protein can tightly bind to antihistaq antibody.…”

Section: Discussionmentioning

confidence: 99%

Characterization of Anti-HER2 scFv Gene Expression as Intracellular Protein in Escherichia coli BL21 (DE3)

Rostinawati

Paramita

Wicaksono

et al. 2020

IJPI

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A single mutation in cyclodextrin glycosyltransferase from Paenibacillus barengoltzii changes cyclodextrin and maltooligosaccharides production

Castillo

Landriel

Sánchez-Costa

et al. 2018

Protein Engineering, Design and Selection

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Cyclodextrin glycosyltransferases (CGTases) are bacterial enzymes that catalyze starch conversion into cyclodextrins, which have several biotechnological applications including solubilization of hydrophobic compounds, masking of unpleasant odors and flavors in pharmaceutical preparations, and removal of cholesterol from food. Additionally, CGTases produce maltooligosaccharides, which are linear molecules with potential benefits for human health. Current research efforts are concentrated in the development of engineered enzymes with improved yield and/or particular product specificity. In this work, we analyzed the role of four residues of the CGTase from Paenibacillus barengoltzii as determinants of product specificity. Single mutations were introduced in the CGTase-encoding gene to obtain mutants A137V, A144V, L280A and M329I and the activity of recombinant proteins was evaluated. The residue at position 137 proved to be relevant for CGTase activity. Molecular dynamics studies demonstrated additionally that mutation A137V produces a perturbation in the catalytic site of the CGTase, which correlates with a 10-fold reduction in its catalytic efficiency. Moreover, this mutant showed increased production of maltooligosaccharides with a high degree of polymerization, mostly maltopentaose to maltoheptaose. Our results highlight the role of residue 137 as a determinant of product specificity in this CGTase and may be applied to the rational design of saccharide-producing enzymes.

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Y93F Substitution of Cyclodextrin Glucanotransferase from Bacillus sp. A2-5a and its Enzyme Characterization

Cited by 2 publications

References 28 publications

Characterization of Anti-HER2 scFv Gene Expression as Intracellular Protein in Escherichia coli BL21 (DE3)

Characterization of Anti-HER2 scFv Gene Expression as Intracellular Protein in Escherichia coli BL21 (DE3)

A single mutation in cyclodextrin glycosyltransferase from Paenibacillus barengoltzii changes cyclodextrin and maltooligosaccharides production

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