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The objective of this study was to substitute cotton pretreatment chemicals with enzymes to create an environmentally friendly process for water and energy savings. In this study, enzyme selection and process optimisation was made in order to increase the glucose content of the desizing liquor of a starch-sized cotton fabric. Results indicated that commercial desizing enzyme formulations of a-amylase enzymes were not appropriate to produce a large quantity of glucose in the desizing bath; the glucose amounts obtained were about 200 mg/l. However the food market enzyme used, an amyloglucodidase/pullanase mixture (amyloglucosidase 186 Units/g, pullanase 395 Units/g), produced approximately 4000 mg/l glucose in the desizing bath after process optimisation. The desizing effect of the amyloglucodidase/pullanase enzyme mixture was compatible with those of commercial desizing enzymes. The results of peroxide generation from glucose and bleaching trials are reported in the second part of the study.

The effect of the granule structure on the methylation of starch was investigated by comparing the substitution patterns of potato starch methylated in granular suspension and in solution to DS 0.3. Substitution patterns were analyzed by successive digestion with α-amylase and amyloglucosidase, fractionation of the resulting malto-oligosaccharide mixture by GPC on a preparative scale, and characterization of the fractions by GLC and MALDI-MS. The mass composition of fractions with intermediate and higher degree of polymerization was indicative of enhanced clustering of substituents in granular methyl starch. On the contrary, the composition of the smaller saccharides was governed by enzyme specificity, which was also reflected in strong deviations in their monomer composition. A sequencing study on selected 'pure' small saccharides confirmed and complemented previous conclusions on enzyme specificity. © 2008 Elsevier Ltd. All rights reserved.

Amyloglucosidase (AMG) and exo-polygalacturonase (exo-PG) were simultaneously produced by two different strains of Aspergillus niger in solid-state fermentation (SSF) using defatted rice-bran as substrate. The effect of Aspergillus niger strain (t0005/007-2 and/or CCT 3312), inoculum type (spore suspension or fermented bran) and addition of inducers (pectin and/or starch) to the culture media was studied using a 3² x 2¹ factorial experimental design. The production of AMG and exo-PG was significantly affected by fungal strain and inoculum type but inducers had no effect. The maximum yields obtained were 1310 U/g dm for AMG using a spore suspension of A. niger CCT 3312 and 50.2 U/g dm for exo-PG production, using A. niger t0005/007-2 and fermented bran as inoculum. The yields obtained represented acceptable values in comparison with data available in the literature and indicated that defatted rice-bran was a good nutrient source.As enzimas amiloglicosidase (AMG) e exo-poligalacturonase (exo-PG) foram produzidas simultaneamente por duas cepas de Aspergillus niger, através de fermentação em estado sólido usando farelo de arroz desengordurado como substrato. Foram avaliados os efeitos da cepa de Aspergillus niger, tipo de inóculo e adição de indutores no meio de cultura, utilizando-se um planejamento experimental fracionário 3² x 2¹. O máximo rendimento obtido foi 1310 U/g ms para a produção de AMG e 50,2 U/g ms para a exo-PG. Comparando-se estes resultados com dados da literatura pode-se dizer que os rendimentos obtidos foram aceitáveis e indicam que o farelo de arroz desengordurado é uma boa fonte de nutrientes. A produção de AMG e exo-PG foi significativamente afetada pelas variáveis cepa de A. niger e tipo de inóculo, enquanto a variável indutor não apresentou influência significativa na produção destas enzimas.

