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Xylanase hyper-producer : the genome of the thermophilic fungus Thermomyces lanuginosusMchunu, Nokuthula Peace 08 August 2014 (has links)
Submitted in complete fulfillment of the requirements for the Degree of Doctor of Technology: Biotechnology, Durban University of Technology, Durban, South Africa. 2014. / The global demand for green technology has created a need to search for microbes that can play an active role in advancing a greener and cleaner future. Microbial enzymes are nature’s keys to life and their efficiency, specificity and environmental-friendliness has lead to their increased use in industrial processes. Thermomyces lanuginosus is a thermophilic fungus that can degrade plant biomass and produces a variety of enzymes that have industrial application. The fungus T. lanuginosus SSBP has been reported in literature to produce the highest level of xylanase among other Thermomyces strains and some of its enzyme s viz., amylase and lipase are already being used. Because of this ability, it has been identified as one of the organisms that can have various industrial applications. Although a few proteins from this fungus have been cloned and used commercially, the vast majority are still unknown. In order to identify new protein candidates and understand their biochemical interactions, the T. lanuginosus genome (DNA) and the transcriptome (mRNA) were sequenced using 454 Roche and Solexa sequencing platforms. Genome and transcriptome data was assembled using Newbler software forming a genome size of 23.3 Mb contained 30 scaffolds. Protein prediction identified 5105 candidates as protein-coding genes and these gene models were supported by expressed sequence tag and transcriptomic data. The annotated data was assembled into metabolic pathways in order to identify functional pathways and validate the accuracy of the annotation process. T. lanuginosus is usually found in composting plant material thus protein related to plant hydrolysis were analysed. The total number of plant biomass-degrading and related proteins that fall into the carbohydrate-active enzyme (CAZy) family was 224. Most of these proteins were similar to proteins found in other filamentous fungi. Surprisingly, T. lanuginosus contained a single gene coding for xylanase which hydrolyses xylan although this organism is well known for being among the highest producers of this enzyme. An important subset of the above group of proteins is the cellulose degrading-proteins as this can be used in biofuel production. Eight candidates belonging to this group were identified, making this fungus significant in the biofuels. Among the eight cellulase candidates, phylogenetic analysis revealed that three of them were closely related to Trichoderma reesei, a well known industrial cellulase-producer. Utilization of cellulase-related compounds was validated by phenotypic microarray experiments, with cellobiose having inducing biomass in T. lanuginosus. Proteins that are involved in high temperature survival are vital for the survival.
of this thermophilic fungus. Interestingly, T. lanuginosus contains 19 heat shocking proteins which are responsible for thermostability. Another adaptation identified in this fungus is the accumulation of trehalose to combat heat stress. Furthermore, T. lanuginosus contains the highest reported number methyltransferases, which have been linked to producing thermostable proteins and higher energy production. Also because of this organism’s ability to grow on composting environments, the assimilation and ability to produce biomass on different carbon sources were analysed using phenotypic microarray technique. The results showed that xylose was the best compound to induce biomass followed by trehalose, maltose and maltotriose. The genomic sequencing of this fungus has provided valuable information that can be used for various biotechnological applications, as well as providing greater insights into its thermostability. Understanding the metabolic pathways involved may allow for manipulation to increase production of these enzymes or cloning into other hosts. This can have an impact in the field of biofuel production and other plant biomass-related processes.
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Improvement of thermostability of a fungal xylanase using error-prone polymerase chain reaction (EpPCR)Pillay, Sarveshni January 2007 (has links)
Thesis (M.Tech.: Biotechnology)-Dept. of Biotechnology, Durban University of Technology, 2007 vi, 92 leaves / Interest in xylanases from different microbial sources has increased markedly in the past decade, in part because of the application of these enzymes in a number of industries, the main area being the pulp and paper industry. While conventional methods will continue to be applied to enzyme production from micro-organisms, the application of recombinant DNA techniques is beginning to reveal important information on the molecular basis and this knowledge is now being applied both in the laboratory and commercially. In this study, a directed evolution strategy was used to select an enzyme variant with high thermostability. This study describes the use of error-prone PCR to modify the xylanase gene from Thermomyces lanuginosus DSM 5826, rendering it tolerant to temperatures in excess of 80°C. Mutagenesis comprised of different concentrations of nucleotides and manganese ions. The variants were generated in iterative steps and subsequent screening for the best mutant was evaluated using RBB-xylan agar plates. The optimum temperature for the activity of xylanases amongst all the enzyme variants was 72°C whilst the temperature optimum for the wild type enzyme was 70°C. Long term thermostability screening was therefore carried out at 80°C and 90°C. The screen yielded a variant which had a 38% improvement in thermostability compared to the wild type xylanase from pX3 (the unmutated gene). Successive rounds of error-prone PCR were carried out and in each round the progeny mutant displayed better thermostability than the parent. The most stable variant exhibited 71% residual activity after 90 minutes at 80˚C. Sequence analysis revealed four single amino acid residue changes that possibly enhanced their thermostabilities. This in vitro enzyme evolution technique therefore served as an effective tool in improving the thermostable property of this xylanase which is an important requirement in industry and has considerable potential for many industrial applications.
