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Cellular and Molecular Basis of Human Biology
Published in Lawrence S. Chan, William C. Tang, Engineering-Medicine, 2019
These small cellular chemicals are responsible for either building up or breaking down cellular components, like DNA, RNA, or proteins. Enzymes catalyze cellular and extracellular reactions either to construct a molecule or to degrade a molecule and they make the chemical reactions go quicker than without the enzymes. They have important role of maintain a balance between providing sufficient components and eliminating excessive components in human body functions in a homeostatic way. Enzymes are generally named according to their functions. For example, the function of RNA polymerase is to synthesize RNA polymer (or RNA strand). DNA ligase functions to joint two pieces of DNA together. The six major groups of human enzymes classified by an international enzyme committee and their functional mechanisms are depicted in Table 3.
Glossary of scientific and technical terms in bioengineering and biological engineering
Published in Megh R. Goyal, Scientific and Technical Terms in Bioengineering and Biological Engineering, 2018
DNA ligase is an enzyme that catalyzes a reaction that links two DNA molecules via the formation of a phospho-diester bond between the 3′ hydroxyl and 5′ phosphate of adjacent nucleotides.
Modeling and evaluation of the sucrose-degrading activity of recombinantly produced oligo-1,6-glucosidase from A. gonensis
Published in Preparative Biochemistry & Biotechnology, 2023
Hakan Karaoglu, Zeynep Dengız Balta
After isolation of the pGEM®-T Easy Vector, including the O-1-6-glucosidase gene, the recombinant plasmid and pET28a (+) vector were restricted by NcoI and EcoRI restriction endonucleases. Digested products were loaded into agarose gel (1%) and then vectors and inserts were isolated from the gel. The vector and insert were ligated at 16 °C for 12 h by using T4 DNA ligase. The plasmid (pETG2SUC2) was transformed into E. coli BL21 (DE3) cells by the CaCl2 method. E. coli BL21/pETG2SUC2 was incubated in 10 mL of medium with kanamycin (0.05 mg/mL) overnight. The fresh culture of E. coli BL21/pETG2SUC2 with an optical density 0.1 (OD600) was re-inoculated into 400 mL of LB with kanamycin and incubated until the optical density reached 0.6-0.9. Then, expression of A. gonensis O-1-6-glucosidase (rAgoSuc2) was induced by the addition of iso-propyl-d-thiogalactopyranoside at a final concentration of 1 mM. Following 4 h of incubation, E. coli BL21/pETG2SUC2 culture was centrifuged (12000 g, 15 min). The precipitated cells were homogenized in 100 mM MOPS buffer (pH 7.0), including 1 µg/mL lysozyme, and lysed by a sonicator (SONIC Vibra Cell) as defined by Karaoglu et al.. The lysate was centrifuged (12000 g, 25 min), and the supernatant was used for further enzyme purification steps.[16]
Heterologous expression of azurin from Pseudomonas aeruginosa in food-grade Lactococcus lactis
Published in Preparative Biochemistry and Biotechnology, 2019
After digesting with PstI and KpnI enzymes (Invitrogen, Waltham, MA), the recovered azurin was inserted into the vector pNZ8149 linearized by the same enzymes. The ligation reaction was carried out at 16 °C for 16 h using T4 DNA Ligase (Invitrogen). The pure ligation product (pNZ8149-azu) was transformed by electroporation into competent L. lactis NZ3900 cells prepared according to the manufacturer’s instructions (MoBiTec, GmbH). Briefly, 40 μL of electrocompetent cells and 2 μL of pure ligation products were placed in a pre-chilled electroporation cuvette on ice. For electroporation, Bio-Rad Gene Pulser (Bio-Rad, Hercules, CA) was used with following adjustments: 2000 V, 25 μF, 200 Ω. After 1 mL of L-M17 broth containing 20 mM MgCl2 and 2 mM CaCl2 was added to the pulsed electroporation cuvette, the cuvette was kept for 5 min on ice and incubated for 1.5 h at 30 °C. For selection of positive transformants, 10, 100, and 900 µL of transformed L. lactis NZ3900 were plated on Elliker medium and incubated for 2 days at 30 °C.[17] The colony PCR was carried out to confirm the colonies containing azurin gene. A yellow colony was scraped with a sterile pipette tip and suspended in 12 µL sterile distilled water. The suspension was incubated for 15 min at 100 °C and then 5 min on ice rapidly. The suspension was used as template DNA in PCR mixture.[18] The nucleotide sequence of azurin was determined by MedSanTek (İstanbul, Turkey).