Identification and characterisation of a Cryptococcus laurentii Abo 510 Phytase

Van Staden, Jason (2004-03)

Thesis (MSc)--University of Stellenbosch, 2004.

Thesis

ENGLISH ABSTRACT: Phosphorus is vital for growth of all life forms and is a fundamental component of nucleic acids, ATP and several other biological compounds. Oilseeds and cereal grains, two major constituents of the diet of animals, contain phytic acid, which is the main storage form of phosphorus in plant cells. Monogastric animals, such as poultry and pigs, are not capable of utilising the bound phosphorus in phytic acid since they do not produce phytase, the essential hydrolysing enzyme. Microbial phytase is therefore added to the animal feed to enhance the availability of phosphorus and thus minimise phosphorus pollution and phosphorus supplementation in diets. For a phytase to be effective in the poultry and swine industry, it needs to be able to release phytic acid phosphorus in the digestive tract, it must be thermostable to resist feed processing and must be inexpensive to produce. One approach for developing an efficient phytase for the animal feed industry is by identifying new phytases from microorganisms, plants and animals. In this study, 11 strains of the genus Cryptococcus were screened for 'phytase activity. Initially, a differential agar plate screening method was employed to determine if any Cryptococcus species were able to express phytase, after which production was confirmed in different liquid media. Cryptococcus laurentii Abo 510 was identified as a strain with significant phytase activity. The C. laurentii Abo 510 strain showed clear zones on the differentialmedia agar plates and the production of phytase at high levels was observed when using wet cells grown in liquid media. The C. laurentii Abo 510 strain produced maximal phytase activity at a relatively high temperature (62°C) and in an acidic pH range (pH 5.0). This phytase also showed a broad substrate specificity that may assist in the release of other phosphate compounds captured in feedstuff. Although the phytase did not require any metal ions for its activity, several metal ions caused inhibition of the phytase activity. The enzyme was stable when exposed to 70°C for up to 180 minutes with only 40% loss in activity. Phosphorus addition to the culture media and enzyme assay solution at concentrations exceeding 500 f.!Minhibited the phytase activity completely. Different carbon sources in the culture media also influenced the phytase activity. The enzyme was determined to be a cell wall-associated phytase with little intracellularactivity.

AFRIKAANSE OPSOMMING: Lewende organismes benodig fosfaat vir groei en oorlewing en fosfaat vorm 'n fundamentele komponent van nukleïensure, ATP en verskeie ander biologiese verbindings. Veevoer bestaan meestal uit twee groot bestanddele, naamlik oliesade en graansoorte wat fitiensuur bevat. Fitiensuur is die vernaamste vorm waarin fosfaat in veevoer gestoor word. Enkelmaagdiere soos pluimvee en varke is nie in staat om die fosfaat van die fitiensuur te benut nie, aangesien hierdie diere nie die geskikte hidrolitiese ensiem, fitase, vir die vrystelling van fosfaat besit nie. 'n Mikrobiese fitase-ensiem word derhalwe by veevoer gevoeg om die fosfaatbeskikbaarheid te verhoog. Sodoende word fosfaatbesoedeling en fosfaataanvullings tot die dieet van diere ook verminder. Vir 'n fitase om effektief in die pluimvee en vark-industrie te wees, moet dit fosfaat vanaf fitiensuur in die spysverteringskanaal vrystel, dit moet behandeling by hoë temperature tydens die veevoervervaardiging oorleef en die ensiem moet goedkoop geproduseer kan word. Een van die benaderings om 'n effektiewe fitase vir die dierevoer-industrie te ontwikkel, is om nuwe fitases in mikroërganismes, plante of diere te identifiseer. In hierdie studie is die fitase-aktiwiteit van 11 stamme van die Cryptococcus genus bepaal. Die seleksie vir die produksie van fitase deur die verskillende Cryptococcus stamme was aanvanklik op differensiële agar plate gedoen en in verskillende vloeisto:finedia bevestig. 'n Cryptococcus laurentii Abo SlOstam is geïdentifiseer as 'n goeie fitase produseerder. Die C. laurentii Abo SlOstam het helder sones op die differensiële media agar plate getoon en die produksie van hoë fitase-aktiwiteit is in nat selle waargeneem na opkweking in vloeisto:finedia. Die C. laurentii Abo 510ras produseer maksimum fitase-aktiwieit by 'n redelike hoë temperatuur (62°C) en in 'n suur pH reeks (pH 5.0). Die fitase het ook 'n wye substraatspesifisiteit wat tot die vrystelling van fosfaat vanaf ander komponente in die veevoer mag bydra. Die fitase het geen metaalione vir sy aktiwiteit benodig nie, maar sekere metaalione het die fitase-aktiwiteit onderdruk. Die ensiem was redelik stabiel by 70°C en het na 180 minute blootstelling slegs 'n 40% verlies in aktiwiteit getoon. Die byvoeging van fosfaat in die kultuurmedium en in die ensiem reaksiemengsel teen konsentrasies bo 500 f.lM, het die fitase aktiwiteit heeltemalonderdruk. Verskeie koolstofbronne het ook 'n effek op die optimale fitase-aktiwiteit getoon. Die fitase ensiem is met die selwand geassosieer en het baie min intrasellulêre aktiwiteit getoon.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/49982
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