Lecture 14, 10/8; Enzymes

Here is the lecture recording for 10/8.

• Exam2 Monday

• • Aerobes – require air. Pseudomonas aeruginosa, micrococcus luteus, molds.
• • Microaerophiles – require a tiny bit of air
• o Campylobacter jejunii – causes gastrointestinal upset
• • Faciltative can go either way
• o No air – fermentation
• o Air – respiration
• o Ex. E. coli - Faciltative anaerobe, great advantage
• • Anaerobe – absence of air
• o Clostridium. Ex. Tetnus
• • Extra CO2 – capneic
• o Neisseria gonorrhoeae
• • pH requirements – most do well at 7 but some can grow all the way to 14 to lower than 1
• o low pH organism thiobacillus thiooxidans and alcaligenes
• • extrememly high salt means they are an obligate high level halophile - archea’s, halobacteria. 15-20% salt. Low level halophiles living at 3.5-4% salt concentration.
• • halo tolerant organisms – staphylococcus aereus (grows on MSA and causes toxic shock)
• • anaerobiosis (growth in the absence of oxygen) discovered by Pasteur
• • Methods
• o Mixed culture – obligate anaerobe and aerobe together. When the aerobe uses up the oxygen then the anaerobe will grow.
• o overlay media – method that keeps oxygen from diffusing in.
• • chemical removal of oxygen: - gas pak apparatus, biobag allows for a little bit of oxygen “campy pouch”
• • pigments in bacteria
• o intracellular – serratia rudigiensia “prodigiosin”
• o extracellular – Pseudomonas aeroginosa “pyocynan” – blue green. Very resistant to antimicrobials. Pseudomonas fluorescent “fluorascenin” yellow green. Both are typically water organisms.
• • Growth handout (handed out 10/1)
• o 0-4 hours – lag phase or adjustment phase. It is metabolically getting ready to grow. If starch is there it will probably try to produce amylase
• o 4-24 hours - log phase or exponential phase – organisms are the healthiest – they are undergoing binary fission. Time it takes for one cell to become two is called “doubling time”.
• o 24-36 hours – plateau or stationary phase, start to die. Runs out of essential nutrients.
• o Death phase – die at logarithmic rate. Most organisms are usually dead 2-3 days after. Except for those organisms that sporilate (hard to kill spore formers).
• o Archea’s tend to grow faster due to high temperatures.
• o specific times for organisms (back sheet of handout from 10/1)
• • Bacillus Cerius
• • E. coli
• • streps are slower because they only have one type of metabolism – fermentation
• • Mycobacterium tuberculosis – very slow. Need to grow it in Lowenstein jenston. Look for it in about a month
• • Treponema pallidum – grow rabbit testes
• • Mycobacterium leprea – grown in armidillo
• • Saccharomyces cervisiae
• o Evaluating growth
• • Viable count – 30-300colonies is a good count. No more (too numerous) no less (to few to count).
• • Direct count - Hep B is a more common blood bourne illness than AIDS
• • Turbidity – as organisms grow they turn the broth turbid. More turbidity more organisms.
• • Chapter 8
• o Metabolism (all the reactions in the cell) composed of two parts (see handout 10/5)
• • Catabolism – break down of complex to simple. Starch to glucose to CO2. All metabolic reactions are mediated by enzymes.
• • Anabolism – taking something simple (like amino acids) and putting them together into proteins. (needs energy – coupled to catabolism)
• • Nucleotides – used to build up DNA, RNA
• o Pasteur and fermentation – anaerobic
• o Metabolism in a nutshell (handout 10/5) =30:00min in recording
  
• • Produce a Protease – breaks down giant proteins into amino acids.
• • Some organisms require CO2 (autotrophs)
• • Pyruvate – most common intermediate in metabolism. We break down sugar to pyruvate with oxygen. Kreb’s cycle. Then the electrons go to the electron transport chain and energy is produced.
• o Fermentation (without oxygen): classical fermentation – alcohols, acids, gases
• Enzymes (handout 10/5) – biocatalysts of the cell
• • Enzyme + substrate = enzyme substrate complex. It then disacociates to enzyme + product and the enzyme goes back and reacts with another substrate. Usually end in “ase”. Exceptions pepsin, lysozyme, etc.
• • Enzymes have high specificity for the substrate ex. Amylase only acts on starch. They also have temperature and pH optima.
• • Either consititive or indusive
• Constitiuve – always present. Enzymes that are found in common pathways.
• Indusive – only present when substrate is present. You would want indusive because you waste less energy.
• We have a mix of both. Commonly used would be consitiutive, if you don’t see the substrate very often it would be indusive.
• • Intracellular enzymes – made and retained in the cell. Enzymes of the common pathways
• • Extracellular enzymes – made in the cell and secreted to break down large molecules that can be utilized in the cell. Amylase, pepsin, trypsen.
• • Most enzymes are protein derivatives.
• o Waves that we get ATP
• • Substrate level – where the phosphate comes off of the substrate level that is usually found in glycolosis.
• • Oxidative phosphorilation – where we get most of our energy. Associated with Kreb’s cycle and cytochrome system.
• • Direct phos. – energy you get from the Krebs cycle
• • Photo phos. – photosynthetic organism
• o (Table in chap 8) If e. coli grows aerobically it gets 38 ATP’s (we only get 36). If it grows anaerobically (ferments) it only gets 2. Respiration is 19x more efficient than fermentation.