Dissertations / Theses on the topic 'Polyphenol oxidase activity (PPO)'

Polyphenol oxidase (PPO) catalyses undesirable darkening in wheat products such as Asian noodles. Genetic variation for PPO activity is characterized in bread wheat. Australian wheat breeding programmes recognize that reduced PPO activity is an important quality target. Despite this interest from breeders, no varieties possessing extremely low and null PPO activity exist. The development of null PPO wheat varieties is dependant on an understanding of the genetic control of the null phenotype. Knowledge of these factors will accelerate efforts to develop them. The inheritance of PPO activity was investigated in two populations that were derived from hybrids between a null PPO genotype and Australian wheat varieties Lang and QAlBis. Observed genetic ratios were consistent with two and three gene control, respectively in these populations. QTL mapping was performed in the QALBis x VAW08-A17 population. The Diversity Array Technology (DArT) approach was employed to genotype the QALBis x VAW08-A17 population. Three highly significant QTLs that control PPO activity were identified on chromosomes 2AL, 2BS and 2DL. Close associations between PPO activity and DArT marker loci wPt-7024, wPt-0094 and wPt-2544 were observed, respectively. Collectively, these loci explained 74% of the observed variation in PPO activity across seasons. Significant QTLs on chromosomes 1B and 3B were also identified that together explained an additional 17% of variation in PPO activity. The relationship between PPO activity and yellow alkaline noodles (YAN) colour stability parameters was investigated in a DM5637*B8 x H45 doubled haploid population. PPO activity and changes in YAN brightness (ΔL* 0-24h) and yellowness (Δb* 0-24h) in both seasons were analysed. Quantitative trait analyses of PPO activity, flour yellowness (b*) and YAN colour stability was also conducted in this population. QTL mapping of variation in PPO activity in the DM5637*B8 x H45 DH population identified a highly significant QTL on chromosome 2AL, which explained 52% of the observed variation across seasons. Regression analysis identified that wPt-7024 was highly significantly associated with PPO activity in this population. A highly significant association between this marker and PPO was also identified in the QALBis x VAW08-A17 population. Collectively, the three identified QTLs (on chromosomes 2AL, 7A and 7B) explained 71% of variation in PPO activity across seasons. A highly significant (P<0.001) QTL on chromosome 2B along with significant (P<0.01) QTLs on the chromosomes 1A, 3B, 4B and 5B were found to control flour yellowness. The QTLs on 2B, 4B and 5B were detected in both seasons analysed and accounted for 90% of variation in flour b* across seasons. The study on YAN colour stability located two highly significant (P<0.001) QTLs and two significant (P<0.01) QTLs that controlled the change in brightness of yellow alkaline noodle. The 2A QTL accounted for 64% of observed variation across seasons. It was in the same location as the PPO QTL and shared a common closest marker wPt-7024. Only one significant QTL for YAN a* (0-24h) was identified. It accounted for 12% of variation across seasons and was only detected in one season. One highly significant (P<0.001) QTL and two significant (P<0.01) QTLs were identified that controlled the change in yellowness of yellow alkaline noodle. The 2A QTL accounted for 68% of observed variation across seasons. The location of this QTL corresponded with that of 2A QTLs for PPO activity and L* of YAN in this study. Furthermore, wPt-7024 was also identified as the marker with the most significant association with L*. The identification of a correlation between the characters and a common location of a highly significant QTL for each of these characters indicates that it is likely that PPO activity is directly responsible for a large proportion of the changes in brightness and yellowness of YAN. QTLs for L* and b* of YAN were detected in a common location on chromosome 1A. However, no corresponding QTL was identified that controls PPO activity, highlighting the complexity of the relationship between these traits. Resistance to three rust pathogens (Puccinia graminis, Puccinia striiformis, and Puccinia triticina) was also investigated in the DM5637*B8 x H45 DH population because they are major yield limiting diseases in wheat. Disease response data at the seedling stage were converted to genotypic scores for rust genes Sr24/Lr24, Sr36, Lr13 and Yr7 to construct a genetic linkage map. No recombination was observed between rust resistance genes Sr36, Lr13 and Yr7 in this DH population. Therefore, these genes mapped in the same position on chromosome 2B. The Lr24/Sr24 locus was incorporated into the chromosome 3D map. Interval mapping analysis identified QTLs on chromosomes 2B, 3B, 4B and 5B that control adult plant resistance (APR) to stripe rust. Two QTLs on chromosomes 2B and 3D were identified that controlled APR to leaf rust in this DH population.