Orientador: Yoon Kil Chang Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos Resumo: As sociedades modernas têm apresentado um crescente interesse por alimentos saudáveis, com teor calórico reduzido e livres de ingredientes, como açúcares, gorduras saturadas e ácidos graxos trans, mas que mantenham as características sensoriais agradáveis dos produtos originais. Este trabalho tem como objetivo apresentar uma alternativa de pão de forma sem adição de açúcar, gordura e emulsificantes, com a utilização de amido modificado por ligações cruzadas (fosfatado), acetilado e pré-gelatinizado de mandioca (AMPM) e das enzimas a-amilase, amiloglucosidase (AMG) e fosfolipase (FOSFO). Para tanto, foi elaborado um planejamento experimental do tipo composto central rotacional com três variáveis independentes: dosagem de amido modificado AMPM (x1), de amiloglucosidase AMG (x2) e fosfolipase FOSFO (x3). Analisaram-se as matérias-primas principais e os pães de forma foram elaborados pelo método de massa direta. Além dos parâmetros L*a*b de coloração do miolo e o volume dos pães após a produção, efetuou-se também a análise da umidade e da textura durante a vida de prateleira, após 24, 72 e 120 horas de estocagem. Dentro das faixas estudadas para as três variáveis, foi possível verificar que nenhuma delas teve influência estatisticamente significativa nas respostas analisadas, apesar do erro puro ter sido adequado para o processo de panificação utilizado, mostrando uma boa reprodutibilidade. Algumas amostras foram submetidas a um painel sensorial de consumidores para avaliação dos parâmetros de sabor e textura, sendo que a amostra com menores concentrações de AMPM, AMG e FOSFO recebeu a menor pontuação. Já as demais amostras apresentaram pontuação estatisticamente igual ao padrão, utilizando gordura, açúcar e emulsificante. De maneira geral, a melhor relação entre a aceitação dos consumidores, o custo da formulação e a porcentagem de retenção de umidade dos pães foram obtidos com a formulação utilizando 1,5% do amido modificado e atividade de fosfolipase de 960 PLU por kg de farinha de trigo Abstract: Modern societies are presenting a growing interest in healthy foods, low calories and free from undesirable ingredients such as sugar, saturated and ¿trans¿ fatty acids but maintaining sensorial characteristics of the original foods. This work intends to present an option of sliced bread with total substitution of sugar, fat and emulsifiers, with the use of a instant cross linked acetylated tapioca starch (AMPM) , a-amylase, amyloglucosidase (AMG) and phospholipase (FOSFO). For this, an experimental design with three independents variable was done: the dosage of modified starch (AMPM ¿ x1), amyloglucosidase (AMG ¿ x2) and phospholipase (FOSFO ¿ x3). The raw materials were analyzed and the bread was produced using straight dough process. Besides the color of the crumb and bread volume after the production the moisture content and texture during the shelf-life (24, 72 and 120 hours) were measured. None of the three variables had statistically significant effects on the results, even though the process showed good reproductibility. However, the same samples were tested in a sensorial panel for taste and texture attributes and the sample with the lower amounts of AMPM, AMG and FOSFO showed the worst score. The remaining samples received the same punctuation as the standard bread. Thus, the recipe with the best combination of cost, consumer acceptance and moisture retention was the bread with 1.5% of modified tapioca starch (AMPM) and phospholipase activity of 960 PLU / kg of wheat flour Mestrado Mestre em Tecnologia de Alimentos

The release of insulin hexamer (39 kD) and amyloglucosidase (AMG, 97 kD), entrapped in spherical Ca–alginate beads, was investigated. While the release of insulin could be described solely by diffusion this was not the case for the 1.6 (r m /r m) larger AMG protein, where rm is the Stokes–Einstein effective molecular radius. Because the alginate bead size was not constant during the release experiments, it was hypothesised that in addition to the diffusional mass transfer, a non-negligible convective flow of liquid in or out of the beads was present due to swelling or shrinkage, respectively. Although it should be expected that the effective diffusion coefficient of AMG is only slightly lower than that of insulin, the results show that the effective diffusions coefficient of AMG was found to be much smaller. In the case of AMG, it was shown that including bead size changes and the resulting convective flow in the numerical model, release could be described more accurately. The numerical model was able to describe the release characteristics from both shrinking, swelling, and non-swelling alginate beads. To evaluate the effect of bead swelling on the protein release rate, a swelling modulus and a release index was defined, describing the different effects on release of smaller and larger proteins. The release of insulin hexamer (39 kD) and amyloglucosidase (AMG, 97 kD), entrapped in spherical Ca–alginate beads, was investigated. While the release of insulin could be described solely by diffusion this was not the case for the 1.6 (r m /r m) larger AMG protein, where rm is the Stokes–Einstein effective molecular radius. Because the alginate bead size was not constant during the release experiments, it was hypothesised that in addition to the diffusional mass transfer, a non-negligible convective flow of liquid in or out of the beads was present due to swelling or shrinkage, respectively. Although it should be expected that the effective diffusion coefficient of AMG is only slightly lower than that of insulin, the results show that the effective diffusions coefficient of AMG was found to be much smaller. In the case of AMG, it was shown that including bead size changes and the resulting convective flow in the numerical model, release could be described more accurately. The numerical model was able to describe the release characteristics from both shrinking, swelling, and non-swelling alginate beads. To evaluate the effect of bead swelling on the protein release rate, a swelling modulus and a release index was defined, describing the different effects on release of smaller and larger proteins.

