Experiment 16: Bacterial Enzymes

what are extracellular exoenzymes

produced by some icrobes

break down large complex molecules starch proteins or lipids via hydrolysis to help them pass through the cell membrane

what are carbohydrases

ex. amylase

hydrolyze polysacs like starch or carbs to simple sugars (mono/disaccharrides) outside cell which si then brought in bc theyre small now

now it is a source of energy and carbon

what are starch hydrolysis assays designed to detect?

amylase, using starch agar which has soluble agar as substrate

if bact secreate starch amylase then it removes starch in the margins of the colonies

what do we use to detect starch -hydroyzing amylase?

iodine, rreacts w starch to give blue violet colour

amylase + —> colourless/clear

amylase - —> plate stains blue violet until edge of colonial growth

what is casein hydrolysis?

assay that tests for cells ability to make protease exoenzymes that turn proteins into short peptides and AA which are taken up by the cell and used as N2 and AA sources

detects specieis that can hydrolyze milk protein casein and thos who cant

how are proteases selective?

about WHERE in the protein they cut, not which protein they cut

casein hydrolysis what do they yse?

skim milk agar plates w cloudy opacity bc casein is substrate

secrete proteases (+) = clearning zone around colony

(-) = no clearning zone

what is lipid hydrolysis?

tests for cells ability to make lipases

break down fats or triglycerides (simple fats made of glycerol and 3 long fatty acid chains) that are too big to enter cell

what is Tween 80?

ex of triglyceride and used to detect organisms that make lipase

screening using tween —> ppt of insoluble crystal around lipase producing colonies

ppt formed when Ca salts are incorporated into mediumw the fatty acid chains

examples of endoenzymes

catalase and oxidase

work in the cell

Energy production via ETC for aerobic and facultative anaerobes may create two potent cellular toxins:

H2O2 and O2- superoxide radical

aerobic and aerotolerant bact make enzumes that detoxify these compounds

catalase works on what?

H2O2, breaks it down into water and oxyegn

oxidase works on what?

superxocxide radical O2-

when do we see bubbles?

bubble sof oxygen obsered when 3% soln of H2O2 is added to catalase positive microorganisms

ETC has crrier molecular called what?????

cytochrome oxidase

transfers electrons to oxygen, reuducing. itto water

how to detetc oxidase positive speixies?

addadd chromogenic reducing agent like tetramehtyl-p-phenylenediamine which acts like an artficla electron donor

chnage colour to purple if oxidized cytochrome oxidase is present

exp 16 learning objecitives

1. detetc interpewt hydrolysis macromols using diff media

2. recognize exo and econo enzymes

3. identify metabolic anc bichemicla and repsratory characterics and pathways

exp 16 materials

1. starch, tween 80, skim mik plates streaked w 3 microorganisms provides

2. e.coli pseodomoas aeorgnosa, bacillus subtilis

3. starch carb pour plate

4. tween80 triglecylaride pour plate

5. casein milk protein pour plate

6. grams iodine

7. oxidase reagent trimethylpphenuldiamine

8. cataslse reagent 3% h2o2

9. filter paper, wooden stick

evidence of amylase carbohydrase actiity?

flood starcg agar plate w iodine = black

amylase + = clearing zones

evidenxe of lipase activity?

tween 80 lipid id disperesed throughout agar in fine amulsion and med = transaprent

hydrolysis of lipids = cloudy/turbid ppt

evidenc eof protease activity?

casein is nitrogenous protein consitient of milk and exists in colloidal state and makes milk white

hydrolzed casein = clearing zone

Results - E.coli

Starch = hydrolysis (-)

Tween80. = lipolysis (-)

Milk = proteolysis (-) weak

Catalase = (+)

Oxidase = (-)

Results - B. subtilis

Starch = hydrolysis (++)

Tween80. = lipolysis (+) weak

Milk = proteolysis (+++)

Catalase = (+)

Oxidase = (-)

Results - P. aeruginosa

Starch = hydrolysis (-)

Tween80. = lipolysis (++)

Milk = proteolysis (++) weak

Catalase = (+)

Oxidase = (+)

evidence. of catalase?

putput drop of h2o2 on colonies on Tween 80 and see if oxygen bubbles occur

evidence od oxidase?

put drop of tetramethyl-p-phenilandediamine o filter paper and use woode stick to sub small abt of abct from milk plate on the drop and see of purple to black colour occurs