Doctor of Philosophy
Polyphenol oxidase (PPO) catalyses undesirable darkening in wheat products such as Asian noodles. Genetic variation for PPO activity is characterized in bread wheat. Australian wheat breeding programmes recognize that reduced PPO activity is an important quality target. Despite this interest from breeders, no varieties possessing extremely low and null PPO activity exist. The development of null PPO wheat varieties is dependant on an understanding of the genetic control of the null phenotype. Knowledge of these factors will accelerate efforts to develop them. The inheritance of PPO activity was investigated in two populations that were derived from hybrids between a null PPO genotype and Australian wheat varieties Lang and QAlBis. Observed genetic ratios were consistent with two and three gene control, respectively in these populations. QTL mapping was performed in the QALBis x VAW08-A17 population. The Diversity Array Technology (DArT) approach was employed to genotype the QALBis x VAW08-A17 population. Three highly significant QTLs that control PPO activity were identified on chromosomes 2AL, 2BS and 2DL. Close associations between PPO activity and DArT marker loci wPt-7024, wPt-0094 and wPt-2544 were observed, respectively. Collectively, these loci explained 74% of the observed variation in PPO activity across seasons. Significant QTLs on chromosomes 1B and 3B were also identified that together explained an additional 17% of variation in PPO activity. The relationship between PPO activity and yellow alkaline noodles (YAN) colour stability parameters was investigated in a DM5637*B8 x H45 doubled haploid population. PPO activity and changes in YAN brightness (ΔL* 0-24h) and yellowness (Δb* 0-24h) in both seasons were analysed. Quantitative trait analyses of PPO activity, flour yellowness (b*) and YAN colour stability was also conducted in this population. QTL mapping of variation in PPO activity in the DM5637*B8 x H45 DH population identified a highly significant QTL on chromosome 2AL, which explained 52% of the observed variation across seasons. Regression analysis identified that wPt-7024 was highly significantly associated with PPO activity in this population. A highly significant association between this marker and PPO was also identified in the QALBis x VAW08-A17 population. Collectively, the three identified QTLs (on chromosomes 2AL, 7A and 7B) explained 71% of variation in PPO activity across seasons. A highly significant (P<0.001) QTL on chromosome 2B along with significant (P<0.01) QTLs on the chromosomes 1A, 3B, 4B and 5B were found to control flour yellowness. The QTLs on 2B, 4B and 5B were detected in both seasons analysed and accounted for 90% of variation in flour b* across seasons. The study on YAN colour stability located two highly significant (P<0.001) QTLs and two significant (P<0.01) QTLs that controlled the change in brightness of yellow alkaline noodle. The 2A QTL accounted for 64% of observed variation across seasons. It was in the same location as the PPO QTL and shared a common closest marker wPt-7024. Only one significant QTL for YAN a* (0-24h) was identified. It accounted for 12% of variation across seasons and was only detected in one season. One highly significant (P<0.001) QTL and two significant (P<0.01) QTLs were identified that controlled the change in yellowness of yellow alkaline noodle. The 2A QTL accounted for 68% of observed variation across seasons. The location of this QTL corresponded with that of 2A QTLs for PPO activity and L* of YAN in this study. Furthermore, wPt-7024 was also identified as the marker with the most significant association with L*. The identification of a correlation between the characters and a common location of a highly significant QTL for each of these characters indicates that it is likely that PPO activity is directly responsible for a large proportion of the changes in brightness and yellowness of YAN. QTLs for L* and b* of YAN were detected in a common location on chromosome 1A. However, no corresponding QTL was identified that controls PPO activity, highlighting the complexity of the relationship between these traits. Resistance to three rust pathogens (Puccinia graminis, Puccinia striiformis, and Puccinia triticina) was also investigated in the DM5637*B8 x H45 DH population because they are major yield limiting diseases in wheat. Disease response data at the seedling stage were converted to genotypic scores for rust genes Sr24/Lr24, Sr36, Lr13 and Yr7 to construct a genetic linkage map. No recombination was observed between rust resistance genes Sr36, Lr13 and Yr7 in this DH population. Therefore, these genes mapped in the same position on chromosome 2B. The Lr24/Sr24 locus was incorporated into the chromosome 3D map. Interval mapping analysis identified QTLs on chromosomes 2B, 3B, 4B and 5B that control adult plant resistance (APR) to stripe rust. Two QTLs on chromosomes 2B and 3D were identified that controlled APR to leaf rust in this DH population.

Nesta dissertação, a polifenol oxidase (PFO) como extrato bruto de abacate (Persea americana) foi imobilizada em filmes de polipirrol (PPI) sintetizados eletroquimicamente utilizando o glutaraldeído (GA) como um agente de ligação entrecruzada. Os filmes PPI e PPI/PFO-GA foram caracterizados por eletroquímica, principalmente voltametria cíclica, sendo avaliadas a eletroatividade e a reversibilidade eletroquímica. A detecção de compostos fenólicos em soluções padrão foi feita por cronoamperometria, tendo um controle sobre a concentração dos compostos. O processo de transferência de massa foi monitorado com uma microbalança de cristal de quartzo eletroquímica. Os resultados indicaram uma boa reprodutibilidade das medidas na detecção dos compostos fenólicos. A estabilidade do biossensor em uma solução tampão manteve-se durante 27 dias, um resultado aceitável já que é encontrado na literatura um tempo de vida estável para sistemas semelhantes em tomo de 30 dias
In this dissertation, polyphenol oxidase (PPO) as crude extract of avocado (Persea americana) was immobilized on electrochemical1y synthesized polypyrrole (PPY) films using glutaraldehyde (GA) as a crosslinking agent. PPY and PPY/PPO-GA films were electrochemical1y characterized, mainly by cyclic voltametry, where electroactivity and electrochemical reversibility were evaluated. The detection of phenolic compounds in standard samples was made by chronoamperometry with a control over the compound concentration. The process of mass transfer was monitored with an electrochemical quartz crystal microbalance (EQCM). Our results indicated a good repeatability of the measurements for the detection of phenolic compounds. The stability of biosensor in a buffer solution has remained for 27 days, a result acceptable since it is found in the literature a time of life stable for similar systems around 30 days

Polyphenol oxidase (PPO) affects both kinds of matters that are nowadays at the forefront of the quality concept: those aspects related to health and those regarding organoleptic perception of food. In this Thesis, PPO activity has been characterized in different fruits and situations, and mathematical models to describe melanin formation from monophenolic and o diphenolic substrates have been developed. In addition, potential toxicity of these melanins on the pancreatic proteases carboxypeptidase A, carboxypeptidase B and trypsin has been studied. The second part of the Thesis covers PPO inactivation by innovative technologies, and the side effects that they cause on different parameters. Ultraviolet-visible irradiation has been modeled, and PPO inactivation by this method has been assessed in model solutions and in apple, pear and grape juices. Moreover, the effects of must irradiation on the quality of wine have also been assessed. In addition, high-hydrostatic pressure effectiveness in inactivating apple PPO was tested, as well as its effects on juices color.