High operational stability and productivity of co-immobilised systems are important aspects for their successful application in industrial processes. A dynamic model is required to describe artificially co- immobilised systems because the time needed to reach steady state normally exceeds the operational life span of these systems. Time dependent intraparticle concentration profiles and macroscopic conversion were modelled to study the operational stability and productivity of these systems theoretically. The model was used to describe experimental results of ethanol production from maltose by a co-immobilised system of amyloglucosidase and Zymomonas mobilis. Furthermore, the influence of the immobilisation procedure with glutaraldehyde and polyethyleneimine could also be studied with and incorporated in the model. From the model it could be derived that co- immobilised systems performing a consecutive reaction evolve towards a steady state, characterised by a constant concentration of the intermediate in the particle if product inhibition is neglected. Such a situation develops independently of the biomass concentration and the radial position, and has important consequences for co-immobilised systems. When the concentration of the intermediate in the bulk liquid is lower than this constant value in the biocatalyst particle, two regions may be distinguished in the particle: an inactive peripheral region without biomass and an active core with a biomass concentration depending on the substrate and immobilised enzyme concentration. Unlike immobilised single cell systems, it is possible to obtain a real steady state and therefore a stable situation for co- immobilised systems. However, a high operational life time could only be achieved at the expense of the productivity of the biocatalyst particle. A stability criterion is derived which agrees very well with the simulation results.

Özet iv ÖZET Bu çalışmada sıcaklığa duyarlı NIPAAm/AAm (n-izopropil akrilamid/akrilamid) kopolimer matrisinde fiziksel olarak tutuklanmış amiloglikosidaz enzimi kullamlarak maltodekstrinden sürekli glikoz şurubu üretimi araştırılmıştır. Çalışma kapsamında öncelikle serbest amiloglikosidaz enziminin en yüksek aktivite verdiği sıcaklık ve pH aralığı tespit edilmiş ve bu optimum koşullarda başlangıç enzim yükünün ve substrat derişiminin D-glikoz ilk üretim hızına (aktiviteye) olan etkisi incelenmiştir. Sonuçta 0.01 mL başlangıç enzim yükü (aktivite bazında, 45.37 g/L/sa) kullamlarak yapılan deneylerde serbest enzimin T = 60 °C 'ta ve pH 4.5 asetat tamponunda en iyi çalıştığı ve bu koşullarda 100 g/L başlangıç substrat derişiminin en yüksek D-glikoz dönüşüm hızım sağladığı tespit edilmiştir. Substratlı ve substratsız ortamda serbest enzimin deaktivasyon hız sabitleri tespit edilmiş ve substrat ortamında deaktivasyon incelendiğinde daha önce literatürde karşılaşılmadığı üzere substrat derişimi ile değişen deaktivasyon hız sabitleri elde edilmiştir. Serbest enzim kinetiğini tespit için bilgisayar ortamında nonlineer regrasyon ile literatürdeki inhibisyonlu/inhibisyonsuz tüm enzimatik hız ifadeleri taranmış ve çalışılan bütün sıcaklıklarda (30-60 °C) en uygun ifadenin substrat inhibisyonu durumunda geçerli olan Haldane kinetiği formunda olduğu bulunmuştur. Bulunan model partikül içi `difüzyon+reaksiyon` çözümünde reaksiyon hız ifadesi olarak kullanılmıştır. Çalışmanın ikinci kısmında farklı NIPAAm/AAm bileşimlerinde partiküller hazırlanarak kopolimer