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Ornithine decarboxylase has been purified to apparent homogeneity from thioacetamide-stimulated calf liver. The purification process, which has been developed to circumvent the lability of the enzyme, employs ion exchange chromatography, gel filtration, hydroxylapatite chromatography, non-denaturing gel electrophoresis, and sulfhydryl affinity chromatography. The enzyme is purified 71,500-fold to a final specific activity of 286,000 pmol/min/mg protein. Non-denaturing gel electrophoresis indicates a single protein present in the final preparation. The enzyme has a Stokes radius of 3.14 nm as indicated by gel filtration and a monomeric molecular weight of 52,000 daltons as indicated by denaturing gel electrophoresis. The K(m) values for ornithine and pyridoxal phosphate are 0.16 mM and 2.5 μM, respectively. Putrescine inhibits the enzyme (Kᵢ 10mM). The existence of three ionic forms of ornithine decarboxylase is suggested by fractionation of the preparation by gradient sievorptive chromatography. Mammalian ornithine decarboxylase is apparently a metalloenzyme. A variety of structurally distinct metal chelators inhibit the enzyme. A non-chelating analog of the most potent chelator, 1,10-phenanthroline, is without effect. The order of efficacy of the chelators suggests the involvement of a metal from the transition series. Incubation of the enzyme with charcoal or Cibacron Blue-Agarose results in a loss of catalytic activity suggesting that the ornithine decarboxylase may also contain a bound nucleotide.

這篇論文研究了幾種基於聚二甲基硅氧烷（PDMS）的微流控芯片，及其對於酶反應和蛋白質結晶的應用。 / 本論文分為兩部份。首先，論文第一部份介紹了兩種微流控芯片。利用到PDMS透氣的性質，第一種微流控芯片是一種基於脫氣PDMS的納升液體進液系統。在第一種微流控芯片的基礎上，我們引進一種可由普通注射器控制的氣閥控制系統，可對反應液體的分配和混合進行更方便和精確的操作。兩種芯片均成功應用於酶反應動力學的測定。在一次實驗中，只需要3~5微升的反應物就可以得到鹼性磷酸酶（alkaline phosphatase）的Michaelis-Menten動力學。 / 論文第二部份研究了“體積效“應對於蛋白質結晶的影響。首先，基於微孔的微流控芯片的蛋白質結晶篩選實驗揭示了在小體積（納升）下，蛋白質的結晶條件比在大體積（微升）下更多。在液滴里的蛋白質結晶實驗結果說明大體積的蛋白質液滴結晶速度更快。最後，蛋白質結晶實驗成功在雙乳液（double emulsion）的內核中進行。 / This thesis describes the design and development of micro dispensing systems for enzyme assay and for protein crystallization. The micro dispensing systems were fabricated by the soft-lithography method with the widely used material poly(dimethylsiloxane) (PDMS), which is gas and water permeable elastomer. / This thesis contains two major parts. In the first part, enzyme assay was performed in two micro dispensing systems, one based on microwells and the other based on pneumatic valves. The complete Michaelis-Menten kinetics measurement of the alkaline phosphatase (AP) with different fluorescein diphosphate (FDP) was achieved in one chip for each system using the fluorescence detection. These micro dispensing systems’ fabrication and operation were simple, and the total sample consumption was about 3~5 μL. / The second part reports the study of the volume effect on protein crystallization. Three micro dispensing systems, the microwell-based, droplet-based and double emulsion-based systems, were developed to perform the protein crystallization. Lysozyme and thaumatin were chosen as the model proteins. First, the protein crystallization screening experiments showed that protein crystallized in more precipitant conditions in the microchip than in conventional microbatch system. Second, the protein crystallization results carried out in droplets showed that protein crystallized faster in larger droplets. Finally, the protein crystallization in double emulsions was demonstrated. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zhou, Xiaohu. