Treść książki
Przejdź do opcji czytnikaPrzejdź do nawigacjiPrzejdź do informacjiPrzejdź do stopki
Microbialbiotechnologyinthelaboratoryandinpractce
(higherinthebaselinecellsthaninprotoplasts)andtherateof
processesinwhichthecellwallisabarriertosubstrateaccess
tointracellularenzymes.Inthiscase,processessuchase.g.
hydroxylationreactionsofsteroidcompoundsarefasterusing
protoplasts.
Thecharacteristicfeaturesofprotoplastsinclude:
•Sphericalshape;
•
Lackofmechanicalresistance-forthisreasonitis
necessarytouselowshakingvalues(below90rpm)during
theprocessofprotoplastreleaseanddelicateconditionsof
protoplastsseparationfromundigestedmyceliumbiomass
(centrifugationrateshouldnotexceed500×g);
•
Sensitivitytoosmoticpressure-protoplastsplaced
inwater,crackwithinseconds.Therefore,during
thereleaseofprotoplastsandinsubsequentstagesof
testing,itisnecessarytouseahypertonicenvironment,
providedbytheuseofosmoticstabilisers.Osmotic
stabilisersareaqueoussolutions(0.4-1.0M)ofinorganic
(e.g.MgSO
4
,CaCl
2
,KCl)ororganic(e.g.mannitol,
sorbitol,sorbose)compounds.Usually,inorganicosmotic
stabilisers;(e.g.0.6-0.8MKCl)areusedtoobtain
protoplastsoffilamentousfungi,whereasthereleaseof
yeastprotoplastsisfavouredbyanorganiccompound
environmentof0.8-1.2M.
Thefirstscientificreportsontheisolationofyeastand
fungiprotoplastscomefromthe1960s.Researchconductedat
thattimefocusedondevelopingefcientmethodsofbacterial
andfungalcellwalldisintegrationtoisolateandcharacterise
intracellularproteinsandgeneticmaterial.Inthefollowing
years,thescopeofresearchusingprotoplastsandspheroplasts
wasextended.
Directionsforthepracticaluseofprotoplastsand
spheroplastsinclude:
•
examinationofthestructureandcourseofcellwallsynthesis
-afterobtainingtheprotoplasts,agradualreversal(the
processofcellwallreconstruction)enablescharacterisation
ofsubsequentstagesofcellwallreconstructionandthe
enzymesinvolvedintheaboveprocess;
•isolationandcharacterisationofcellorganelles;
54