La polifenol oxidasa (PPO) afecta a ambdós tipus de qüestions que són actualment al capdavant del concepte de qualitat: els aspectes relacionats amb la salut i els relatius a la percepció organolèptica dels aliments. En aquesta Tesi, l'activitat de la PPO s'ha caracteritzat en diferents fruites i situacions, i s'han desenvolupat models matemàtics per descriure la formació de melanina a partir de substrats monofenòlics i o difenòlics. A més, s'ha estudiat la toxicitat potencial d'aquestes melanines sobre les proteases pancreàtiques carboxipeptidasa A, carboxipeptidasa B i tripsina. La segona part de la Tesi tracta la inactivació de la PPO per tecnologies innovadores, i els efectes secundaris que causen en diferents paràmetres. La irradiació ultraviolada-visible ha estat modelitzada, i la inactivació de la PPO per aquest mètode s'ha avaluat en solucions model i en sucs de poma, pera i raïm. A més, els efectes de la irradiació del most sobre la qualitat del vi també han estat avaluats. En darrer lloc, s'estudià l'eficàcia de l'alta pressió hidrostàtica en la inactivació de la PPO de poma, així com els seus efectes sobre el color dels sucs.
La polifenol oxidasa (PPO) afecta a ambos tipos de cuestiones que se encuentran actualmente al frente del concepto de calidad: los aspectos relacionados con la salud y los relativos a la percepción organoléptica de los alimentos. En esta Tesis, la actividad de la PPO se ha caracterizado en diferentes frutas y situaciones, y se han desarrollado modelos matemáticos para describir la formación de melanina a partir de sustratos monofenólicos y o difenólicos. Además, se ha estudiado la toxicidad potencial de estas melaninas sobre las proteasas pancreáticas carboxipeptidasa A, carboxipeptidasa B y tripsina. La segunda parte de la Tesis trata la inactivación de la PPO por tecnologías innovadoras, y los efectos secundarios que causan en diferentes parámetros. La irradiación ultravioleta-visible ha sido modelizada, y la inactivación de la PPO por este método se ha evaluado en soluciones modelo y en zumos de manzana, pera y uva. Además, los efectos de la irradiación del mosto sobre la calidad del vino también han sido evaluados. Por último, se estudió la eficacia de la alta presión hidrostática en la inactivación de la PPO de manzana, así como sus efectos sobre el color de los zumos.

Neste trabalho, foram fabricados biossensores amperométricos a partir do uso da polifenol oxidase (PFO), obtida do abacate como fonte enzimática, imobilizada em filmes de polipirrol (PPI) e que foram eletrossintetizados em meio de três diferentes eletrólitos de suporte (NaCl e NaClO4 e NaDDS). O método de imobilização enzimática foi o da adsorção, sendo a solução de enzima adicionada à solução com o pirrol e o eletrólito durante o processo de eletropolimerização. Os filmes de PPI/PFO foram caracterizados por técnicas eletroquímicas, principalmente por voltametria cíclica. A detecção de compostos fenólicos (catecol e pirogalol) foi realizada pela técnica de cronoamperometria após se variar a concentração do analito. A morfologia dos filmes foi estudada por microscopia de força atômica (AFM), sendo observado que a presença da enzima no filme polimérico assim como o uso de diferentes eletrólitos de suporte levou a diferenças na superfície dos filmes. Além disto, verificou-se que o biossensor construído a partir do uso do NaCl, apresentava uma resposta mais eficiente, ou seja, ele foi capaz de detectar catecol e pirogalol em um menor limite de detecção.
In this study amperometric biosensors were manufactured from the use of polyphenol oxidase (PPO) obtained from avocado as a source of enzyme immobilized in polypyrrole (PPY) films that were electrosynthesized with three different support electrolytes (NaCl, NaClO4 and NaDDS). The method of enzyme immobilization was the adsorption. The PPO was added in the solution containing pyrrole and electrolyte during electropolymerization. The PPY/PPO films were characterized by electrochemical techniques mainly by cyclic voltammetry. Detection of phenolic compounds (catechol and pyrogallol) was performed by the technique of chronoamperometry after varying the concentration of the analyte. The morphology of the films was studied by the atomic force microscopy (AFM) and observed that the presence of the enzyme in the polymer film and the use of different electrolytes support led to differences in the surface of films. However it was found that the biosensor constructed from the use of NaCl showed more efficient response and it was able to detect catechol and pyrogallol in a lower limit of detection.

Vacuum microwave (VM) blanching decreases enzymatic food browning at low temperatures (between ~40-55°C) when compared to conventional processing methods. The impact and reaction of microwave blanching on polyphenol oxidase (PPO) of the Russet potato was investigated and compared with the effects of conventional convective heating. In order to closely examine PPO kinetics in a microwave field and to establish a PPO assay for use in further investigations, potato puree plus polyvinylpolypyrrolidone (PVPP) was centrifuged and filtered to produce a treatment ready suspension. Whole potatoes were cut into French fry strips and blanched using a pilot-sized V M dehydrator. This was compared to a steam kettle blanch to determine the blanch effects on physical form. Finally, two different microwave treatments were utilized along with a heated water bath in order to compare the microwave heated potato suspension samples with conventional heating. Microwave treatments used a 700 W, 2450 MHz vacuum microwave oven and an Ethos Synth (ES-MR) microwave reactor set at 2450 MHz, which dropped the microwave power level from 700 W to below 100 W after reaching the desired temperature. Experiments in all three treatments were conducted under controlled temperatures between 40°C and 70°C. The decimal reduction values (D-value) of PPO were reduced by heating in vacuum microwave treatments as compared to heating in a water bath at temperatures between 40°C and 57°C. At these temperatures, the D-values, z-values and activation energies (E[sub a]) for the PPO model reaction were significantly lower (p < 0.05) for the VM and the ES than the heated water bath. The results suggest the existence of an alternative inactivation mechanism for PPO when heated in a microwave field compared to a water bath at low processing temperatures.
Land and Food Systems, Faculty of
Graduate

碩士
實踐大學
食品營養與保健生技學系碩士班
101
The objectives of this study were trying to understand properties of Day Lily polyphenol oxidase and the effect of different processing on products browning. In order to control over the quality of dried products. First, compare the fresh and commercially available dry Day Lily of physical and chemical properties. Second, simulations using hot water, microwave, steam with blanching and combined with optimal concentration of sulfiting agent. To understand the differences in the physical and chemical properties after drying and color change during storage. Finally, to explore the characteristics of Day Lily polyphenol oxidase and understanding its correlation with enzyme browning. The results showed that the water content between 86 to 89% and 10 to 17%, pH values between 5.26 to 5.30 and 4.39 to 5.19. Commercially available samples mostly add sulfur dioxide, and content between 700 to 20,000 ppm. L, b value is increased with residual SO2 and both low in no sulfur products. Sulfur dioxide residue and color value will increased with soaking time and soaking concentration in processing and the crispness will higher when the sulfur dioxide residue increase in rehydrated Day Lily products. Day Lily polyphenol oxidase activity will decrease with increasing microwave energy density. The group with hot water 85℃ blanching decrease significantly. After blanching, steam treatment ineffective, but blanching with 85℃ or microwave energy density 4 treatment and soaking sulfur dioxide solution with 12,000 ppm for two hours, the dried products color is more better after one year and the color are similar with the commercially available products. To extracted the crude polyphenol oxidase enzyme solution from fresh Day Lily with citric acid - disodium hydrogen phosphate buffer solution, and use catechol as a substrate to determination of the enzyme activity. The results show that the optimal pH for polyphenol oxidase activity was about 5.0, and had a temperature optimum of 35 ℃ and enzyme inactivated after heat treatment at 80 ℃／25 minutes. When the temperature at 25-65 ℃, the Z value is 52.63℃ and when the temperature at 70-85℃, the Z value is 7.89℃. To determinate the distribution of the enzyme activity by longitudinal section into petals, stamens and cross-section into the head, the middle, the tail. The results show that the petals activity was significantly higher than the stamens and the head is significantly higher than the tail and middle. The optimal water activity for polyphenol oxidase activity was about 0.6 - 0.7. The effect of catechol concentrations ranging, straight lines were obtained for the Lineweaver-Burk plots. The 3.7 mM of Km and 41.3 μM/min of Vmax. Compared with other plants Km values with catechol substrate, the polyphenol oxidase from Day Lily is more active.