matrisin şişme-kasılma kinetiği incelenmiş ve dinamik şişme ve kasılma için suyun partikül içindeki etkin difüzyon katsayıları 10`5 cm2/s mertebesinde hesaplanmıştır. Enzimin endüstriyel uygulamada kullanılan çalışma sıcaklığı olan 60 °C 'ta faz geçişi gösteren matrisin % NIPAAm değeri 80 (w/w) olarak tespit edilmiştir. Amiloglikosidaz enziminin NIPAAm/AAm kopolimer matrisine immobilizasyonu çalışmaları gerçekleştirilmiş, en yüksek ve kararlı enzim aktivitesini veren partikül hazırlama koşullan tespit edilmiştir. Sonuçta; 40 mL dağıtma fazında kullanılan başlatıcı (KPS) ve CaCİ2 miktarı sırasıyla 0.4 g ve 1.2 g olduğunda ve 3.6 mL monomer fazında kullanılan çapraz bağlayıcı (MBAAm); 0.033 g/mL, hızlandırıcı (TEMED); 0.3 mL, sodyum aljinat; 0.03 g, NIPAAm monomeri; 0.394 g, AAm monomeri 0.094 g, enzim yükü; 0.8 mL olduğunda elde edilen 2.7 mm çapındaki (+ 4 °C 'ta) partiküller gerek yüksek görünür aktivite ve gerekse kararlılık açısından en uygun bulunmuştur. Bu koşullarda hazırlanan partiküllerin gözlenir aktivitesinin gerçek immobilize enzim aktivitesinin % 14 'ü olduğu tespit edilmiştir. SEM ve FTIR analizinden elde edilen sonuçlar NIPAAm/AAm kopolimer matrisinin oldukça gözenekli bir iç yapıya sahip olduğu ve immobilize edilen amiloglikosidaz enziminin fiziksel bir tutuklama yönteminden beklenildiği üzere kimyasal olarak matrisle etkileşmediğini göstermiştir. Bu çalışmada serbest ve immobilize enzimin aktivasyon enerjileri sırasıyla 13.24 ve 7.36 kcal/gmol olarak tespit edilmiştir. BuÖzet sonuç immobilize enzimin sıcaklık değişimine serbest enzim kadar duyar olmadığım göstermiştir. Ayrıca immobilize enzim partiküllerinin sürekli beslemeli karıştırmalı reaktörde (SBKR) farklı alıkonma zamanlarında (0.51-12 sa) ve işletim sıcaklıklarında (30-60 °C) D-glikoz üretim performansı incelenmiş ve maltodekstrinin farklı sıcaklıklarda partikül içi etkin difüzyon katsayıları 10`6 cm2/s mertebesinde hesaplanmıştır. SBKR 'de enzim immobilize partiküllerin D-glikoz üretim kararlılığı incelenmiş ve sıcaklık döngüsü uygulanan sistemde kasılma-şişme hareketinin sistem performansı üzerindeki etkileri araştırılmıştır. Sonuçta sıcaklık döngüsü uygulamasının amaçlanan performans artışım hiçbir koşulda veremediği görülmüştür. Bu amaca yönelik olarak hesaplanan görünür Thiele modülü değerleri (0.9-3) çalışılan bütün sıcaklıklarda prosesin kimyasal reaksiyon kontrollü olmadığını ancak baskın bir difüzyon kontrolünün de olmadığım göstermiştir. Farklı bir reaktörde immobilize partiküllerin performansının izlenmesi amacıyla dolgulu kolonda farklı alıkonma zamanlarında çalışılmış ve ayrıca kolonda D-glikoz üretim kararlılığı incelenmiştir. Enzim immobilize partiküllerin kararlılığının (~300 saat) incelendiği SBKR ve dolgulu kolon deneylerinden elde edilen sonuçlar immobilizasyonun enzim deaktivasyonunu önemli derecede yavaşlattığım ve aynı koşullarda serbest enzimin çok kısa sürede deaktive olduğunu göstermiştir. Literatürde NIPAAm/AAm kopolimer matrisinde tutuklu amiloglikosidaz enzimi kullanarak SBKR ve dolgulu kolonda maltodekstrinden sürekli glikoz şurubu üretimini araştıran ve sıcaklık döngüsü uygulamasının reaktör performansına etkisini inceleyen başka bir çalışma yoktur. Bu çalışmada sıcaklık döngüsünün uygulaması, enzim kararlılığının ve performansının artırılmasına yönelik deneysel optimizasyon, serbest enzim ve desteğe ait kinetik ve kütle aktarım katsayılarının bulunması, substrat ve deaktive olan enzimin kullanım zorluğuna rağmen