Fungal enzymes and microbial systems for industrial processing

De Villiers, Tania (2008-03)

Thesis (PhD)--Stellenbosch University, 2008.

Thesis

ENGLISH ABSTRACT: This study strives to improve two current industrial processes by making them more cost effective through the use of hydrolytic enzymes or microbial systems. The first process targeted is the industrial conversion of starch to ethanol. In the second process, hydrolytic enzymes are applied to the manufacturing of instant coffee. The engineering of microbial systems to convert starch to bio-ethanol in a one-step process may result in large cost reductions in current industrial processes. These reductions will be due to decreased heating energy requirements, as well as a decrease in money spent on the purchase of commercial enzymes for liquefaction and saccharification. In this study, a recombinant Saccharomyces cerevisiae strain was engineered to express the wild-type Aspergillus awamori glucoamylase (GA I) and α-amylase (AMYL III) as well as the Aspergillus oryzae glucoamylase (GLAA) as separately secreted polypeptides. The recombinant strain that secreted functional GA I and AMYL III was able to utilise raw corn starch as carbon source, and converted raw corn starch into bio-ethanol at a specific production rate of 0.037 grams per gram dry weight cells per hour. The ethanol yield of 0.40 gram ethanol per gram available sugar from starch translated to 71% of the theoretical maximum from starch as substrate. A promising raw starch converter was therefore generated. In the second part of this study, soluble solid yields were increased by hydrolysing spent coffee ground, which is the waste generated by the existing coffee process, with hydrolytic enzymes. Recombinant enzymes secreted from engineered Aspergillus strains (β-mannanase, β-endoglucanase 1, β-endo-glucanase 2, and β-xylanase 2), enzymes secreted from wild-type organisms (β-mannanases) and commercial enzyme cocktails displaying the necessary activities (β-mannanase, cellulase, and pectinase) were applied to coffee spent ground to hydrolyse the residual 42% mannan and 51% cellulose in the substrate. Hydrolysis experiments indicated that an enzyme cocktail containing mainly β-mannanase increased soluble solids extracted substantially, and a soluble solid yield of 23% was determined using the optimised enzyme extraction process. Soluble solid yield increases during the manufacturing of instant coffee will result in; (i) an increase in overall yield of instant coffee product, (ii) a decrease in amount of coffee beans important for the production of the product, and (iii) a reduction in the amount of waste product generated by the process.

AFRIKAANSE OPSOMMING: Hierdie studie poog om twee huidige industriële prosesse te verbeter deur die prosesse meer kosteeffektief met behulp van hidroltiese ensieme en mikrobiese sisteme te maak. Die eerste industrie wat geteiken word, is die omskakeling van rou stysel na etanol, en die tweede om hidrolities ensieme in die vervaardiging van kitskoffie te gebruik. Die skep van mikrobiese sisteme om rou-stysel in ’n ’een-stap’ proses om te skakel na bio-etanol sal groot koste besparing tot gevolg hê. Hierdie besparings sal te wyte wees aan die afname in verhittingsenergie wat tydens die omskakelingsproses benodig word, asook ’n afname in die koste verbonde aan die aankoop van duur kommersiële ensieme om die stysel na fermenteerbare suikers af te breek. In hierdie studie is ’n rekombinante Saccharomyces cerevisiae-gis gegenereer wat die glukoamilase (GA I) and α-amilase (AMYL III) van Aspergillus awamori, asook die glukoamilase van Aspergillus oryzae (GLAA) as aparte polipeptide uit te druk. Die rekombinante gis wat die funksionele GA I en AMYL III uitgeskei het, was in staat om op die rou-stysel as koolstofbron te groei, en het roustysel na bio-etanol teen ’n spesifieke tempo van 0.037 gram per gram droë gewig biomassa per uur omgeskakel. Die etanolopbrengs van 0.40 gram per gram beskikbare suiker vanaf stysel was gelykstaande aan 71% van die teoretiese maksimum vanaf stysel as substraat. ’n Belowende gis wat roustysel kan omskakel na bio-etnaol was dus geskep. In die tweede deel van hierdie studie is die opbrengs in oplosbare vastestowwe vermeerder deur die koffie-afval wat tydens die huidige industrieële proses genereer word, met hidrolitiese ensieme te behandel. Rekombinante ensieme afkomstig vanaf Aspergillus-rasse (β-mannanase, β-endoglukanase 1, β-endo-glukanase 2 en β-xilanase 2), ensieme deur wilde-tipe organismes uitgeskei (β-mannanase), asook kommersiële ensiempreparate wat die nodige ensiemaktiwiteite getoon het (β-mannanase, sellulase en pektinase) is gebruik om die oorblywende 42% mannaan en 51% sellulose in koffie-afval te hidroliseer. Hidrolise eksperimente het getoon dat ’n ensiempreparaat wat hoofsaaklik mannanase bevat, die oplosbare vastestofopbrengs grootliks kan verbeter, met ’n verhoogde opbrengs van 23% tydens geöptimiseerde ensiembehandelings. ’n Verhoogde opbrengs in oplosbare vastestowwe tydens die vervaardiging van kitskoffie sal die volgende tot gevolg hê: (i) ’n toename in totale opbrengs van kitskoffie produk, (ii) ’n afname in die hoeveelheid koffiebone wat vir die produksie ingevoer moet word, en (iii) ’n afname in die hoeveelheid afval wat tydens die vervaardigingsproses produseer word.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/21457
This item appears in the following collections: