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M. Tech. (Department of Biotechnology, Faculty of Applied and Computer Sciences), Vaal University of Technology. / During one of our preliminary studies in 2015, metagenomic DNA extracted from the goat rumen was sequenced and the in silico mining of the biorefining enzyme showed the presence of significant number of different biocatalysts, such as amylases (E.C 3.2.1.1), xylanases (E.C 3.2.1.8), pectinases (E.C 3.2.1.15) and cellulases (E.C 3.2.1.4). Hence, a subsequent study was conducted which is aimed at extracting metagenomic DNA from the goat rumen, constructing the metagenomic library using pCC2-FOS™ plasmid vector (Epicentre®), and eventually screening the constructed library for potential novel amylases using soluble starch as a substrate. Accordingly, rumen digesta was aseptically collected from four compartments of each goat and pulled before extraction of metagenomic DNA. The conventional CTAB protocol was modified to extract the metagenomic DNA from the rumen digesta. As a result, high molecular weight DNA was obtained and used to construct the metagenomic fosmid library. Since the host (Escherichia coli EPI 300-T1r) supplied with CopyControlHTP Fosmid Library Production Kit has background amylase expression we opted for a knockout E. coli strain with deleted starch hydrolysis (amylase expression) pathway. The library was subsequently screened for the presence of amylase isoforms using soluble starch as a substrate. Therefore, for the purpose of this study, four fosmids clones showing amylase activity were selected, recombinant vector isolated and MiSeq-sequenced. Out of four recombinant proteins, only one (pET30a(+)-amy-vut12) was successfully expressed. Subsequently, pET30a(+)-amyvut12 was further characterize physicochemically. Interestingly, the recombinant enzyme showed maximum activity in the pH and temperature ranges of 6.0 - 8.0 and 70 - 90oC, respectively. Hence, this implies that novel recombinant protein has sound activity from acidic to alkaline pH range and potently thermostable. Further work should be done to optimize and improve the solubility of three other recombinant proteins (pET30a(+)-amy-vut2, pET30a(+)- amy-vut9 and pET30a(+)-amy-vut14) studied, which might harbour important traits. Most importantly, immobilization as well as crystallographic studies of pET30a(+)-amy-vut12 and downstream applications should further be investigated.

Call number: LD2668 .T4 1985 G875 / Master of Science

The staling of baked products remains a significant cause of economic loss due to the loss of enjoyment seen as crumb firming occurs. The aims of the current project have been to investigate the stability of amylases in bakery formulations. In addition, the impact of partial hydrolysis products of starch on staling is investigated. Specific assays were used to measure ƒÑ-amylase and ƒÒ-amylase, in the presence of the other potentially interfering activity. ƒÑ-Amylase activity levels appeared to gradually increase during the proofing stages and then to decline upon heating of the dough. However, the activity remaining in the final baked loaf was readily measurable indicating that not all of the enzyme had been inactivated. Free and total ƒÒ-amylase activities were also measured and most was found to be in the free form. ƒÒ-Amylase was unstable with only relatively low activities remaining in the final baked loaf. It appears that of the two amylolytic enzymes, ƒÑ-a mylase is sufficiently stable that it may exert some impact on the crumb characteristics in the freshly baked product and during subsequent storage. In order to assess the likelihood that amylolysis is of significance to crumb characteristics, HPLC was used to analyse aqueous extracts for sugars. Commercial flours were found to contain low levels of sugars with maltose being the predominant sugar present. A number of commercial breads were also analysed and the composition found to vary between the different samples. Typically maltose was present at higher levels than the other sugars. When experimental loaves were analysed, the patterns showed that other sugars declined during proofing whereas maltose remained at readily measurable levels. Upon baking and subsequent storage the amounts of maltose increased. These results are consistent with the findings that some amylolytic activity remains in the baked product. In the third phase of this study, a potential means of investigating the role of particular carb ohydrates in product textures and staling rates was examined. The approach of spray drying was used to prepare microencapsulated maltodextrin. The encapsulating agents used were based upon rice starch and guar galactomannan. When these microcapsules were incorporated into the breadmaking formulation and baked, it appeared that softer crumb characteristics were achieved. The data also indicates an effect of delay in the staling rates. In a preliminary evaluation of the potential of two X-ray scattering methods, it was found that both techniques appear useful. The differences seen for samples of bread crumb analysed at various stages of storage did not show large differences in the intensity patterns. Of the two approaches, small angle analysis (SAXS) appears to show greater potential for application in ongoing studies of staling. In conclusion, cereal grain ƒÑ-amylase may be more stable during breadmaking than previously thought. There appears to be an increase in the level of some low molecular weight sugars in the final, baked product. Microencapsulation may offer a useful technique for the study of the role of specific carbohydrates during baking and storage of breads.

