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Cellulose liquefaction under mild conditionsSabade, Sanjiv B. (Sanjiv Balwant) January 1983 (has links)
No description available.
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Implante dérmico acelular sintético no subcutâneo e na superfície da pele de cobaiasNatsuaki, Kryscia Leiko [UNESP] 16 July 2015 (has links) (PDF)
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000865259.pdf: 3255498 bytes, checksum: 5ab3f0da34d9a2202c8baaada085b6cc (MD5) / A Nanoskin® é uma película de celulose bacteriana produzida pela bactéria Acetobacter xylinum,por meio de um processo biotecnológico. É composta por uma rede de nanofibrilas cuja estrutura cria uma extensa superfície, a qual permite a retenção de grande quantidade de água e importantes modificações em seu formato, sem perder suas características estruturais. Objetivo: visando acrescentar novas possibilidades para a reconstrução da pálpebra, em seus folhetos profundo ou superficial, o presente estudo foi desenvolvido com o objetivo de avaliar se a película de Nanoskin® poderia ser uma opção. Método: foram utilizadas 40 cobaias, do sexo masculino, que receberam fragmentos de Nanoskin® na região dorsal, em dois estudos, um direcionado para a utilização da Nanoskin nos tecidos profundos, quando o biomaterial foi colocado no subcutâneo e outro no qual a Nanoskin foi colocada na superfície da pele. Em ambos os estudos foram utilizados dois tipos de Nanoskin: grupo 1 (G1), no qual foi utilizada película de Nanoskin® (2X2 cm) sem recobrimento de gelatina no subcutâneo ou na superfície da pele e o grupo 2 (G2), que recebeu implante de Nanoskin® (2X2 cm) com revestimento de gelatina no subcutâneo e na superfície da pele. O grupo controle foi obtido com colocação de enxerto de pele de espessura total no tamanho de 2X2 cm, contíguo ao implante de Nanoskin®, em todos os animais de G1 e de G2 do experimento que estudou o biomaterial na superfície da pele. Cinco animais de cada grupo foram eutanasiados em quatro momentos experimentais: 7 dias (M1), 30 dias (M2), 90 dias (M3) e 180 dias (M4). Foram realizadas avaliações morfométricas do implante das lâminas histológicas, exame histológico e exame ultraestrutural. Resultados: a Nanoskin® quando implantada no subcutâneo não foi encontrada em um animal de M3 e em cinco animais de M4. Nos momentos M3, M4 e M5, houve separação entre as suas lamelas. Houve... / The Nanoskin® is a bacterial cellulose film produced by the bacteria Acetobacter xylinum by means of a biotechnological process. It is composed by a network of nanofibrils whose structure creates a large surface area, which allows the retention of a large amount of water and significant changes in its shape without losing its structural characteristics. Purpose: aiming to add new possibilities for the reconstruction of the eyelid, in its superficial or deep lamellae, this study was developed in order to assess whether the Nanoskin® film could be an option. Method: 40 male guinea pig were used, that received Nanoskin® fragments in the dorsal region, in two studies, one directed to the use of Nanoskin® in deep tissue when the biomaterial was placed subcutaneously and another in which the Nanoskin® was placed on skin surface. In both studies we used two types of Nanoskin: group 1 (G1), which was used Nanoskin® film (2x2 cm) without gelatin coating on the subcutaneous or on the skin surface, and Group 2 (G2), which received Nanoskin® implant (2X2 cm) with gelatin coating on the subcutaneous and on the skin surface. The control group was obtained with full-thickness skin graft placement on the size of 2X2 cm Nanoskin® adjacent to the implant in all animals in G1 and G2 experiment that studied the biomaterial on the surface of the skin. Five animals from each group were euthanized at four experimental times: 7 days (M1), 30 days (M2), 90 days (M3) and 180 days (M4). Morphometric assessments of the implant and the histological slides were held, histological and ultrastructural examination. Results: the Nanoskin® when implanted subcutaneously was not found in one animal from M3 and in five animals from M4. In moments M3, M4 and M5, there was separation between their lamellae . There was a significant inflammation at the beginning of the experiment which reduced the following times, and formation of a pseudocapsule around the ...
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Cellulose liquefaction under mild conditionsSabade, Sanjiv B. (Sanjiv Balwant) January 1983 (has links)
No description available.
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The development of a soft and disposable cellulosic product by partial oxidation of cotton with oxides of nitrogenJohnson, Stuart January 1947 (has links)
M.S.
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In Vitro Determination of the Cellulose-Decomposing Rates of Twelve Denton County, Texas SoilsHeather, Carl D. 08 1900 (has links)
In this study twelve types of top soil were collected under aseptic conditions. The cellulose-decomposing rates of these were compared in order to determine the relative rates in the cellulose-decomposing potential of the microorganisms involved. Furthermore, this investigation is designed to acquire pertinent information on the rate at which natural cellulose materials are returned to available plant food.