ticari proseste kullanılanla aynı seçilmesi çalışmayı literatürde orjinal hale getirmekte ve endüstriyel uygulama olanağım artırmaktadır. Anahtar kelimeler: Amiloglikosidaz, maltodekstrin, sıcaklık duyarlı polimer, n-izopropil akrilamid, difüzyon ve reaksiyon modeli, etkin difüzyon katsayısı, sürekli glikoz üretimi. Abstract vi ABSTRACT In this study, the production of glucose syrup from maltodextrin using amyloglucosidase enzyme entrapped in a thermo-responsive NIPAAm/AAm (n-isopropylacrylamide/acrylamide) copolymer matrix was investigated. Firstly, optimum temperature and pH range for free enzyme activity were determined and then effects of initial enzyme load and substrate concentration on the initial D-glucose production rate (activity) were examined at these optimum conditions. As a result, experiments conducted with 0.01 mL initial free enzyme load (45.37 g/L/h, in terms of activity) indicated that free enzyme gave the optimum activity at T=60 °C and pH 4.5 acetate buffer, and 100 g/L initial substrate concentration provided the highest D-glucose conversion rate at these optimum conditions. Deactivation rate constants for the free enzyme used in substrate-free and substrate- containing medium were determined and for the latter one deactivation rate constants as a function of substrate concentration -not reported in literature before- were obtained. For the determination of free enzyme kinetics, all enzymatic rate expressions in literature with or without inhibition were searched with nonlinear regression analysis through Systat® canned program and Haldane kinetics for substrate inhibition gave the best fit for the experimental data at all temperatures studied (30-60 °C). This model was used in the `diffusion+reaction` solution within the particle as the reaction rate expression. In the second part of the study, particles having different NIPAAm/AAm compositions were prepared and swelling-shrinking kinetics of copolymer matrix were investigated. Effective diffusion coefficients of water in the particle for the dynamic swelling and shrinking cases were determined with the order of magnitude 10` cm /s. NIPAAm % value of copolymer matrix having the LCST (lower critical solution temperature) of 60 °C, corresponding to the industrial operating temperature of amyloglucosidase, was found as 80 % (w/w). Entrapment of amyloglucosidase in the NIPAAm/AAm copolymer matrix was realized and then particle preparation conditions for the highest and most stable enzyme activity were determined. As a result, particles (2.7 mm in diameter, at 4 °C) prepared by using 40 mL continuous phase having 0.4 g initiator (KPS) and 1.2 g CaCb, and 3.6 mL monomer phase having 0.3 mL accelerator (TEMED), 0.9 mL cross-linker (from the solution of 0.033 g MBAAm /mL), 0.03 g sodium alginate, 0.394 g NIPAAm, 0.096 g (AAm) and 0.8 mL enzyme provided the optimum observable enzyme activity. Observable activity of the immobilized enzyme was determined as 14 % of the real enzyme activity immobilized. FTIR and SEM analysis of enzyme-free and enzyme immobilized particles indicated that NIPAAm/AAm copolymer matrix has very macroporous internal structure and enzyme molecules were entrapped only physically within the macroporous matrix and there was no chemical interaction between enzyme and matrix.Abstract vii In this study activation energies of free and immobilized enzyme were determined as 13.24 and 7.36 kcal/gmol, respectively. This result indicated that