Differential Tests II Catalase, Oxidase, DNase, and Starch Hydrolysis PDF

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This lecture covers the content of Differential Tests II: Catalase, Oxidase, DNase, and Starch Hydrolysis. Lecture led by Noelle Visser....

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Differential Tests II: Catalase, Oxidase, DNase, and Starch Hydrolysis Tests for Aerobic Respiration ● 1) Catalase ○ Electron transport chain ○ One carrier molecule in the electron chain transport called flavoprotein can bypass next carrier and transfer electrons directly to oxygen ■ Creates highly potent cellular toxin - hydrogen peroxide ■ Organisms that produce these must have a way to break them down ■ Catalase converts hydrogen peroxide into water and gaseous oxygen (results in bubbles) ● 2) Oxidase ○ Oxidase test was designed to identify presence of cytochrome c oxidase which is part of complex 4 in ETC ■ Final transfer of electrons from cytochrome c to oxygen ■ Testing for use of oxygen as final electron acceptor ○ Cytochrome C oxidase catalyze reduction of cytochrome c by a chromogenic reducing agent called tetramethyl-p-phenylenediamine (TMPD- chemical indicator) that results in blue color ■ TMPD (colorless) + Cytochrome C (oxidised) ■ TMPD (deep blue) + Cytochrome C (reduced)

Test of Hydrolysis ● Hydrolysis is the use of water to split complex molecules ○ These are chemical reactions called hydrolytic reactions ● Enzymes used in hydrolysis are hydraulic enzymes ○ Enzymes are usually proteins that greatly increase the rate of a chemical reaction ○ Hydrolytic enzymes can be extracellular or intracellular ● If the enzymes work inside the cell, they are intracellular ● If the enzymes are secreted and act on substrate in the environment they are extracellular (exoenzymes)

DNase Test

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DNase is an enzyme that catalyzes the hydrolysis of DNA into smaller DNA fragments called oligonucleotides ○ DNA is a very long chain (polymer) of building blocks (monomers) called nucleotides ○ The DNase enzyme is extracellular and acts to break down long DNA molecules that are too large to get into the cell ● DNase Agar Plates: Contain DNA as a substrate and methyl green dye as an indicator ● Methyl Green forms a complex with polymerized DNA (a long strand of DNA) and produces an aqua color ● When polymerized DNA is broken down by the DNase, there is no longer a complex to form the aqua color and it results in a clearing around the bacterial growth

Starch Hydrolysis Test ● Starch is a polysaccharide made up of glucose subunits ○ Can be a linear (amylose) or branched (amylopectin) ○ Usually a mixture of both with amylopectin being predominant ● Starch is too large to pass through cell membrane, so it must be broken down into smaller glucose and maltose fragments ○ Enzymes are therefore extracellular (exoenzymes) ○ The enzymes break starch into smaller units by hydrolysis ■ Amylase (for linear polysaccharides) ■ Oligo-1, 6-glucosidase (for branch points in polysaccharide) ● Starch plates contain: scratch as a substrate...