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Expression and characterization of an intracellular cellobiose phosphorylase in Saccharomyces cerevisiaeSadie, Christa J. (Christiena Johanna) 03 1900 (has links)
Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Cellulose, a glucose polymer, is considered the most abundant fermentable polymer
on earth. Agricultural waste is rich in cellulose and exploiting these renewable
sources as a substrate for ethanol production can assist in producing enough
bioethanol as a cost-effective replacement for currently used decreasing fossil fuels.
Saccharomyces cerevisiae is an excellent fermentative organism of hexoses;
however the inability of the yeast to utilize cellulose as a carbon source is a major
obstruction to overcome for its use in the production of bio-ethanol. Cellobiose, the
major-end product of cellulose hydrolysis, is hydrolyzed by -glucosidase or
cellobiose phosphorylase, the latter having a possible metabolic advantage over
-glucosidase. Recently, it has been showed that S. cerevisiae is able to transport
cellobiose. The construction of a cellulolytic yeast that can transport cellobiose has
the advantage that end-product inhibition of the extracellular cellulases by glucose
and cellobiose is relieved. Furthermore, the extracellular glucose concentration
remains low and the possibility of contamination is decreased.
In this study the cellobiose phosphorylase gene, cepA, of Clostridium stercorarium
was cloned and expressed under transcriptional control of the constitutive PGK1
promoter and terminator of S. cerevisiae on a multicopy episomal plasmid. The
enzyme was expressed intracellulary and thus required the transport of cellobiose
into the cell. The fur1 gene was disrupted for growth of the recombinant strain on
complex media without the loss of the plasmid. The recombinant strain,
S. cerevisiae[yCEPA], was able to sustain aerobic growth on cellobiose as sole
carbon source at 30°C with Vmax = 0.07 h-1 and yielded 0.05 g biomass per gram
cellobiose consumed. The recombinant enzyme had activity optima of 60°C and
pH 6-7. Using Michaelis-Menten kinetics, the Km values for the colorimetric substrate
p-nitrophenyl-b-D-glucopyranoside (pNPG) and cellobiose was estimated to be 1.69
and 92.85 mM respectively. Enzyme activity assays revealed that the recombinant
protein was localized in the membrane fraction and no activity was present in the
intracellular fraction. Due to an unfavourable codon bias in S. cerevisiae, CepA
activity was very low. Permeabilized S. cerevisiae[yCEPA] cells had much higher
CepA activity than whole cells indicating that the transport of cellobiose was
inadequate even after one year of selection. Low activity and insufficient cellobiose transport led to an inadequate glucose supply for the yeast resulting in low biomass
formation. Cellobiose utilization increased when combined with other sugars
(glucose, galactose, raffinose, maltose), as compared to using cellobiose alone. This
is possibly due to more ATP being available for the cell for cellobiose transport.
However, no cellobiose was utilized when grown with fructose indicating catabolite
repression by this sugar.
To our knowledge this is the first report of a heterologously expressed cellobiose
phosphorylase in yeast that conferred growth on cellobiose. Furthermore, this report
also reaffirms previous data that cellobiose can be utilized intracellularly in
S. cerevisiae. / AFRIKAANSE OPSOMMING: Sellulose, ‘n homopolimeer van glukose eenhede, word beskou as die volopste
suiker polimeer op aarde. Landbou afval produkte het ‘n hoë sellulose inhoud en
benutting van diè substraat vir bio-etanol produksie kan dien as ‘n koste-effektiewe
aanvulling en/of vervanging van dalende fossielbrandstof wat tans gebruik word. Die
gis, Saccharomyces cerevisiae, is ‘n uitmuntende organisme vir die fermentasie van
heksose suikers, maar die onvermoë van die gis om sellulose as koolstofbron te
benut is ‘n groot struikelblok in sy gebruik vir die produksie van bio-etanol.
Sellobiose, die hoof eindproduk van ensiematiese hidrolise van sellulose, word
afgebreek deur -glukosidase of sellobiose fosforilase. Laasgenoemde het ‘n
moontlike metaboliese voordeel bo die gebruik van -glukosidase vir sellobiose
hidrolise. Daar was onlangs gevind dat S. cerevisiae in staat is om sellobiose op te
neem. Die konstruksie van ‘n sellulolitiese gis wat sellobiose intrasellulêr kan benut,
het die voordeel dat eindproduk inhibisie van die ekstrasellulêre sellulases deur
sellobiose en glukose verlig word. Verder, wanneer die omsetting van glukose vanaf
sellobiose intrasellulêr plaasvind, word die ekstrasellulêre glukose konsentrasie laag
gehou en die moontlikheid van kontaminasie beperk.