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Expression of a modified xylanase in yeastMchunu, Nokuthula Peace January 2009 (has links)
Submitted in fulfillment for the requirement of a Degree of Master of Technology: Biotechnology, in the Department of Biotechnology and Food Technology, Faculty of Applied Sciences, Durban University of Technology, Durban, South Africa, 2009. / Protein engineering has provided a key for adapting naturally-occurring enzymes for industrial processes. However, several obstacles have to be overcome after these proteins have been adapted, the main one being finding a suitable host to over-express these recombinant protein. This study investigated Saccharomyces cerevisiae, Pichia pastoris and Escherichia coli as suitable expression hosts for a previously modified fungal xylanase, which is naturally produced by the filamentous fungus, Thermomyces lanuginosus. A xylanase variant, NC38, that was made alkaline-stable using directed evolution was cloned into four different vectors: pDLG1 with an ADH2 promoter and pJC1 with a PGK promoter for expression in S. Cerevisiae, pBGP1 with a GAP promoter for expression in P. pastoris and pET22b(+) for expression in E. Coli BL21 (DE3). S. Cerevisiae clones with the p DLG1-NC38 combination showed very low activity on the plate assay and were not used for expression in liquid media as the promoter was easily repressed by reducing sugars used during production experiments. S. cerevisiae clones carrying pJC1-NC38 were grown in media without uracil while P. Pastoris clones were grown in YPD containing the antibiotic, zeocin and E. Coli clones were grown in LB with ampicillin. The levels of xylanase expression were then compared between P. Pastoris, S. cerevisiae and E. coli. The highest recombinant xylanase expression was observed in P. Pastoris with 261.7U/ml, followed by E.coli with 47.9 U/ml and lastly S. cerevisiae with 13.2 U/ml. The localization of the enzyme was also determined. In the methylotrophic yeast, P. Pastoris, the enzyme was secreted into the culture media with little or no contamination from the host proteins, while the in other hosts, the xylanase was located intracellularly. Therefore in this study, a mutated alkaline stable xylanase was successfully expressed in P. Pastoris and was also secreted into the culture medium with little or no contamination by host proteins, which favours the application of this enzyme in the pulp and paper industry. / National Research Foundation
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Overexpression and partial characterization of a modified fungal xylanase in Escherichia coliWakelin, Kyle January 2009 (has links)
Submitted in complete fulfillment for the Degree of Master of Technology (Biotechnology)in the Department of Biotechnology and Food Technology, Faculty of Applied Sciences, Durban University of Technology, Durban, South Africa, 2009. / Protein engineering has been a valuable tool in creating enzyme variants that are capable of withstanding the extreme environments of industrial processes. Xylanases are a family of hemicellulolytic enzymes that are used in the biobleaching of pulp. Using directed evolution, a thermostable and alkaline stabl xylanase variant (S340) was created from the thermophilic fungus, Thermomyces lanuginosus. However, a host that was capable of rapid growth and high-level expression of the enzyme in large amounts was required. The insert containing the xylanase gene was cloned into a series a pET vectors in Escherichia coli BL21 (DE3) pLysS and trimmed from 786 bp to 692 bp to remove excess fungal DNA upstream and downstream of the open reading frame (ORF). The gene was then re-inserted back into the pET vectors. Using optimized growth conditions and lactose induction, a 14.9% increase in xylanase activity from 784.3 nkat/ml to 921.8 nkat/ml was recorded in one of the clones. The increase in expression was most probably due to the removal of fungal DNA between the vector promoter and the start codon. The distribution of the xylanase in the extracellular, periplasmic and cytoplasmic fractions was 17.3%, 51.3% and 31.4%, respectively. The modified enzyme was then purified to electrophoretic homogeneity using affinity chromatography. The xylanase had optimal activity at pH 5.5 and 70°C. After 120 min at 90°C and pH 10, S340 still displayed 39% residual activity. This enzyme is therefore well suited for its application in the pulp and paper industry. / National Research Foundation
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Evaluation of commercial enzymes for the bioprocessing of Rooibos teaCoetzee, Gerhardt 04 1900 (has links)
Thesis (MSc) -- University of Stellenbosch, 2005. / ENGLISH ABSTRACT: The Rooibos tea plant (Aspalathus linearis) is indigenous to South Africa and occurs
only in the Western Cape's Cedarberg region. Rooibos tea is produced from the
leaves and fine stems of the plant. The tea is normally prepared by brewing the leaves
and consuming the liquor. However, the Rooibos plant is not only used to prepare
tea; the plant extracts are also used in various neutraceutical and pharmaceutical
products, including health drinks, iced tea, soaps and moisturising creams.