The color of wheat end-products depends on flour characteristics, including polyphenol oxidase (PPO) level, in addition to processing. The purpose of this study was to evaluate the methodologies used to determine PPO activity, to compare the effects of genotype and environment on PPO, and to determine the relationship of PPO to end-product discoloration. A set of double haploid experimental lines, including red and white kernel genotypes and agronomical checks, were grown at four locations in western Canada during two years. PPO activity was analyzed in grain and flour by the oxygen consumption method with catechol as substrate, and by spectrophotometric methods. Color values and discoloration after 24 hours were determined in salted (Udon) and alkaline (Kansui) type noodle sheets. A simple fast spectrophotometric whole kernel method using tyrosine as a substrate was well correlated with results of the oxygen consumption method (r = 0.813, P = 0.001). Milling and flour properties of the samples were affected significantly by the environment. (Abstract shortened by UMI.)

碩士
國立嘉義大學
應用化學系研究所
98
Polyphenol oxidase or tyrosinases(PPO) are enzymes with a dinuclear copper center, only when copper ions to be the case as a cofactor will have activity. Structure of the active site of the enzyme is composed of copper which is bound by six or seven histidine residues and a single cysteine residue. Although the enzyme seems to be of almost universal distribution in animals, plants, fungi and bacteria. Much is still unknown about its biological function and structural character, especially in plants. In this study, the recombinant PPO from sweet potato leaves was first prepared by using recombinant DNA and protein expression technique. And the structure correlated with enzyme activity was elucidated by Raman spectroscopy. Three different types of recombinant PPO (PPO34, PPO43, PPO69) exhibit highly similarity in amino acid sequence. Both PPO34 and PPO69 exhibited similar enzyme activity (equivalent kcat/km value) although the exhibited difference in the sequence site of 209, where Trp in PPO34 was replaced by Arg in PPO69. Based on the Raman result, α-helix takes most part in the secondary structure of PPO as shown by the ratio of α-helix, β-Sheet and random coil, 44％, 36％, 19％ in PPO34 and 48％, 34％, 18％ in PPO69, respectively. In addition, tyrosine residues of PPO was favored in exposed state and was insensitive to the presence of Cu2+ in the enzyme. However, Trp was sensitive to the presence of Cu2+, which would lead Trp in a buried state. Keyword: FT-Raman; Polyphenol oxidas

碩士
國立屏東科技大學
熱帶農業暨國際合作系所
97
The series of studies were conducted to elucidate the changes of physico-chemical characteristics, polyphenol oxidase (PPO) and peroxidase(POD) activities, and total phenolic compounds during fruit growth and development in three cultivars of smooth loofah (Luffa cylindrical Roem.) fruits (cv. ‘Kaohsiung 2’, ‘Merit’ and ‘Tainung No. 1’). Moreover, the correlation between enzymatic browning, polyphenol oxidase (PPO) and peroxidase (POD) activities, and total phenolic compounds in three cultivars of smooth loofah fruit were investigated. It is possible to determine the optimum stage for harvest from the data. Last but not least, it was also made to find out the best storage temperature for smooth loofah fruit, effects of different storage temperatures (5, 10, and 15°C respectively) on fruit storage life, and symptom of chilling injury was investigated. The curves of length, width, fresh weight and volume in the three cultivars of smooth loofah showed a linear growth. According to length, width, fresh weight, peel color, total soluble solid content, titratable acidity, and sugar/acid ratio in fruits, these cultivars attained their specific character within 9-11 days after anthesis. The degree of green and yellow of three cultivars of peel increased and the degree of green and yellow of fruits pulp decreased during the growth and development of the cultivars. The fruit polyphenol oxidase (PPO) activity of ‘Kaohsiung 2’, ‘Merit’and ‘Tainung No. 1’ increased with 5-11th days