In hierdie studie was die sellobiose fosforilase geen, cepA, van Clostridium
stercorarium gekloneer en uitgedruk onder transkripsionele beheer van die
konstitutiewe PGK1 promoter en termineerder van S. cerevisiae op ‘n multikopie
episomale plasmied. Die ensiem is as ‘n intrasellulêre proteïen uitgedruk en het dus
die opneem van die sellobiose molekuul benodig. Die disrupsie van die fur1 geen
het toegelaat dat die rekombinante ras op komplekse media kon groei sonder die
verlies van die plasmied. Die rekombinante ras, S. cerevisiae[yCEPA], het aërobiese
groei by 30°C op sellobiose as enigste koolstofbron onderhou met mmax = 0.07 h-1 en
‘n opbrengs van 0.05 gram selle droë gewig per gram sellobiose. Die rekombinante
ensiem het optima van 60°C en pH 6-7 gehad. Die K m waardes vir die kolorimetriese
substraat pNPG en sellobiose was 1.69 en 92.85 mM onderskeidelik. Ondersoek
van die ensiem aktiwiteit het getoon dat die rekombinante proteïen gelokaliseer was
in die membraan fraksie en geen aktiwiteit was teenwoordig in die intrasellulêre
fraksie nie. CepA aktiwiteit was laag as gevolg van ‘n lae kodon voorkeur in S.
cerevisiae. Verder het geperforeerde S. cerevisiae[yCEPA] selle aansienlik beter CepA aktiwiteit getoon as intakte selle. Hierdie aanduiding van onvoldoende
transport van sellobiose na binne in die sel tesame met die lae aktiwiteit van die
CepA ensiem het gelei tot onvoldoende glukose voorraad vir die sel en min biomassa
vorming. Sellobiose verbruik het toegeneem wanneer dit tesame met ander suikers
(glukose, galaktose, raffinose, maltose) gemeng was, heelwaarskynlik deur die
vorming van ekstra ATP’s vir die sel wat ‘n toename in sellobiose transport teweeg
gebring het. Fruktose het egter kataboliet onderdrukking veroorsaak en sellobiose
was nie benut nie.
Sover ons kennis strek, is hierdie die eerste verslag van ‘n heteroloë sellobiose
fosforilase wat in S. cerevisiae uitgedruk is en groei op sellobiose toegelaat het.
Verder, bewys die studie weereens dat S. cerevisiae wel sellobiose kan opneem.
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Cloning of a novel Bacillus pumilus cellobiose-utilising system : functional expression in Escherichia coliVan Rooyen, Ronel, 1976- 12 1900 (has links)
Thesis (MScAgric)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Cellulose, a ~-1,4-linked polymer of glucose, is the most abundant renewable carbon source
on earth. It is well established that efficient degradation of cellulose requires the
synergistic action of three categories of enzymes: endoglucanases (EG), cellobiohydrolases
(CBH) and ~-glucosidases. ~-Glucosidases are a heterogenous group of enzymes that
display broad substrate specificity with respect to hydrolysis of cellobiose and different
aryl- and alkyl-ê-u-glucosides. They not only catalyse the final step in the saccharification
of cellulose, but also stimulate the extent of cellulose hydrolysis by relieving the cellobiose
mediated inhibition of EG and CBH. The ability to utilize cellobiose is widespread among
gram-negative, gram-positive, and Archaea bacterial genera. Cellobiose phosphoenolpyruvate-
dependent phosphotransferase systems (PTS) have been reported in various
bacteria, including: Bacillus species.
In this study, we have used a cellobiose chromophore analog, p-nitrophenyl-
~-D-glucopyranoside (pNPG), to screen a Bacillus pumilus genomic library for cellobiose
utilization genes that are functionally expressed in Escherichia coli. Cloning and
sequencing of the most active clone with subsequent sequence analysis allowed the
identification of four adjacent open reading frames. An operon of four genes (celBACH),
encoding a cellobiose phosphotransferase system (PTS): enzyme II (encoded by celB, celA
and celC) and a ó-phospho-f-glucosidase (encoded by celH) was derived from the sequence
data. The amino acid sequence of the celH gene displayed good homology with
~-glucosidases from Bacillus halodurans (74.2%), B. subtilis (72.7%) and
Listeria monocytogenes (62.2%). .As implied by sequence alignments, the celH gene
product belongs to family 1 of the glycosyl hydrolases, which employ a retaining
mechanism of enzymatic bond hydrolysis.
In vivo PTS activity assays concluded that the optimal temperature and pH at which the
recombinant E. coli strain hydrolysed pNPG were pH 7.5 and 45°C, respectively.