Although the tea plant contains native enzymes responsible for the colour and aroma
development of Rooibos tea, the disruption and maceration of the plant material
during processing is insufficient to allow these enzymes proper access to the
substrates responsible for Rooibos tea's characteristics. The current processing of
Rooibos tea is also time consuming and is done under uncontrolled conditions,
leading to unnecessary loss in aroma and antioxidant content. The addition of
enzymes could improve the maceration of the plant material, shorten the processing
time and improve the extraction of aroma, colour and antioxidant components.
During this study, 16 commercially available microbial enzymes were evaluated on
three different Rooibos substrates for the improvement of aroma and colour
development, as well as the extraction of soluble solids (SS) and total polyphenols
(TP). Thirteen enzymes were evaluated on spent tea for the enhanced extraction of
soluble solids and to determine the best candidates for further evaluation on fermented
and green Rooibos tea. Seven of the enzymes improved the yield in SS from spent
tea. Up to 232% improvement was obtained, depending on the type of enzyme and
dosage applied.
The best six enzyme preparations were further evaluated on fermented Rooibos tea.
For Depol™ 670L at 20 ul/g tea, the laboratory treatment increased the yield in SS by
44%, while small-scale industrial simulations increased the SS by 26%. However, an
increase in the yield in SS was usually accompanied by a decrease in the %TP/SS
ratio, indicating that mainly inactive compounds were extracted. Based on the results
with the commercial enzymes, twelve "synthetic" enzyme cocktails, consisting of different combinations of commercial enzymes were designed, of which three
cocktails released increased amounts of SS without decreasing the %TP/SS ratio
significantly.
Thirteen enzymes were evaluated on dried and freshly cut green Rooibos tea, with
three enzymes (Depol™ 670L, Pectinex Ultra SP-L and Depol™ 692L) increasing the
yield in SS between 21% and 66%, and the TP content between 11% and 47%.
Laccase was the best candidate in improving colour development from green tea, with
the improvement being slightly better at 50°C than at 40°C.
All the "synthetic" cocktails containing laccase improved the colour extract of all
three substrates evaluated, but also significantly decreased the TP and antioxidant
content. However, lower dosages of laccase resulted in colour development with little
loss in the antioxidant content. Due to the promising results obtained with the
treatments of Rooibos tea with laccases, it was decided to clone and express the
laccase gene (lacA) of Pleurotus ostreatus into Aspergillus niger. The gene was
successfully transformed into A. niger, but the expression of the recombinant gene
was not effective. / AFRIKAANSE OPSOMMING: Die Rooibostee plant (Aspalathus linearisi is inheems tot Suid-Afrika en kom slegs in
die Sederberg-omgewing in die Wes-Kaap voor. Rooibostee word van die blare en
fyn stingels van die plant geproduseer. Die tee word normaalweg voorberei deur die
blare in kookwater te laat trek en dan die aftreksel te drink. Die Rooibos plant word
nie net gebruik om tee te maak nie; die tee ekstrak word ook gebruik vir verskeie
neutraseutiese en farmaseutiese produkte, insluitende gesondheidsdrankies, ystee,
seep en bevogtigingsrome.