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 87-91). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgements --- p.iv / Table of Contents --- p.vi / List of figures --- p.viii / Chapter Chapter 1 --- Introduction to microfluidics --- p.1 / Chapter 1.1 --- Fabrication of microfluidic devices --- p.2 / Chapter 1.1.1 --- PDMS-based microfluidic device --- p.2 / Chapter 1.1.2 --- PMMA microfluidic device --- p.6 / Chapter 1.1.3 --- Assembly of the microfluidic device --- p.8 / Chapter 1.2 --- Degassed PDMS pumping method --- p.9 / Chapter 1.3 --- Droplet-based microfluidics --- p.13 / Chapter 1.4 --- Thesis organization --- p.16 / Chapter Chapter 2 --- Micro dispensing systems for enzyme assay --- p.17 / Chapter 2.1 --- Introduction to enzyme assay --- p.17 / Chapter 2.1.1 --- Introduction to micro platforms for enzyme assay --- p.17 / Chapter 2.1.2 --- Introduction to enzyme kinetics and Michaelis-Menten kinetics --- p.21 / Chapter 2.2 --- Experimental --- p.26 / Chapter 2.2.1 --- Design of the micro dispensing systems --- p.26 / Chapter 2.2.2 --- Fabrication of the microfluidic devices --- p.30 / Chapter 2.2.3 --- Reagents and operation --- p.32 / Chapter 2.3 --- Results and discussion --- p.32 / Chapter 2.3.1 --- Microwell-based dispensing system --- p.32 / Chapter 2.3.2 --- Micro dispensing system based on pneumatic valves --- p.40 / Chapter 2.4 --- Conclusions --- p.45 / Chapter Chapter 3 --- Micro dispensing systems for protein crystallization --- p.47 / Chapter 3.1 --- Introduction to protein crystallization --- p.47 / Chapter 3.1.1 --- Principle of protein crystallization --- p.49 / Chapter 3.1.2 --- From macrofluidics to microfluidics --- p.51 / Chapter 3.2 --- Experimental --- p.56 / Chapter 3.2.1 --- Design of the micro dispensing systems --- p.56 / Chapter 3.2.2 --- Fabrication of the microfluidic devices --- p.58 / Chapter 3.2.3 --- Reagents and operation --- p.60 / Chapter 3.2.4 --- Layer-by-layer modification --- p.61 / Chapter 3.3 --- Results and discussion --- p.65 / Chapter 3.3.1 --- Demonstration of the micro dispensing systems --- p.65 / Chapter 3.3.2 --- Protein crystallization screening results --- p.69 / Chapter 3.3.3 --- Protein crystallization in droplets --- p.73 / Chapter 3.3.4 --- Protein crystallization in w/o/w double emulsions --- p.77 / Chapter 3.4 --- Conclusions --- p.80 / Appendix --- p.82 / References --- p.87

published_or_final_version / Biological Sciences / Master / Master of Philosophy

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: This study describes: a) the optimization of the current industrial-scale extraction and purification of Horseradish peroxidase from horseradish at BBI Enzymes, focussing on: a. Raw material quality, b. Extraction, c. Ultra-filtration, d. Salt fractionation, e. Diafiltration, f. Ion Exchange Chromatography, b) developing an new in-process microtitre plate calorimetric assay, c) characterization of main groups of HRP relevant to BBI Enzymes by SDS-PAGE- and HPLC analysis. / AFRIKAANSE OPSOMMING: Hierdie studie beskryf: a) die optimisering van die huidige industriële-skaal ekstraksie en suiwering van peperwortelperoksidase vanuit peperwortel by BBI Enzymes, deur te focus op: a. Rou material kwaliteit, b. Ekstraksie, c. Ultra-filtrasie, d. Sout fraksionering, e. Diafiltrasie, f. Ioon-uitruilchromatografie b) Ontwikkeling van ‘n nuwe in-proses mikro-titer gebaseerde kalorimetriese toetsmetode c) die karakterisering van die hoof groepe peperwortel-peroksidase belangrik vir BBI Enzymes.