Unfortunately, at 45°C the CelBACH-associated activity of the recombinant strain was only
stable for 20 minutes. It was also shown that the enzyme complex is very sensitive to glucose. Since active growing cells metabolise glucose very rapidly this feature is not a
significant problem.
Constitutive expression of the B. pumilus celBACH genes in E. coli enabled the host to
efficiently metabolise cellobiose as a carbon source. However, cellobiose utilization was
only achievable in the presence ofO.01% glucose. This phenomenon could be explained by
the critical role of phosphoenolpyruvate (PEP) as the phosphate donor in PTS-mediated
transport. Glucose supplementation induced the glycolytic pathway and subsequently the
availability of PEP. Furthermore, it could be concluded that the general PTS components .
(enzyme I and HPr) of E. coli must have complemented the CelBACH system from
B. pumilus to allow functionality of the celBACH operon, in the recombinant E. coli host. / AFRIKAANSE OPSOMMING: Sellulose (' n polimeer van p-l,4-gekoppelde glukose) is die volopste bron van hernubare
koostof in die natuur. Effektiewe afbraak van sellulose word deur die sinnergistiese
werking van drie ensiernklasse bewerkstellig: endoglukanases (EG), sellobiohidrolases
(CBH) en P-glukosidases. p-Glukosidases behoort tot 'n heterogene groep ensieme met 'n
wye substraatspesifisiteit m.b.t. sellobiose en verskeie ariel- and alkiel-ê-n-glukosidiesc
verbindings. Alhoewel hierdie ensieme primêr as kataliste vir die omskakeling van
sellulose afbraak-produkte funksioneer, stimuleer hulle ook die mate waartoe sellulose
hidroliese plaasvind deur eindprodukinhibisie van EG en CBH op te hef. Sellobiose word
algemeen deur verskeie genera van die gram-negatiewe, gram-positiewe en Archae
bakterieë gemetaboliseer. Die sellobiose-spesifieke fosfoenolpirovaatfosfotransportsisteem
(PTS) is reeds is in verskeie bakterië, insluitende die Bacillus spesies,
beskryf.
In hierdie studie word die sifting van 'n Bacillus pumilus genoombiblioteek m.b.V. 'n
chromofoor analoog van sellobiose, p-nitrofeniel-p-o-glukopiranosied (pNPG), vir die
teenwoordigheid van gene wat moontlike sellobiose-benutting in Escherichia coli kan
bewerkstellig, beskryf. Die DNA-volgorde van die mees aktiewe kloon is bepaal en
daaropvolgende analiese van die DNA-volgorde het vier aangrensende oopleesrame
geïdentifiseer. 'n Operon (celBACH), bestaande uit vier gene, wat onderskeidelik vir die
ensiem II (gekodeer deur celB, celA en celC) en fosfo-B-glukosidase (gekodeer deur celH)
van die sellobiose-spesifieke PTS van B. pumilus kodeer, is vanaf die DNA-volgorde
afgelei. Die aminosuuropeenvolging van die celH-geen het goeie homologie met
P-glukosidases van Bacillus halodurans (74.2%), B. subtilis (72.7%) en
Listeria monocytogenes (62.2%) getoon. Belyning van die DNA-volgordes het aangedui
dat die celH geenproduk saam met die familie 1 glikosielhidrolases gegroepeer kan word.
Hierdie familie gebruik 'n hidrolitiese meganisme waartydens die stoigiometriese posisie
van die anomeriese koolstof behou word. PTS-aktiwiteit van die rekombinante E. coli ras, wat die celBACH gene uitdruk, is in vivo
bepaal. Die optimale temperatuur en pH waarby die rekombinante ras pNPG hidroliseer, is
onderskeidelik pH 7.5 en 45°C. Alhoewel die ensiernkompleks baie sensitief is vir glukose,
is dit nie 'n wesenlike probleem nie, omdat aktief groeiende E. coli selle glukose teen 'n
baie vinnige tempo benut.
Die celBACH operon het onder beheer van 'n konstitiewe promotor in E coli die
rekombinante gasheer in staat gestelom sellobiose as 'n koolstofbron te benut. Die
benutting van sellobiose word egter aan die teenwoordigheid van 'n lae konsentrasie
glukose (0.01 %) gekoppel. Hierdie verskynsel dui op die kritiese rol van fosfoenolpirovaat
(PEP) as die fosfaatdonor gedurende PTS-gebaseerde transport. Glukose speel waarskynlik
'n rol in die indusering van glikoliese, en sodoende die produksie van PEP as tussenproduk.
Verder kan afgelei word dat die algemene PTS komponente (ensiem I en HPr) van E. coli
die B. pumilis CelBACH-sisteem komplementeer en derhalwe funksionering van die
celBACH operon in E. coli toelaat.