Ten spyte daarvan dat die teeplant sy eie ensieme vir die kleur en aroma ontwikkeling
van Rooibostee bevat, is die verbreking en maserasie van die plantmateriaal tydens
prosessering onvoldoende om die ensieme genoeg toe gang tot die substrate
verantwoordelik vir die kenmerkende eienskappe van Rooibostee te gee. Die huidige
prosessering van Rooibostee is ook tydrowend en geskied onder onbeheerde
toestande, wat tot 'n onnodige verlies in aroma en antioksidante lei. Die toevoeging
van ensieme kan die afbraak van die plantmateriaal verbeter, die behandelingsproses
verkort en die aroma, kleur en antioksidant inhoud van ekstrakte verbeter.
Tydens hierdie studie is 16 kommersieel-beskikbare mikrobiese ensieme op drie
verskillende Rooibos substrate vir die verbetering van aroma, kleur en ekstraksie van
oplosbare vastestowwe (SS) en totale polifenole (TP) getoets. Dertien ensieme is op
oorskot tee vir die verbeterde ekstraksie van oplosbare vastestowwe geevalueer,
waama die beste kandidate vir evaluering op gefermenteerde en ongefermenteeede
Rooibostee gekies is. Sewe ensieme het die SS vanaf oorskot tee verhoog. Tot 232%
verhoging is waargeneem, afhangende van die tipe ensiem en die dosis wat gebruik is.
Die beste ensiern preparate IS verder op gefermenteerde Rooibostee geevalueer,
Labarotoriurn behandelings met Depol™ 670L teen 20 ul/g tee het die SS inhoud met
44% verhoog, terwyl die kleinskaalse industriele simulasie die SS inhoud met 26%
verhoog het. 'n Verhoging in SS het egter gewoonlik met 'n afname in die %TP/SS
verhouding gepaard gegaan, wat aandui dat hoofsaaklik onaktiewe stowwe vrygestel
IS. Na aanleiding van die resultate met die kommersiele ensieme, is twaalf "sintetiese" ensiemmengsels met verskillende ensiemkombinasies getoets, waarvan
drie mengsels ook meer SS vrygestel het met byna geen verlaging in die %TP/SS
verhouding nie.
Dertien ensieme was op gedroogde en vars gekerfde groen Rooibostee getoets met
drie ensieme (Depol™ 670L, Pectinex Ultra SP-L en Depol™ 692L) wat die SS met
tussen 21% en 66%, en die TP inhoud met tussen 11% en 47% verhoog het. Lakkase
was die beste kandidaat vir die verbetering van kleur ontwikkeling by groen
Rooibostee met die verbetering effens beter by 50°C as by 40°C.
Al die "sintetiese" ensiem mengsels wat lakkase bevat het, het die kleur by al die
verskillende substrate verbeter, maar het ook die TP en antioksidant inhoud aansienlik
verlaag. Laer lakkase dosisse het goeie kleurontwikkeling tot gevolg gehad met
minimale verlies in die antioksidant inhoud. Vanwee die goeie resultate wat met die
lakkase behandelings verkry is, is daar besluit om die lakkase geen (lacA) van
Pleurotus ostreatus te kloneer en in Aspergillus niger uit te druk. Die geen is
suksesvol in A. niger getransformeer, maar die uitdrukking daarvan was nie effektief
nie.
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The use of enzymes for increased aroma formation in wineStidwell, Tanya Gwendryth 12 1900 (has links)
Thesis (MSc)--University of Stellenbosch, 2001. / ENGLISH ABSTRACT: Monoterpene alcohols (monoterpenols) play an important role in the flavour and aroma of
grapes and wine. This is especially applicable to wines of a muscat variety, but these
flavour compounds are also present in other non-muscat grape varieties, where they
supplement other varietal flavours and aromas. These monoterpenols can be found in grapes and wine as free, volatile and odorous molecules, as well as in flavourless, nonvolatile
glycosidic complexes. These complexes most often occur as
6-0-a-L-arabinofuranosyl-p-D-glucopyranosides (vicianosides), 6-0-P-D-xylopyranosyl-
P-D-gluco-pyranosides (primverosides), 6-0-P-D-glucopyranosyl-p-D-glucopyranosides
(gentio-biosides ), 6-0-a-L -rhamnopyra nosyl-p-D-g lucopyra nos ides (rutinos ides), or
6-0-p-D-apiofuranosyl-p-D-glucopyranosides of mainly linalool, geraniol, nerol, a-terpineol
and hotrienol. These precursors are, however, hydrolyzed only to a limited extent by
endogenous glycosidases during the fermentation process, as they exhibit very low activity
in wine conditions.
