การควบคุมการแสดงออกของยีนอัลคาไลน์โปรติเอสในรา Aspergillus oryzae สายพันธุ์ 4a

Regulation of the alkaline protease gene expression in Aspergillus oryzae strain 4a

Name: รวิสรา รื่นไวย์

Organization : มหาวิทยาลัยเทคโนโลยีพระจอมเกล้าธนบุรี

keyword: Aspergillus oryzne

; Alkaline protease

; Gene regulation

Abstract: Aspergillus oryzae has played a crucial role in many traditional Japanesefermentation industries because of its ability to secrete large amounts of varioushydrolytic enzymes such as amylases and proteases. This advantage served A. oryzaeas a favorable host for both of homologous and heterologous protein production.However, the presence of proteases in the culture fluid was a barrier to theproduction of extracellular proteins in A. oryzae. The understanding of regulation ofthe alkaline protease (alpA) gene expression, which represents the main extracellularprotease, would allow one to up-regulate or down-regulate the protease production.In A. oryzae strain 4a, studies of basic features of regulation of the alpA geneexpression by nutrients and ambient pH were carried out both at the transcriptionallevel and at the enzymatic activity level. During the first 24 h of cultivation in acomplete medium, northern blot analysis indicated that the alpA gene did not expressand extremely low enzyme activity was detected. To clarifL the effect of nutrientsand PH on the alpA gene expression, mycelia grown in a complete medium for 24 hwere transferred to a minimal salt medium (MSM) containing appropriatesupplements. The alpA gene was fully derepressed when 24 h old cultures weretransferred into MSM without supplement of carbon and nitrogen sources. The alpAgene was partially derepressed when lactose was used as a sole carbon source inMSM medium because of lactose was metabolized more slowly than glucose. Inglucose medium (MSM+glucose), extracellular pH was dropped since glucoseconsumption resulting in acidified pH and alpA transcript was not detected. ExternalpH was therefore controlled to 6.5 in order to provoke the expression of the alpAgene and enzyme production. It appeared that the controlled pH did not override therepressive effect of glucose. Ammonia and histidine, nitrogen sources in MSMneither repressed nor promoted the expression of alpA gene. On the other hand, onlytrace amounts of alkaline protease were detected when cysteine was added in MSM.Addition of low concentration (0.1%) of peptone and yeast extract enhanced theenzyme production when compared with MSM culture. Repression by glucose didnot alleviate by combination with non-repressing nitrogen. The presence of both non -repressing carbon and nitrogen sources in the culture derepressed but did notpromote alpA gene expression. In order to study optimal pH for alpA geneexpression, pH of MSM was maintained individually between pH 3.0 to 9.0.Controlling pH of minimal medium dramatically decreased the level of alpA mRNAand enzyme production. The alpA gene expression in MSM culture at pH 3 wascompletely repressed. At pH 5, 7, 8 and 9 the low levels of alpA were detected.Analysis of the alpA 5' non-coding sequence (up to 1,376 nucIeotides upstream fromstart codon) revealed the presence of several putative CREA-, AREA- and PACC-binding sites known to mediate carbon, nitrogen and pH regulation in many otherfilamentous fungi. However, no specifically DNA binding proteins could beidentified within the particular DNA fragments of interest (-1045 to -5 13).

มหาวิทยาลัยเทคโนโลยีพระจอมเกล้าธนบุรี

Address: กรุงเทพมหานคร (Bangkok)

Email: info.lib@mail.kmutt.ac.th

Name: Dr.taweerat Vichitsoonthonkul

Role: Supervisor

Created: 2544

Issued: 2005-10-21

Modified: 2006-05-24

วิทยานิพนธ์/Thesis

ISBN: 9746285009

CallNumber: BIT334

tha

DegreeName: วิทยาศาสตรมหาบัณฑิต

Level: ปริญญาโท

Descipline: เทคโนโลยีชีวภาพ

Grantor: มหาวิทยาลัยเทคโนโลยีพระจอมเกล้าธนบุรี

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