The effects of b-glucan on corn tortilla texture were evaluated. Barley flour (9.7% b-glucan) was substituted at 2.5, 5 and 10% for dry masa flour in corn tortillas. Texture was evaluated after 4 hr and up to 7 d storage at 4°C. Substitution of 2.5-10% barley flour significantly improved tortilla texture. Combined effects of barley flour (0-2.5%), maltogenic amylase (0-1650MAU) and carboxymethylcellulose (0-0.5%) were evaluated using surface response methodology. Barley flour increased rollability, pliability, energy dissipated and reduced rupture force and final stiffness. Overall, maltogenic amylase decreased rupture force and Young's modulus but decreased rupture distance, rollability and pliability at levels above 825 MAU. CMC improved rollability, pliability, and rupture distance. The best response was found using barley flour and CMC with 825 MAU, where rollability, pliability, rupture distance and energy dissipated increased while rupture force, Young's modulus and final stiffness decreased. A 70% barley b-glucan concentrate combined with amylase (550 MAU) or CMC (0-0.5%) was evaluated in corn tortillas. Amylase combined with b-glucan did not improve texture. Tortillas with b-glucan and CMC had significantly improved pliability, rollability, final stiffness and energy dissipated. Texture measurements analysis showed that depending on the stage of storage, objective and subjective methods correlate differently. Subjective and objective measurements of texture were not correlated at 4 hr storage. At the end of storage, pliability had significant correlations with stress relaxation measurements, but rollability had higher correlation coefficients with extensibility measurements. Pliability had higher R2 and lower coefficients of variation compared to rollability. Sensory evaluation was conducted using reheated 14-day-old tortillas of control, 825 MAU with 0.25% CMC, 0.12% b-glucans, 0.18% b-glucan with 0.375% CMC, and 0.24% b-glucan with 0.25% CMC. All tortillas had similar appearance, flexibility, gumminess, flavor and overall quality. Softness and chewiness of treatments with 0.12% b-glucan or 0.24% b-glucan with 0.25% CMC were similar to control. Other tortillas were significantly tougher and chewier. b-glucan may be the active ingredient in barley flour that modifies firming of corn tortillas during storage. Barley flour is inexpensive and effectively improves texture of corn tortillas.