The monoterpenols can be released from their sugar moieties by one of two
methods: either an acid or an enzymatic hydrolysis. The enzymatic hydrolysis mechanism
is fully understood, and the process functions in two successive steps: firstly, depending on
the precursor, the glycosidic linkage is cleaved by an a-L-arabinofuranosidase, an
a-L-rhamnosidase, a p-D-xylosidase, or a p-D-apiosidase. The second step involves the
liberation of the monoterpene alcohol by a p-glucosidase. This enzymatic hydrolysis does
not influence the intrinsic aromatic characteristics of the wine, as opposed to acid
hydrolysis.
As the endogenous grape glycosides of Vitis vinifera and the yeast Saccharomyces
cerevisiae show very low activity towards these aromatic precursors during the handling of
the juice and winemaking processes, the focus has increasingly fallen on introducing
exogenous p-glucosidases to wines and juices. Genes encoding p-glucosidases and
a-L-arabinofuranosidases have been cloned from various organisms, including bacteria,
fungi and yeasts. However, the activities and properties of these enzymes are not always
suitable for exploitation under winemaking conditions, where a low pH, low temperatures,
and high ethanol and glucose concentrations prevail. A genetically engineered wine yeast
strain of S. cerevisiae that expresses glycosidases that are active in these conditions would
be useful in improving the flavour and aroma of wines, thereby adding to the complexity
and value of the wine.
Two p-glucosidase genes, BGL 1 and BGL2 from Saccharomycopsis fibufigera, were
subcloned into two Escherichia coli-yeast shuttle vectors. A dominant selectable marker
gene (SMR1) was also inserted onto these plasmids. These plasmids were designated pBGL 1 (containing the BGL 1 gene) and pBGL2 (containing the BGL2 gene) respectively.
Introduction of the two plasmids into two strains of S. cerevisiae then followed. A laboratory
strain, L1278, was transformed to confirm the effective secretion of the expressed protein.
An industrial yeast strain, VIN13, was subsequently transformed by making use of the
selectable marker (resistance against sulfometuron). Enzyme assays with the synthetic
substrate p-nitrophenol-j3-D-glucopyranoside (pNPG) were performed to determine the
activity of the j3-glucosidases over a period of days, as well as at certain temperatures and
pH values. The stability of the enzymes was also investigated.
These recombinant yeasts were able to degrade the pNPG efficiently. They showed
promising results concerning pH optima, with a substantial amount of activity found at the
pH levels as found in the wine environment. There was also a slight increase in specific
activity at lower temperatures. The recombinant yeast strains were also tested in smallscale
fermentations. Three wines were made, of which two were from white cultivars
(Chenin blanc and GewOrtztraminer) and one from red (Pinotage). Results obtained from
micro-extraction from the finished wines showed that the terpenol content did increase,
although this was not the only wine component influenced. Other flavour compounds also
showed increases, especially the esters. This also played a role in the flavour increase in
the wine.
Future work would include optimizing the available results. This would entail the
addition of another glycosidic enzyme, such as a-L-arabinofuranosidase, to the genome of
the wine yeast to aid the further breakdown of glycosidic bonds. The cloning or engineering
of a j3-glucosidase enzyme that is more active at low temperatures would also yield better
results and release even more of the aroma of the wine. / AFRIKAANSE OPSOMMING: Monoterpeenalkohole (monoterpenole) speel 'n belangrike rol in die geur en aroma van
druiwe en wyn. Dit is veral van toepassing op wyne van Muskaat-varieteite, maar hierdie
geurkomponente is ook teenwoordig in ander nie-Muskaat druifsoorte, waar dit bydra tot
die varieteitsqeur en aroma. Hierdie monoterpenole kom voor in druiwe as vry, vlugtige en
aromatiese molekules, of as geurlose, nie-vlugtige glikosidies-gebonde komplekse. Hierdie
komplekse is meestal in die vorm van 6-0-a-L-arabinofuranosiel-~-D-glukopiranosiede, 6-
O-~-D-xilopiranosiel-~-D-glukopiranosiede (primverosiede), 6-0-~-D-glukopiranosiel-~-Dglukopiranosiede
(gentiobiosiede), 6-0-a-L-ramno-pyranosiel-~-D-glukopiranosiede
(rutinosiede), of 6-0-~-D-apiofuranosiel-~-D-glukopirano-siede van hoofsaaklik linalool,
geraniol, nerol, a-terpineol en hotrienol. Hierdie geurvoorlopers word egter slegs tot In
beperkte mate tydens die proses van fermentasie deur die endogene glikosidase ensieme
gehidroliseer, aangesien hulle baie min aktiwiteit toon onder wynbereidingstoestande.