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Genetic engineering of the yeast Saccharomyces cerevisiae to ferment cellobioseVan Rooyen, Ronel, 1976- 03 1900 (has links)
Dissertation (PhD)--Stellenbosch University, 2007. / PCT patent registered: https://www.google.com/patents/WO2009034414A1?cl=en&dq=pct/ib2007/004098&hl=en&sa=X&ei=b7AxUsSZK4jB0gWi14HgCQ&ved=0CEkQ6AEwAg
USA: https://www.google.com/patents/US20110129888?dq=pct/ib2007/004098&ei=b7AxUsSZK4jB0gWi14HgCQ&cl=en / USA patent registered: https://www.google.com/patents/US20110129888?dq=pct/ib2007/004098&ei=b7AxUsSZK4jB0gWi14HgCQ&cl=en / ENGLISH ABSTRACT: The conversion of cellulosic biomass into fuels and chemicals has the potential to positively
impact the South African economy, but is reliant on the development of low-cost conversion
technology. Perhaps the most important progress to be made is the development of “consolidated
bioprocessing” (CBP). CBP refers to the conversion of pretreated biomass into desired
product(s) in a single process step with either a single organism or consortium of organisms and
without the addition of cellulase enzymes. Among the microbial hosts considered for CBP
development, Saccharomyces cerevisiae has received significant interest from the biotechnology
community as the yeast preferred for ethanol production. The major advantages of S. cerevisiae
include high ethanol productivity and tolerance, as well as a well-developed gene expression
system. Since S. cerevisiae is non-cellulolytic, the functional expression of at least three groups
of enzymes, namely endoglucanases (EC 3.2.1.4); exoglucanases (EC 3.2.1.91) and
β-glucosidases (EC 3.2.1.21) is a prerequisite for cellulose conversion via CBP. The endo- and
exoglucanases act synergistically to efficiently degrade cellulose to soluble cellodextrins and
cellobiose, whereas the β-glucosidases catalyze the conversion of the soluble cellulose hydrolysis
products to glucose. This study focuses on the efficient utilization of cellobiose by recombinant
S. cerevisiae strains that can either hydrolyse cellobiose extracellularly or transport and utilize
cellobiose intracellularly.
Since it is generally accepted that S. cerevisiae do not produce a dedicated cellobiose
permease/transporter, the obvious strategy was to produce a secretable β-glucosidase that will
catalyze the hydrolysis of cellobiose to glucose extracellularly. β-Glucosidase genes of various
fungal origins were isolated and heterologously expressed in S. cerevisiae. The mature peptide
sequence of the respective β-glucosidases were fused to the secretion signal of the
Trichoderma reesei xyn2 gene and expressed constitutively from a multi-copy yeast expression
vector under transcriptional control of the S. cerevisiae PGK1 promoter and terminator. The
resulting recombinant enzymes were characterized with respect to pH and temperature optimum,
as well as kinetic properties. The maximum specific growth rates (μmax) of the recombinant
strains were compared during batch cultivation in high-performance bioreactors. S. cerevisiae
secreting the recombinant Saccharomycopsis fibuligera BGL1 enzyme was identified as the best
strain and grew at 0.23 h-1 on cellobiose (compared to 0.29 h-1 on glucose). More significantly, was the ability of this strain to anaerobically ferment cellobiose at 0.18 h-1 (compared to 0.25 h-1
on glucose).
However, extracellular cellobiose hydrolysis has two major disadvantages, namely glucose’s
inhibitory effect on the activity of cellulase enzymes as well as the increased risk of
contamination associated with external glucose release. In an alternative approach, the secretion
signal from the S. fibuligera β-glucosidase (BGL1) was removed and expressed constitutively
from the above-mentioned multi-copy yeast expression vector. Consequently, the BGL1 enzyme
was functionally produced within the intracellular space of the recombinant S. cerevisiae strain.
A strategy employing continuous selection pressure was used to adapt the native S. cerevisiae
disaccharide transport system(s) for cellobiose uptake and subsequent intracellular utilization.
RNA Bio-Dot results revealed the induction of the native α-glucoside (AGT1) and maltose
(MAL) transporters in the adapted strain, capable of transporting and utilizing cellobiose
intracellularly. Aerobic batch cultivation of the strain resulted in a μmax of 0.17 h-1 and 0.30 h-1
when grown in cellobiose- and cellobiose/maltose-medium, respectively. The addition of
maltose significantly improved the uptake of cellobiose, suggesting that cellobiose transport (via
the combined action of the maltose permease and α-glucosidase transporter) is the rate-limiting
step when the adapted strain is grown on cellobiose as sole carbon source. In agreement with the
increased μmax value, the substrate consumption rate also improved significantly from
0.25 g.g DW-1.h-1 when grown on cellobiose to 0.37 g.g DW-1.h-1 upon addition of maltose to the
medium. The adapted strain also displayed several interesting phenotypical characteristics, for
example, flocculation, pseudohyphal growth and biofilm-formation. These features resemble
some of the properties associated with the highly efficient cellulase enzyme systems of
cellulosome-producing anaerobes.