by Lau King-Kwan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 119-142). / ACKNOWLEDGMENTS --- p.I / ABSTRACT --- p.II / ABBREVIATIONS --- p.IV / TABLE OF CONTENTS --- p.V / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter CHAPTER 2 --- PURIFICATION OF ADH-I & ADH-C --- p.25 / Chapter CHAPTER 3 --- "PURIFICATION & IDENTIFICATION OF ""ADH-ACTIVATING"" PROTEASE" --- p.60 / Chapter CHAPTER 4 --- ACTIVATION OF ADH-I BY COMMERCIAL PROTEASE & BY ACETIMIDYLATION --- p.90 / Chapter CHAPTER 5 --- CONCLUSION --- p.114 / REFERENCES --- p.118

Orientador: Elias Basile c / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica / Made available in DSpace on 2018-08-09T16:09:35Z (GMT). No. of bitstreams: 1 Godoi_PatriciaHelenade_M.pdf: 865544 bytes, checksum: 0a09a500bcd26f04fe7be64851f3522a (MD5) Previous issue date: 2007 / Resumo: Bromelina é uma enzima de origem vegetal obtida de diversas espécies da família Bromeliaceae, presente na casca, no talo e no fruto do abacaxi. O Brasil encontra-se entre um dos maiores produtores mundiais de abacaxi, ocupando o terceiro lugar no ranking mundial. O desenvolvimento de novas técnicas de extração e purificação da bromelina vem sendo bem explorado, entretanto, tornouse necessário um estudo de estabilidade desta protease, proporção pela qual a bromelina conserva sua conformação estrutural ou sua atividade quando sujeita à estocagem, isolamento e purificação ou várias outras manipulações físicas ou químicas, incluindo autodigestão, outras enzimas proteolíticas e aquecimento. Tornou-se de fundamental importância saber em que condições a bromelina se mantém estável, ou seja, ativa e por qual período de tempo, já que a meta mais significante da enzimologia aplicada é obter compostos úteis para biocatálise. Este trabalho apresenta um estudo das condições de pH e temperatura nas quais a Bromelina P.A em solução aquosa em concentração próxima ao do suco extraído da polpa da fruta, mantém-se ativa, ou seja, não desnaturada. A atividade enzimática da bromelina em solução foi medida através da hidrólise da caseína e a condição de pH e temperatura mais próxima da ideal foi determinada / Abstract: Bromelain is a vegetable enzyme found in many species of Bromeliaceae family, its present in pineapple skins, stem and fruit. Brazil is one of the world¿s largest producers of pineapples, its production being the third one in the world. The development of new extraction and purification processes of bromelain have been studied, however, its necessary a enzyme stabilization investigation, state that bromelain remains its structure or biological activity when stored, isolated, purified or any other manipulation, included autodigestion, proteolytic enzymes and heating. It became very important to know the conditions and the time which bromelain remain stabilized, active. In applied enzymology, the most significant goal is to achieve useful compounds by biocatalysis. This work presents a study about pH and temperature conditions which a bromelain aqueous solution, in the same concentration of a pineapple fruit extract, remains with biological activity. The bromelain aqueous solution enzyme activity was tested across the casein hydrolysis and the ideal pH and temperature was determinated. / Mestrado / Sistemas de Processos Quimicos e Informatica / Mestre em Engenharia Química

Orientadores: Elias Basic Tambourgi, Priscila Gava Mazzola / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica / Made available in DSpace on 2018-08-21T20:42:46Z (GMT). No. of bitstreams: 1 Bresolin_IaraRochaAntunesPereira_D.pdf: 1407939 bytes, checksum: 1259333d5753554819357dfada6831b4 (MD5) Previous issue date: 2013 / Resumo: A bromelina é uma enzima proteolítica encontrada em tecidos vegetais como casca, talo, fruta e folhas de espécies da família Bromeliaceae, incluindo o abacaxi (Ananas comosus). É uma enzima conhecida por seus efeitos terapêuticos, sendo usadas em tratamentos de vários problemas de saúde, como desordens digestivas, feridas, queimaduras e inflamações. Este trabalho teve como objetivo a purificação da enzima bromelina de casca de abacaxi e sua posterior aplicação em bases dermatológicas. Foi demonstrado que é possível purificar bromelina extraída de casca de abacaxi, um resíduo da indústria alimentícia, através de um processo de recuperação e purificação, incluindo precipitação por sulfato de amônio (40- 80%), seguido de dessalinização e liofilização, com 75% de recuperação da atividade. Cromatografia de troca iônica com dietilaminoetil-agarose (DEAE-Sepharose) separou polissacarídeos da enzima e esta foi recuperada na etapa de eluição, mantendo sua atividade enzimática. A enzima foi incorporada em creme e loção Lanette, gel de