Die monoterpenole kan op een van twee wyses van hul suikermolekules vrygestel
word: 'n suurhidrolise, of ensiematiese hidrolise. Die ensiematiese hidroliseproses word
baie goed begryp en behels twee opeenvolgende stappe: eerstens, afhangende van die
aard van die voorloper, word die glikosidiese verbinding deur In a-L-arabinofuranosidase, In
a-L-ramnosidase, In ~-D-xilosidase, of 'n ~-D-apiosidase gebreek. In die tweede stap word
die monoterpeenalkohol deur In ~-glukosidase vrygestel. Hierdie ensiematiese
afbraakproses verander nie die intrinsieke aromatiese kenmerke van die wyn, soos wat met
suurhidrolise die geval is nie.
Omdat die endogene glikosidases van Vitis vinifera en die van die gis
Saccharomyces cerevisiae baie lae aktiwiteit ten opsigte van die aromatiese voorlopers
gedurende die hantering van die druiwesap en wynmaakprosesse toon, val die fokus al hoe
meer op die inkorporering van eksogene ~-glukosidases in wyn en sappe. Gene wat vir ~-
glukosidases en a-L-arabinofuranosidases kodeer, is al vanuit verskeie organismes
gekloneer, insluitende bakteriee, fungi en giste. Die aktiwiteite en kenmerke van hierdie
ensieme is egter nie altyd wenslik vir hul gebruik in wyn nie, aangesien dit In omgewing is
met 'n lae pH, lae temperatuur, en hoe etanolvlakke en glukose-konsentrasies. In geneties
veranderde wyngis van S. cerevisiae wat in staat is om glikosidases uit te druk wat onder
hierdie kondisies aktief is, sal baie handig te pas kom in die verbetering van die geur en
aroma van wyne, om daardeur die kompleksiteit en die waarde van die wyn te verhoog.
Twee ~-glukosidasegene, BGL 1 en BGL2 vanaf die gis Saccharomycopsis
fibuligera , is in twee afsonderlike Esccherichia coli-gis-pendelplasmiede gesubkloneer. In
Dominante selekteerbare merkergeen (SMR1) is ook in hierdie plasmiede gekloneer.
Hierdie plasmiede word onderskeidelik pBGL 1 (met die BGL 1-geen) en pBGL2 (bevattende die BGL2-geen) genoem. Hierdie twee plasmiede is hierna apart na twee rasse van
S. cerevisiae getransformeer. Eerstens is 'n laboratoriumras, L1278, getransformeer om te
bevestig dat effektiewe sekresie en uitdrukking van die proteTen plaasvind. Hierna is 'n
industriele gisras, VIN13, getransformeer deur gebruik te maak van die selektiewe merker
(bestandheid teen sulfometuron). Ensiem-bepalings met behulp van die sintetiese
substraat p-nitrofeniel-p-O-glukopiranosied (pNPG) is gedoen om die aktiwiteit van die
p-glukosidqses oor 'n aantal dae te bepaal, asook om die aktiwiteit by sekere temperature
en pH-vlakke te meet. Die stabiliteit van die ensieme is ook bepaal.
Hierdie rekombinante giste was in staat om pNPG effektief af te breek. Hulle het
belowende resultate betreffende die pH-optima getoon, met 'n aansienlike hoeveelheid
aktiwiteit by die pH-vlakke soos dit in die wynomgewing voorkom. Daar was ook 'n effense
verhoging in die ensieme se aktiwiteite by laer temperature. Die rekombinante gisrasse is
ook in kleinskaalse wynfermentasies gebruik. Drie verskillende wyne is gemaak, waarvan
twee wit kultivars was (Chenin blanc en GewOrtztraminer) en een 'n rooi kultivar (Pinotage).
Resultate wat deur die mikro-ekstraksie van die voltooide wyne verkry is, het getoon dat die
terpenolinhoud wei verhoog het, alhoewel dit nie die enigste wynkomponente was wat
beinvloed is nie. Ander geurkomponente het ook 'n verhoging in konsentrasie getoon, veral
die esters. Hierdie verbindings het ook 'n rol in die verhoging van geur in die wyne gespeel.