Submitted by Geyciane Santos (geyciane_thamires@hotmail.com) on 2015-07-03T14:46:16Z No. of bitstreams: 1 Dissertação - Lanna Lôbo de Araújo.pdf: 1424509 bytes, checksum: e22f493ad51c66d665b74c545edb8568 (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2015-07-13T15:41:29Z (GMT) No. of bitstreams: 1 Dissertação - Lanna Lôbo de Araújo.pdf: 1424509 bytes, checksum: e22f493ad51c66d665b74c545edb8568 (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2015-07-13T15:45:47Z (GMT) No. of bitstreams: 1 Dissertação - Lanna Lôbo de Araújo.pdf: 1424509 bytes, checksum: e22f493ad51c66d665b74c545edb8568 (MD5) / Made available in DSpace on 2015-07-13T15:45:47Z (GMT). No. of bitstreams: 1 Dissertação - Lanna Lôbo de Araújo.pdf: 1424509 bytes, checksum: e22f493ad51c66d665b74c545edb8568 (MD5) Previous issue date: 2012-09-03 / FAPEAM - Fundação de Amparo à Pesquisa do Estado do Amazonas / There some decades the search for alternative energetic sources is mundialy on the focus. For environmental reasons economic and geopolitical this search has been strained and one of the sources of alternative energy that awakens the trend to replace fossil fuels, is the ethanol that is a cleaner fuel. Even more, companies, governments and researchers has been focused their reachers to it. The obtained alcohol by biomass fermentation is called "bioethanol". Brazil's position about the full use of biomass is very privileged because of its greatest world’s biodiversity, an intense solar radiation, abundance water, climate’s diversity and biomass from biomass products on a large scale, Brazil presents itself as the most advanced country in this technology once it is the currently largest bioethanol producer in the world. In this context the goal of this study is presenting the ethanol’s production viability from a efficiently hydrolyzate of cassava starch Manhiot esculenta Crantz which was obtained with the recombinant amylases (α-amylase and glucoamylase), resulting from the previous work developed in the research group by OLIVEIRA (2009) and CARMO (2010). The hydrolyzate was fermented by the yeast Saccharomyces cerevisiae commercial yeast on systems known as fermentometers. The hydrolyzate resulted in a 20 gL-1 ethanol concentration in 6 h, with hydrolysis efficiency of exceeding 57% volumetric productivity 3.33 g/L-1h-1 and yield factor of substrate consumption of 0.29 gg-1. / Há algumas décadas a busca por fontes energéticas alternativas está em foco no circuito mundial. Por questões ambientais, econômicas e geopolíticas essa procura tem se acirrado, e uma das fontes alternativas de energia, que desperta essa tendência em substituir os combustíveis fósseis é o etanol; que é um combustível menos poluente. E cada vez mais empresas, governos e pesquisadores têm voltado seu foco de pesquisa para ele. O álcool obtido por fermentação de biomassas é denominado “bioetanol”. A posição do Brasil em relação ao aproveitamento integral das biomassas é bastante privilegiada pelo fato do mesmo possuir a maior biodiversidade do planeta; uma intensa radiação solar; água em abundância; diversidade de clima e pioneirismo na produção de bicombustíveis da biomassa em larga escala, o Brasil apresenta-se como o país mais avançado nessa tecnologia já que atualmente é o maior produtor de bioetanol do mundo. Neste contexto, o estudo em questão teve como objetivo apresentar a viabilidade da produção de etanol a partir de um hidrolisado eficiente de fécula de mandioca Manihot esculenta Crantz que foi obtido com a utilização de amilases recombinantes, sendo uma α-amilase clonada de Bacillus licheniformis e expressa em Bacillus subtilis e uma glucoamilase clonada de Aspergillus awamori e expressa em Pichia pastoris, advindas de trabalhos anteriormente desenvolvidos em nosso grupo de pesquisa. O hidrolisado obtido foi fermentado pela levedura Saccharomyces cerevisiae comercial em sistemas conhecidos como fermentômetros. O hidrolisado resultou em uma concentração de 20 gL-1 de etanol em 6 h, com eficiência de hidrólise igual a 57%, produtividade volumétrica 3,33 gL-1h-1 e fator de rendimento em substrato consumido de 0,29 gg-1.

Alpha-amylase synthesis can be induced in wheat and barley half-seeds by addition of gibberellic acid (GA) to the incubation medium. In maize, induction in de-embryonated kernels by exogenous GA has been reported in some studies but not others. Alpha-amylase induction was investigated in maize by measuring activity in extracts from whole and de-embryonated kernels incubated with and without GA during germination. Alpha-amylase activity was first detected on the 3rd day of germination in whole kernels and GA-treated endosperms and on the 4th day in the controls. Thereafter both whole kernels and GA-treated endosperms followed approximately the same time course in α-amylase activity with the control lagging a day behind. Studies indicated that maximum α-amylase activity occurred on the 7th day in whole kernels and GA-treated endosperms and the 8th in control endosperms. Maize α-amylase was purified using differential solubility, column chromatography, glycogen precipitation and polyacrylamide gel electrophoresis, of these, the best purification method was glycogen precipitation. Maize α-amylase exhibited isozymes. The isozyme patterns were qualitatively similar in all samples and throughout incubation. Wheat and barley α-amylase isozymes have been divided into two groups on the basis of a number of characteristics. Genetics analysis revealed these isozymes to be the result of two multigene families. To shed light on the genetic basis of the maize α-amylase isozymes, physicochemical characterization was initiated. Studies of pH and temperature profiles and optima showed no differences between maize isozymes. The pH optima was pH 5 and the temperature optima was about 37°C. / Master of Science