Recombinant S. cerevisiae strains that can either hydrolyse cellobiose extracellularly or transport
and utilize cellobiose intracellularly. Both recombinant strains are of particular interest when the
final goal of industrial-scale ethanol production from cellulosic waste is considered. However,
the latter strain’s ability to efficiently remove cellobiose from the extracellular space together
with its flocculating, pseudohyphae- and biofilm-forming properties can be an additional
advantage when the recombinant S. cerevisiae strain is considered as a potential host for future
CBP technology. / AFRIKAANSE OPSOMMING: Die omskakeling van sellulose-bevattende biomassa na brandstof en chemikalieë beskik oor die
potensiaal om die Suid-Afrikaanse ekonomie positief te beïnvloed, indien bekostigbare
tegnologie ontwikkel word. Die merkwaardigste vordering tot dusvêr kon in die ontwikkeling
van “gekonsolideerde bioprosessering” (CBP) wees. CBP verwys na die eenstap-omskakeling
van voorafbehandelde biomassa na gewenste produkte met behulp van ‘n enkele organisme of ‘n
konsortium van organismes sonder die byvoeging van sellulase ensieme. Onder die mikrobiese
gashere wat oorweeg word vir CBP-ontwikkeling, het Saccharomyces cerevisiae as die voorkeur
gis vir etanolproduksie troot belangstelling by die biotegnologie-gemeenskap ontlok. Die
voordele van S. cerevisiae sluit in hoë etanol-produktiwiteit en toleransie, tesame met ‘n goed
ontwikkelde geen-uitdrukkingsisteem. Aangesien S. cerevisiae nie sellulose kan benut nie, is die
funksionele uitdrukking van ten minste drie groepe ensieme, naamlik endoglukanases (EC
3.2.1.4); eksoglukanases (EC 3.2.1.91) en β-glukosidases (EC 3.2.1.21), ‘n voorvereiste vir die
omskakeling van sellulose via CBP. Die sinergistiese werking van endo- en eksoglukanases
word benodig vir die effektiewe afbraak van sellulose tot oplosbare sello-oligosakkariede en
sellobiose, waarna β-glukosidases die finale omskakeling van die oplosbare sellulose-afbraak
produkte na glukose kataliseer. Hierdie studie fokus op die effektiewe benutting van sellobiose
m.b.v. rekombinante S. cerevisiae-rasse met die vermoeë om sellobiose ekstrasellulêr af te breek
of dit op te neem en intrasellulêr te benut.
Aangesien dit algemeen aanvaar word dat S. cerevisiae nie ‘n toegewyde sellobiosepermease/
transporter produseer nie, was die mees voor-die-hand-liggende strategie die produksie
van ‘n β-glukosidase wat uitgeskei word om sodoende die ekstrasellulêre hidroliese van
sellobiose na glukose te kataliseer. β-Glukosidase gene is vanaf verskeie fungi geïsoleer en
daaropvolgend in S. cerevisiae uitgedruk. Die geprosesseerde peptiedvolgorde van die
onderskeie β-glukosidases is met die sekresiesein van die Trichoderma reesei xyn2-geen verenig
en konstitutief vanaf ‘n multikopie-gisuitdrukkingsvektor onder transkripsionele beheer van die
S. cerevisiae PGK1 promotor en termineerder uitgedruk. Die gevolglike rekombinante ensieme
is op grond van hul pH en temperatuur optima, asook kinetiese eienskappe, gekarakteriseer. Die
maksimum spesifieke groeitempos (μmax) van die rekombinante rasse is gedurende aankweking in
hoë-verrigting bioreaktors vergelyk. Die S. cerevisiae ras wat die rekombinante Saccharomycopsis fibuligera BGL1 ensiem uitskei, was as the beste ras geïdentifiseer en kon teen
0.23 h-1 op sellobiose (vergeleke met 0.29 h-1 op glukose) groei. Meer noemenswaardig is the ras
se vermoë om sellobiose anaërobies teen 0.18 h-1 (vergeleke met 0.25 h-1 op glukose) te
fermenteer.