Carbopol e creme e loção da Chemyunion na concentração de 5 mg de enzima por mL de base dermatológica. Essas bases foram submetidas ao teste de centrifugação e teste de estabilidade acelerada durante 90 dias a 25ºC (com e sem a incidência de luz solar), 37ºC e 4°C, a fim de avaliar a estabilidade da bromelina nas bases dermatológicas. As formulações permaneceram estáveis quanto a suas características organolépticas (aspecto, cor, odor, sensibilidade ao tato) apenas quando mantidas a 4°C, com atividade remanescente de 84,9%, 73,8%, 95,5%, 77,7% e 72,3% após 90 dias de teste em creme e loção Lanette, gel de Carbopol e creme e loção da Chemyunion, respectivamente. Baseando-se nestes resultados, foi possível purificar bromelina de casca de abacaxi e incorporá-la em bases dermatológicas mantendo se sua atividade mais estável quando estas bases foram mantidas em geladeira a 4ºC / Abstract: Bromelain is a proteolytic enzyme found in vegetable tissues like peel, stem, fruit and leaves of the Bromeliaceae family including pineapple (Ananas comosus). Bromelain is known for its therapeutic effects, being useful in the treatment of several health problems such as digestive disorders, burns and inflammation. This work aimed the purification of the enzyme bromelain from pineapple peel for potential therapeutic application in dermatological bases. It was shown that it is possible to purify bromelain extracted from pineapple peel, a waste in food industry, in a downstream processing, including ammonium sulfate precipitation (40-80%), followed by desalting and freeze-drying with a 75% activity recovery. Ion exchange chromatography on diethylaminoethyl-agarose (DEAE-Sepharose) was able to separate polysaccharides from the enzyme, which was recovered in the elution step, maintaining its enzymatic activity. The enzyme was then incorporated in Lanette cream and lotion, Carbopol gel and Chemyunion cream and lotion at a concentration of 5 mg enzyme per mL of dermatological bases. These bases were subjected to centrifugation test and accelerated stability test during 90 days at 25°C (with and without sunlight), 37°C and 4°C, in order to evaluate bromelain stability in a dermatologic formulation. The formulations were stable as its organoleptic characteristics (appearance, color, smell and sensitivity to touch) only when kept at 4ºC with activity remaining 84.9%, 73.8%, 95.5%, 77.7 % and 72.3% after 90 days of testing in Lanette cream and lotion, Carbopol gel and Chemyunion cream and lotion, respectively. Based on the results, it was possible to purify bromelain from pineapple peel and to incorporate it in dermatological bases maintaining the activity stabler when these bases were kept in refrigerator at 4ºC / Doutorado / Sistemas de Processos Quimicos e Informatica / Doutora em Engenharia Quimica

The termina1 enzyme in the linoleoyl-CoA desaturase enzyme complex, delta-6-desaturase was implied in the control of cell proliferation in cancer cells. One of the aims of this study was to isolate the terminal enzyme. It was decided that in order to isolate this enzyme it was first necessary to isolate the entire complex and then to enzymatically solubilise the first two components of the complex i e cytochrome b5 reductase and cytochrome b5 from the complex resulting in a pure delta-6-desaturase . The first two components were isolated and purified using simplified and easily reproducible methodologies which could be utilised in the final purification of delta-6- desaturase. The entire enzyme complex, linoleoyl-CoA desaturase was also isolated in a pure form and this pure complex was used to attempt to isolate delta-6-desaturase. The terminal enzyme was isolated with some cytochrome b5 still bound to it. The methods used had proven to be successful and with some modifications should yield a pure enzyme. Zinc and GLA were known to play a role in the inhibition of cancer cell proliferation and zinc was hypothesised to inhibit cell growth by stimulating the activity of the linoleoyl-CoA desaturase enzyme complex which is involved in the regulation of cell proliferation. GLA is the product of the reaction that this enzyme complex catalyses and GLA has been shown to inhibit cancer ce ll growth. The effect of GLA on cell growth and linoleoyl-CoA desaturase activity was thus investigated. Results showed that both zinc and GLA inhibited cell growth and that the combined addition of zinc and GLA generally resulted in the inhibition of cell growth and the activation of linoleoyl-CoA desaturase activity in the BL-6 cells while having a less pronounced effect on the LLCMK cells. The results of this study support the hypothesis that zinc may be a cofactor of linoleoyl-CoA desaturase.