A series of experiments was conducted to evaluate the effects of amylase supplementation upon the endogenous amylase levels and growth performance of male turkey poults. [In the first experiment, a multi-enzyme supplement containing amylase and an additional protease supplement were added to diets of low (24%) and high (28%) protein content. Enzyme supplements Significantly improved performance of birds on the low, but not on the high protein diet. Even with the improvement from enzyme addition, the performance of poults fed the low protein diet was not equal to poults fed the high protein diet. The second experiment was designed to evaluate the effect of varying levels of amylase and xylanase on bird performance, and to determine optimal level of enzyme inclusion in the basal corn-soybean meal diet. Feed utilization or growth of birds did not differ between the amylase diets, however 200 units of amylase per kilogram of feed produced numerically optimal growth and feed efficiency values. Xylanase inclusion greater than 160 units per kilogram of feed appeared to be detrimental to bird growth and feed efficiency. In the third experiment, endogenous amylase levels were measured to determine if supplemental amylase produced a quantitative, qualitative, or inhibitory effect upon the endogenous amylase activity. Amylase supplementation was found to be additive to the endogenous levels and did not inhibit endogenous activity. Amylase supplemented diets decreased body weight and feed efficiency loss due to weighing and handling stress on the birds when compared to the control and xylanase diets. Villi lengths within the jejunal and ileal sections of the small intestine were longer during the first three weeks for amylase supplemented birds when compared to intestinal villi of either the control or xylanase fed poults. The fourth experiment was conducted to compare the effect on growth and feed utilization of the serial addition of various enzyme preparations to corn-soybean poult diets. In retrospect, due to the serial application of the supplements, assessment of the benefits of a given supplement within a composite application was futile. Amylase, however, was present within the composite supplements fed to poults with the highest growth performance values. These experiments suggest that the addition of amylase to conventional corn-soybean meal turkey poult starter diets can be effective in improving growth and feed utilization during the first few weeks. The addition of amylase supplements to basal diets did not inhibit the poult endogenous amylase activity levels and were associated with increased intestinal villus length corresponding to increased utilization and absorption of nutrients. Xylanase appeared to be a detrimental enzyme additive to corn-soybean meal diets fed to male poults. / Ph. D.

Nucleic Acid Sequence Based Amplification (NASBA) is a powerful in vitro technique for amplification. NASBA is routinely used in many fields of microbiology, including food microbiology, and most recently in the identification of forensic body secretions (saliva, tears, sweat, semen, vaginal secretions). NASBA has many advantages over the traditional Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) including speed, high sample throughput and increased sensitivity. Proper selection of the sequence of importance and the designe of NASBA primers precisely for that sequence are the two most critical steps for any NASBA assay. Proper designe of NASBA primers includes important considerations such as product (amplicon) length, the addition of a T7 RNA polymerase promoter sequence at the 5’end of one primer, and a 3’AT rich region. Primer designing is, therefore, laborious and error-prone. Currently, no such software is available that facilitates primer designing for NASBA. In this study Java-based software for designing NASBA primers was developed which will enable rapid and specific NASBA primer designing for gene expression studies. The designed program focused on scripting minimum Java coding lines to reduce the time and storage space. Two codes were scripted for this software, a pseudo-code (for Java Program Developers) and Byte-code (for Windows operating system). Our results showed that Java is an efficient tool in searching sequences of interest within a gene (or mRNA), allowing for NASBA primers to be designed more quickly and effortlessly. The program has maximum memory storage capacity and allows the users to retrieve old data with reference to date or time. To test the practicality of the newly developed program, gene sequences of salivary mucin and amylase were examined to facilitate extraction of novel RNA tandem repeat element NASBA markers for human saliva forensic identification. Tandem repeats of non-coding portion with in the of human genome are highly polymorphic and considered best for forensic use. Currently, only 13 Short Tandem Repeat (STR) markers are available for forensic case work, which are not enough to establish a definitive link between the victim and suspects. Identification and validation of a new human body fluid tandem repeat markers is cruicial for achieving high through put results and to exonerate the innocent. / Access to thesis permanently restricted to Ball State community only / Department of Biology

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv. / Härtill 5 uppsatser.