temperature sensibility of immobilized enzyme was not as much as free enzyme. Finally, D-glucose production performance of enzyme immobilized particles in a continuously fed stirred tank reactor (CFSTR) for different retention times (0.51-12 h) and operating temperatures (30-60 °C) were investigated and effective diffusion coefficients of maltodextrin at these operating temperatures were determined with the order of magnitude 10`6 cm2/s. D-glucose production stability of enzyme immobilized particles was examined and the effect of shrinking-swelling behaviour on the system performance was investigated in a periodically changing temperature cycle. As a result, application of temperature cycling did not provide expected performance enhancement for all cases studied. Calculated observable Thiele modulus values (0.9-3) indicated that the process was neither chemical reaction controlled nor dominantly diffusion controlled. Performance (at different retention times) and stability of enzyme immobilized particles were investigated also in a packed bed reactor. Experiments conducted as long as ~300 h using CFSTR and packed bed for the investigation of stability of enzyme immobilized particles indicated that immobilization slowed down the deactivation of enzyme significantly and free enzyme under the same conditions deactivated in a very short period of time. In literature there is no publication reporting the investigation of continuous glucose production from maltodextrin by using amyloglucosidase enzyme entrapped in a thermo-responsive copolymer matrix and effect of temperature cycling on the reactor performance. Application of periodic temperature cycling, experimental optimization for the enhancement of performance and stability, determination of kinetic and mass transfer coefficients of free enzyme and immobilized enzyme matrix, selection of commercial substrate and enzyme in spite of difficult handling make this study original in literature and give opportunity for the industrial use. Keywords: Amyloglucosidase, maltodextrin, thermo-responsive polymer, n-isopropyl acrylamide, diffusion and reaction model, effective diffusion coefficient, continuous glucose production. 230

Fermentation of grain in the rumen and the digestion of starch in the small intestine are influenced by both animal and grain characteristics. Two assays were established to determine the importance of grain characteristics on fermentation and enzyme digestion. To simulate microbial fermentation in the rumen finely milled samples of grain were incubated in rumen fluid and the end products of fermentation and starch disappearance were measured. The second assay was based on amylase and amyloglucosidase and was established to simulate starch digestion in the small intestine. The assays identified large differences between and within grains with respect to both susceptibility of grain to microbial fermentation and enzyme digestibility of starch. Ranking grains on the basis of total acid production identified expected differences between grains. Lactic acid was positively correlated to total acid production, which implies that strategies aimed at increasing the rate of fermentation of grain inevitably increase the risk of rumen acidosis. The assays identified a sorghum cultivar with a higher rate of fermentation and enzyme digestibility compared to other sorghum varieties tested. Triticale was also identified as a grain with a high enzyme digestibility of starch.