Toekomstige werk sal die beskikbare resultate verder optimaliseer. Dit sal insluit die
byvoeging van nog 'n glikosidiese ensiem, soos a.-L-arabinofuranosidase, tot die genoom
van die wyngis, om verdere afbraak van glikosidiese verbindings teweeg te bring. Die
klonering of verandering van 'n p-glukosidase-ensiem met verhoogde aktiwiteit by laer
temperature sal ook beter resultate toon en meer geur in die wyn kan vrystel.
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Fungal enzymes and microbial systems for industrial processingDe Villiers, Tania 03 1900 (has links)
Thesis (PhD)--Stellenbosch University, 2008. / 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.
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Sensory and chemical analysis of 1997 Oregon Pinot noir enzyme treated winesGoldberg, Naomi 04 December 1998 (has links)
Pinot noir has a reputation for lower color stability than other red wine varieties.
Because it has relatively low anthocyanin and phenolic content and lacks acylated
anthocyanin pigments compared to other red vinifera varieties, color extraction and
stability are particularly important. Varying the processing during fermentation of red
wine can produce high quality wines. Pectolytic enzymes are used in wine processing for
many purposes from increasing juice yield and filtering rates to improving color and
phenolic extraction.
Macerating enzymes used in this study, Scottzyme Color Pro (Scott
Laboratories), Scottzyme Color X (Scott Laboratories), Lallzyme EX (Lallemand), GB
Rapidase EX Color (Gist Brocades), and Vinozyme G (Cellulo) were added to 1997
Oregon Pinot noir must prior to fermentation to observe color, aroma and flavor changes.
These commercial enzymes have been reported to increase color and improve aroma and
flavor of red wines. The effect of these enzymes had not previously been investigated on
Oregon Pinot noir but the manufacturers reported increased polymeric phenols, polymeric
anthocyanins, tannins, color stability, red hue and saturation of red wine varieties. These
enzymes have varying manufacturer recommended usage levels and it is not known how the dosage levels and the enzymes themselves affect Oregon Pinot noir. Sensory
evaluations of these wines, at a high and low dosage level, were conducted through free-choice
profiling by winemakers and descriptive analysis from a trained panel. In addition,
chemical analyses were performed and related to sensory panel results.
Overall the addition of these enzymes to Oregon Pinot noir produced wines with greater
purple, red descriptors and higher color intensity than the control wine from trained
descriptive panel and winemaker panel results. In aroma, the enzyme treated wines were
higher in vegetative and earthy descriptors compared to the control. GB Rapidase EX
Color (Gist Brocades) was higher in bitterness flavor compared with other samples.
Low enzyme wine treatments separated wine samples more from the control then high
enzyme wine treatments. The color and appearance, aroma and flavor axes of the profile
maps were not significant in the high enzyme treated wines as determined from the
winemaker panel. Furthermore, the winemaker panel found acidity the only aroma or
flavor descriptor significant in the high dosage ANOVA results. Whereas six descriptors
in the low enzyme ANOVA results were significant in separating aroma and flavor
samples. Except for Lallzyme EX (Lallemand) treated wine, the hunter colorimeter results
showed all low enzyme treated wines were significantly (p<0.05) more red-purple (lower
hues) than the high enzyme treated wines. / Graduation date: 1999
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The chitinolytic enzyme system of the compost-dwelling thermophilic fungus Thermomyces lanuginosusZhang, Meng January 2014 (has links)
Submitted in complete fulfillment for the Degree of Master of Technology (Biotechnology), Durban University of Technology, Durban, South Africa, 2014. / Chitin, a highly insoluble 1,4- -linked polymer of N-acetyl- -D-glucosamine, is the second-most abundant bio-polysaccharide in nature after cellulose. Most chitinolytic fungi are known to produce more than one kind of chitinase. The recent sequencing of the Thermomyces lanuginosus SSBP genome by our group has revealed four putative family 18 chitinases. In this study, three novel chitinase genes (chitl, chit2 and chit3) and the previously reported chit4 gene were cloned from Thermomyces lanuginosus SSBP and their gene structures were analysed. chit3, encoding a 36.6 kDa protein, and chit4, encoding a 44.1 kDa protein, were successfully expressed in Pichia pastoris. The recombinant Chit3 and Chit4 enzymes exhibited optimum activity at pH 4.0 and