Ekstrasellulêre sellobiose-hidroliese het twee groot nadele, naamlik glukose se onderdrukkende
effek op die aktiwiteit van sellulase ensieme, asook die verhoogde risiko van kontaminasie wat
gepaard gaan met die glukose wat ekstern vrygestel word. ’n Alternatiewe benadering waarin die
sekresiesein van die S. fibuligera β-glucosidase (BGL1) verwyder en konstitutief uitgedruk is
vanaf die bogenoemde multi-kopie gisuitrukkingsvektor, is gevolg. Die funksionele BGL1
ensiem is gevolglik binne-in die intrasellulêre ruimte van die rekombinante S. cerevisiae ras
geproduseer. Kontinûe selektiewe druk is gebruik om die oorspronklike S. cerevisiae
disakkaried-transportsisteme vir sellobiose-opname and daaropvolgende intrasellulêre benutting
aan te pas. RNA Bio-Dot resultate het gewys dat die oorspronklike α-glukosied (AGT1) en
maltose (MAL) transporters in die aangepaste ras, wat in staat is om sellobiose op te neem en
intrasellulêr te benut, geïnduseer is. Aërobiese kweking van die geselekteerde ras het gedui dat
die ras teen 0.17 h-1 en 0.30 h-1 groei in onderskeidelik sellobiose en sellobiose/maltose-medium.
Die byvoeging van maltose het die opname van sellobiose betekenisvol verbeter, waarna
aangeneem is dat sellobiose transport (via die gekombineerde werking van die maltose permease
en α-glukosidase transporter) die beperkende stap gedurende groei van die geselekteerde ras op
sellobiose as enigste koolstofbron is. In ooreenstemming hiermee, het die substraatbenuttingstempo
ook betekenisvol toegeneem van 0.25 g.g DW-1.h-1, gedurende groei op
sellobiose, tot 0.37 g.g DW-1.h-1 wanneer maltose by die medium gevoeg word. Die
geselekteerde ras het ook verskeie interessante fenotipiese kenmerke getoon, byvoorbeeld
flokkulasie, pseudohife- en biofilm-vorming. Hierdie eienskappe kom ooreen met sommige van
die kenmerke wat met die hoogs effektiewe sellulase ensiem-sisteme van sellulosomeproduserende
anaerobe geassosieer word.
Hierdie studie beskryf die suksesvolle konstruksie van ‘n rekombinante S. cerevisiae ras met die
vermoë om sellobiose ekstrasellulêr af te breek of om dit op te neem en intrasellulêr te benut.
Beide rekombinante rasse is van wesenlike belang indien die einddoel van industriële-skaal
etanolproduksie vanaf selluloseafval oorweeg word. Die laasgenoemde ras se vermoë om
sellobiose effektief uit die ekstrasellulêre ruimte te verwyder tesame met die flokkulasie, pseudohife- en biofilm-vormings eienskappe kan ‘n addisionele voordeel inhou, indien die
rekombinante S. cerevisiae ras as ‘n potensiële gasheer vir toekomstige CBP-tegnologie oorweeg
word.
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Cellulolytic and hemicellulolytic enzymes of flammulina velutipes.January 1994 (has links)
by Cheung Pui Yi. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 124-135). / Abstract --- p.ii / Acknowledgements --- p.iv / List of Tables --- p.viii / List of Figures --- p.ix / List of Abbreviations --- p.xiii / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- General Background --- p.1 / Chapter 1.2 --- Occurrence and Structure of Cellulose --- p.1 / Chapter 1.3 --- Occurrence and Structure of Hemicelluloses --- p.4 / Chapter 1.4 --- Biodegradation of Cellulose and Hemicelluloses --- p.4 / Chapter 1.4.1 --- Cellulolytic and Hemicellulolytic Microorganisms --- p.4 / Chapter 1.4.2 --- Enzymes Involved in Cellulose Degradation --- p.10 / Chapter 1.4.2.1 --- "Endo-1,4-β-glucanases" --- p.12 / Chapter 1.4.2.2 --- "Exo-1,4-β-glucanases" --- p.14 / Chapter 1.4.2.3 --- β-Glucosidases --- p.16 / Chapter 1.4.2.4 --- Oxidative Enzymes --- p.18 / Chapter 1.4.3 --- Synergistic Action between Cellulolytic Enzymes --- p.19 / Chapter 1.4.4 --- Enzymes Involved in Hemicellulose Degradation --- p.21 / Chapter 1.4.4.1 --- "Endo-1,4-β-xylanases" --- p.22 / Chapter 1.4.4.2 --- β-Xylosidases --- p.24 / Chapter 1.4.4.3 --- Other Xylanolytic Enzymes --- p.24 / Chapter 1.4.5 --- Synergistic Action between Hemicellulolytic Enzymes --- p.25 / Chapter 1.5 --- Flammulina velutipes --- p.26 / Chapter 1.6 --- Aims of the Present Investigation --- p.27 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Organism --- p.28 / Chapter 2.2 --- Culture Medium --- p.28 / Chapter 2.3 --- Determination of the Optimal Growth pH of Flammulina velutipes --- p.29 / Chapter 2.4 --- "Preparation of Inoculum, Cultivation and Harvest of Fungal Cultures" --- p.30 / Chapter 2.5 --- Enzyme Assays --- p.30 / Chapter 2.5.1 --- "Exo-1,4-β-glucanase" --- p.30 / Chapter 2.5.2 --- "Endo-1,4-β-glucanase" --- p.31 / Chapter 2.5.3 --- "Endo-1,4-β-xylanase" --- p.34 / Chapter 2.5.4 --- Extracellular β-Glucosidase --- p.36 / Chapter 2.5.5 --- Cell-Associated β-Glucosidase --- p.38 / Chapter 2.5.6 --- Extracellular β-Xylosidase --- p.38 / Chapter 2.5.7 --- Cell-Associated β-Xylosidase --- p.38 / Chapter 2.6 --- Determination of Optimal Temperatures for Cellulolytic and Xylanolytic Enzymes --- p.39 / Chapter 2.7 --- Determination of the Optimal pH for Enzyme Reaction --- p.39 / Chapter 2.8 --- Protein Determination --- p.39 / Chapter 2.9 --- Determination of Enzyme Induction Patterns --- p.42 / Chapter 2.10 --- Elucidation of Cellulase Production Patterns in F. velutipes --- p.43 / Chapter 2.10.1 --- Native Polyacrylamide Gel Electrophoresis --- p.43 / Chapter 2.10.2 --- Activity Staining for Endoglucanases --- p.43 / Chapter 2.10.3 --- Activity Staining for β-Glucosidases --- p.44 / Chapter 2.10.4 --- Protein Staining --- p.44 / Chapter 2.10.5 --- Preparative Polyacrylamide Gel Electrophoresis --- p.44 / Chapter 2.10.6 --- Separation of Proteins and Partial Purification of Different Cellulase Species after Preparative Polyacrylamide Gel Electrophoresis --- p.45 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Determination of the Optimal pH for Fungal Growth --- p.46 / Chapter 3.2 --- Determination of the Optimal Temperature for Cellulolytic and Xylanolytic Enzyme Activity --- p.48 / Chapter 3.3 --- Determination of the Optimal pH for Enzyme Reaction --- p.64 / Chapter 3.4 --- Time Course Experiments on the Production of Cellulolytic and Hemicellulolytic Enzymes --- p.72 / Chapter 3.4.1 --- Production of Cellulolytic Enzymes --- p.72 / Chapter 3.4.2 --- Production of Hemicellulolytic Enzymes --- p.77 / Chapter 3.5 --- Determination of Enzyme Induction Patterns --- p.82 / Chapter 3.5.1 --- Induction of Exoglucanase Production --- p.82 / Chapter 3.5.2 --- Induction of Endoglucanase Production --- p.84 / Chapter 3.5.3 --- Induction of Extracellular β-Glucosidase Production --- p.86 / Chapter 3.5.4 --- Induction of β-Xylanase Production --- p.88 / Chapter 3.5.5 --- Induction of Extracellular β-Xylosidase Production --- p.90 / Chapter 3.5.6 --- Changes in Extracellular Protein Levels in DMS Media Supplemented with Different Substrates --- p.92 / Chapter 3.5.7 --- Changes in Reducing Sugar Levels in DMS Media Supplemented with Different Substrates --- p.94 / Chapter 3.6 --- Partial Purification of Different Cellulases Species Produced by Flammulina velutipes --- p.96 / Chapter 3.6.1 --- Native Polyacrylamide Gel Electrophoresis --- p.96 / Chapter 3.6.2 --- Activity Staining for Endoglucanases --- p.96 / Chapter 3.6.3 --- Activity Staining for β-Glucosidases --- p.96 / Chapter 3.6.4 --- Assay of Cellulolytic Enzymes after Preparative Polyacrylamide Gel Electrophoresis --- p.101 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Optimal Conditions for Cellulolytic and Hemicellulolytic Enzymes of F. velutipes --- p.105 / Chapter 4.1.1 --- Optimal Temperature for Enzymic Reaction --- p.105 / Chapter 4.1.2 --- Optimal pH for Enzymic Reaction --- p.106 / Chapter 4.2 --- Production of Cellulolytic and Hemicellulolytic Enzymes --- p.109 / Chapter 4.2.1 --- Production of Cellulolytic Enzymes --- p.109 / Chapter 4.2.2 --- Production of Hemicellulolytic Enzymes --- p.110 / Chapter 4.3 --- Enzyme Induction Patterns --- p.111 / Chapter 4.4 --- Partial Purification of Different Cellulase Species Produced by Flammulina velutipes --- p.116 / Chapter 4.5 --- Conclusion --- p.121 / Chapter 4.6 --- Further Studies --- p.123 / List of References --- p.124
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Biodegradation of cellulose acetate reverse osmosis membranesBell, Pamela Elizabeth January 1981 (has links)
No description available.
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