In this study the effect of processing conditions during the manufacture of pelleted animal feed is related to the degree of gelatinization of tapioca starch as measured by the amyloglucosidase method (SGDAGS) and Differential Scanning Calorimetry (DSC). The process conditions used were related to some physical quality characteristics of the pelleted feeds as well. Physical pellet quality was evaluated for hardness and durability, which incorporated tests that are also used in the feed industry. The processing line used in the experiment consisted of a conventional barrel type conditioner where steam pressure (100–180 kPa) and tap water addition were varied (0–52 g kg−1 of the feed debit). Subsequently, expander processing was carried out and the screw speed of the expander was varied (60–140 rpm) as well as the amount of dissipated (expander) motor power (1.3–5.7 kW). Response surface regression methodology was used to assess the directions and relative magnitude of changes in processing conditions on starch modification, physical pellet quality and some system variables. The results show that no combination of independent factor levels can be found that satisfies one common maximum or one common minimum value for all of the dependent variables tested. Hardness and durability values of the pellets were most affected by steam pressure and the amount of expander motor power used, whereas starch modification was most affected by steam pressure and water addition. © 1999 Society of Chemical Industry

II ÖZET Bu çalışmada Aspergillus niger küf mantarından amiloglukosidaz [Glukoamilaz (1,4-a-D-ghıkan glukohidrolaz) enzimi elde edilmiş, çözünür ve immobilize formdaki enzimin kinetik aktivitesi saptanmıştır. Aspergillus niger malt agar besi ortamına ekildikten sonra, amiloglukosidaz üretimi için uygun ortam hazırlanmış ve uygun konstantrasyondaki amiloglukosidaz nişasta, un ve uygun bir tampon içeren biyoreaktöre ilave edilmiştir. 5 gün boyunca çözeltide Fehling testi ile glukoz, Lugol testi ile de nişasta kalitatif olarak tayin edilmiştir. 5. günün sonunda nişastanın parçalanması tamamlandıktan sonra enzim iki kısma ayrılmıştır. Birinci kısımda enzim %70 lik amonyum sülfat doygunluğunda çöktürülüp sodyum asetat tamponuna karşı diyalizlenmiştir. İkinci kısımdaki enzim ise aktif kömüre immobilize edilmiştir. Çözünür ve immobilize formdaki enzim için enzim kinetiği çalışmaları yapılmış, enzim aktivitesi, spesifik aktivite ve Kn, değerleri saptanmıştır. m SUMMARY In this study amyloglucosidase [Glucoamylase (1,4-a-D-glucan glucohydrolase] was prepared from fungus which is called Aniger. Its activity was calculated in soluble and immobilized form. After A.niger was cultivated in the malt agar, suitable media was provided for amyloglucosidase to grow and reproduce. Bioreactor containing starch, flour and a suitable buffer was innoculated with a suitable concentration of A.niger. For a five days the solution was tested for glucose by Fehling test and for starch by Lugol test. The degradation of starch was completed in 5 days then the enzyme was divided into two parts. In the first part the enzyme was precipitated by 70% ammonium sulphate saturation then dialyzed against acetate buffer. In the second part the enzyme was immobilized to activated charcoal. Kinetic studies were done for soluble and immobilized forms of the enzyme. The enzymatic activity the specific activitiy and Km were determined. 30

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Submitted by Guilherme Lemeszenski (guilherme@nead.unesp.br) on 2013-08-22T19:02:14Z No. of bitstreams: 0 Made available in DSpace on 2013-08-22T19:02:14Z (GMT). No. of bitstreams: 0 Previous issue date: 2000-03-01 Made available in DSpace on 2013-09-30T20:02:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2000-03-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T15:16:09Z No. of bitstreams: 0 Made available in DSpace on 2014-05-20T15:16:09Z (GMT). No. of bitstreams: 0 Previous issue date: 2000-03-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) A simple method for the preparation of silica with controlled pore size, for use as a support for the immobilization of enzymes, is described in this article. Using sodium silicate and hydrochloric acid, a microporous silica was obtained that was then submitted to a hydrothermal treatment, resulting in macroporous silica suitable for enzyme immobilization. Suitability of the macroporous silica as a support depends on the method chosen for its preparation, which will determine pore volume and the effect of hydrothermal treatment on pore size. The pore volume of the support was 0.8-0.9 cc/g and the average pore size, controlled by the hydrothermal treatment, was in the range of 16 to 75 nm. The enzyme amyloglucosidase was used for the immobilization studies. UNESP IQ Depto. de Bioquímica e Tecnologia Química UNICAMP Faculdade de Engenharia Química Depto. de Tecnologia de Polímeros FUEM Depto. de Engenharia Química UNESP IQ Depto. de Bioquímica e Tecnologia Química