5.0 and at 40oC and 50°C, respectively. Chit3 was stable at 40oC and retained 71% of its activity at 50°C after 60 min, while Chit4 was stable at 50°C and retained 56% of its activity at 60°C after 30 min. Both enzymes produced chitobiose as the major product using colloidal chitin, chitooligosaccharides and shrimp shell powder as substrates. Of the fungal strains tested, Chit3 displayed antifungal activity against Penicillium sp. and Aspergillus sp. This is the first report on the multi-chitinolytic system of T. lanuginosus and enzyme characterization has shown the potential of the enzymes to be used in degradation of the under-utilized bio-resource chitin. / PDF Full-text unavailable. Please refer to hard copy for Full-text / M
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Cultivo submerso de Aspergillus empregando óleos vegetais visando a síntese de lipases para aplicação industrial. /Tacin, Mariana Vendrasco. January 2018 (has links)
Orientador: Valéria de Carvalho Santos Ebinuma / Coorientador: Ariela Veloso de Paula / Coorientador no exterior: Jose M. Palomo / Banca: Tales Alexandre da Costa e Silva / Banca: Marcdelo Chuei Matsudo / Banca: Fernando Masarin / Banca: Ana Lúcia Martiniano Nasser / Resumo: As lipases são enzimas amplamente aplicadas em processos industriais. Sua obtenção por fungos filamentosos apresenta algumas vantagens em relação às outras fontes e por isso estudos de incremento de sua produção vêm sendo realizados. Aliado ao processo de obtenção, técnicas de imobilização de enzimas podem melhorar sua estabilidade em relação à temperatura e pH, e diminuir custos associados à sua aplicação devido à possibilidade de reaproveitamento da enzima imobilizada nos processos biocatalíticos. Objetivo: este trabalho teve como objetivo estudar a produção de lipases pela cepa Aspergillus sp. DPUA 1727, a imobilização da referida enzima em suporte octil-sepharose, sua caracterização na forma livre e imobilizada e a aplicação da enzima imobilizada na obtenção de ésteres. Métodos: a produção da enzima foi realizada por 96 horas em cultivo submerso, avaliando-se diferentes fontes de carbono (óleos de soja, oliva, semente de uva e de algodão) como substratos indutores da produção enzimática. Posteriormente, estudou-se a imobilização da enzima em suporte octil-sepharose® por adsorção através de interação hidrofóbica, após o meio fermentado ter sido submetido à 4 ciclos de extração do óleo residual da fermentação com n-hexano. O processo de imobilização da enzima foi realizado em cascata em 4 ciclos com a finalidade de sobrecarga da enzima no suporte. Os testes de estabilidade foram feitos por 24 horas, nas temperaturas de 30 à 70ºC e pH de 3 a 9 com a enzima livre e imobilizad... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Lipases are enzymes widely applied in industrial processes. They can be obtained by filamentous fungi which presents some advantages in relation to other sources and for this reason, studies aiming to increase its production have been performed. Moreover, the process of enzyme immobilization can improve the enzyme stability in relation to temperature and pH, and reduce the costs associated with its application due to the possibility of immobilized enzyme reuse in biocatalytic processes. The objective of this work was to study the production of lipases by Aspergillus sp. DPUA 1727, the immobilization of lipases produced in octyl-sepharose support and make the enzyme characterization in its free and immobilized form. Furthermore, the immobilized enzyme was applied to obtain the ester isoamyl propionate. Methods: the enzyme production was carried out for 96 hours in submerged culture, with different carbon sources (soybean, olive, grape seed, and cotton oils) as substrates. Subsequently, the enzyme immobilization in octyl-sepharose® support by adsorption through hydrophobic interaction was studied ( to this experiment the fermented medium was submitted to 4 cycles to extract the residual oil with n-hexane). The enzyme immobilization process was carried out in cascade employing 4 cycles with the purpose of overloading the enzyme in the support. Stability studies were done for 24 hours, at temperatures from 30 to 70ºC and pH from 3 to 9 with the enzyme-free and immobilized. As the last step of this work, the immobilized enzyme was applied in the preparation of the ester isoamyl propionate. Results: In the production stage, the best condition to obtain the enzyme... (Complete abstract click electronic access below) / Doutor
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