Orientador: Eleonora Cano Carmona / Banca: Aline Aparecida Pizzirani Kleiner / Banca: Helia Hamuri Sato / Banca: Rosa dos Prazeres M.F. Inocentes / Banca: Marcia Regina Brochetto Braga / Resumo: Enzimas degradadoras de xilana, principal componente da hemicelulose, têm sido utilizadas em várias aplicações biotecnológicas, sendo que em alguns processos é necessário o uso de enzimas purificadas. Aplicações comerciais para as enzimas xilanolíticas envolvem a hidrólise enzimática da xilana, que está presente nos resíduos agrícolas e agroindustriais, sendo convertido a xilose e outros açúcares, que podem ser utilizados como substratos em processos fermentativos para a obtenção de proteínas celulares, combustíveis líquidos e outras substâncias químicas. A utilização destas enzimas também diminui a liberação de agentes poluentes em determinados efluentes, como da indústria de polpa de celulose. Xilanases e β- xilosidases são produzidas principalmente por bactérias e fungos, sendo que em geral, os fungos as produzem em níveis mais elevados. O gênero Penicillium apresenta espécies já caracterizadas como boas produtoras destas enzimas. Uma linhagem deste gênero, isolada de solo brasileiro, na região da Mata Atlântica e identificada como Penicillium sclerotiorum destacou-se por produzir xilanase em níveis elevados. O objetivo deste trabalho consistiu na avaliação da influência das condições de cultivo sobre a produção do complexo xilanolítico produzido por P. sclerotiorum, na caracterização físico-química desse sistema, bem como purificação e caracterização bioquímica de seus principais componentes. Por meio da determinação das condições ótimas de produção e da caracterização deste complexo enzimático foi possível estabelecer metodologias eficientes de purificação de xilanases e uma β-xilosidase. Através da caracterização físico-química das enzimas purificadas, foi possível avaliar seu potencial biotecnológico, visando futuras aplicações em processos industriais. / Abstract: Xylan degrading enzymes, the main component of hemicellulose, have been used in various biotechnological applications, and in some cases the use of purified enzymes is necessary. Commercial applications of xylanolytic enzymes involve the enzymatic hydrolysis of xylan, which is present in agricultural and agro-industrial wastes, and can be converted to xylose and other sugars, which can be further used as substrates in fermentation processes to obtaining cellular protein, liquid fuels and other chemicals. The utilization of these enzymes also decreases the release of certain pollutants in wastewater, as in the pulp and paper industry. Xylanases and β-xilosidases are mainly produced by bacteria and fungi, and in general, the fungi produce them at higher levels. The genus Penicillium presents species already characterized as good producers of these enzymes. One strain of this genus isolated from Brazilian soil in the Mata Atlântica region and identified as Penicillium sclerotiorum attracted attention by producing xylanase in high levels. The objective of this study was to evaluate the influence of culture conditions on the production of the xylanolytic complex produced by P. sclerotiorum to characterize physical and chemical properties of this system as well to purify and biochemical characterize its main components. By determining optimal conditions for production and by characterizing this enzymatic complex it was possible to establish efficient methodologies for purification of xylanases and one β-xylosidase. Through their physical and chemical characterization, it was possible to evaluate their biotechnological potential for future applications in industrial processes. / Doutor