after anthesis. The peel polyphenol oxidase activity of ‘Kaohsiung 2’ fruit peaked on the 3rd day after anthesis, and the peel polyphenol oxidase activity of ‘Merit’ and ‘Tainung No.1’ fruits peaked on the 11th day after anthesis. The pulp peroxidase (POD) activity of ‘Kaohsiung 2’, ‘Merit’ and ‘Tainung No. 1’decreased with the fruits’ growth and development. The content of total phenolic compounds of the three smooth loofah cultivars peaked on the 5th day after anthesis, and the total phenolic compounds in pulp increased on 7-11th days after anthesis. Besides, the total phenolic compounds were highest in ‘Tainung No. 1’ fruit pulp than ‘Kaohsiung 2’ and ‘Merit’ throughout the growth and development of the fruits. The pulp lightness and white index of ‘Tainung No. 1’ was significantly decreased, and ‘Tainung No. 1’ fruit pulp browning existed except for‘Kaohsiung 2’ and ‘Merit’ after cutting at ambient temperature. The pulp PPO activity, POD activity, and total phenolic compounds were highest for‘Tainung No. 1’ and lowest for ‘Kaohsiung 2’. The fruit PPO and POD activity of the three cultivars increased with the increase of chilling injury index, and the chilling injury occurred earliest in‘Tainung No. 1’ than ‘Kaohsiung 2’ and ‘Merit’ during 5°C storage. There were no symptoms of chilling injuries during 15°C storage of three cultivars.The peel PPO activity of the three cultivars increased with the increase of storage duration and the surface yellowing at 25°C. Results of the comparison of the storage life of three smooth loofah cultivars showed that storage life was longest at 15°C than 5 and 25°C respectively. Moreover, the storage life at 5° and 25°C were lowest in ‘Tainung No. 1’ than ‘Kaohsiung 2’.

碩士
國立中興大學
生物科技學研究所
105
Lignocelluloses contain cellulose and hemicellulose which can be hydrolyzed into simple sugars suitable for the production of biofuels by fermentation. However, cellulose is wrapped by lignin, in turn bound covalently to hemicelluloses; this complex structure makes lignocelluloses hard to be degraded. A strain of Serratia sp. from a piece of rotting wood was isolated using agar plates with alkaline lignin as the sole carbon source in this study. This strain grows fast in alkaline lignin-containing minimal medium. 0.5 g/L alkaline lignin and 0.125 g/L bagasse were used as inducers to induce the oxidative enzyme activity of Serratia sp.. After three day culture, the cell was disrupted, the supernatant was collected by centrifugation and the activity was analyzed by using ABTS as the substrate. The activity was 69 U/L. The hydrolysis of sugarcane bagasse by Trichoderma reesei cellulases (Tre cellulases) in the presence or absence of the Serratia crude enzyme was analyzed. At pH 6.0, the addition of 50 mU Serratia enzyme into 1.8 U Tre cellulases increase the content of reducing sugar by 2.7 times compared with the reaction with Tre cellulases alone. The enzyme preparation after purification steps of ammonium sulfate precipitation, gel filtration chromatography and anion exchange chromatography still contain many proteins bands on SDS-PAGE. Serratia sp. has a polyphenol oxidase CueO that was thought to be the enzyme promoting the hydrolysis activity of Tre cellulases. The CueO gene was cloned into pETDuet-1 vector and the recombinant protein was expressed in E. coli BL21 (DE3). Nonetheless, the purified recombinant Serratia CueO was not able to promote the hydrolysis of bagasse catalyzed by Tre cellulases as did the crude Sarratia enzyme preparation. On the other hand, the recombinant CueO had the ability to decolorize several dyes. At pH 6.0, aniline blue and methyl blue could be decolorized 91% and 92% after an overnight reaction. At pH 8.0, remazol brilliant blue R was decolorized 62% after an overnight reaction, showing that Serratia CueO has the potential for bleaching of the dyes.

碩士
國立中興大學
昆蟲學系
93
Effect of formulation of hormone (NAA 10-20 ppm, 6-BA 5-10 ppm, GA 5-10 ppm), fertilizers (Humic acid, Seaweed extract, urea and Hyporex II®) and seed soaking duration on 5-day and12-day seedling survivals and plant growth (height of plants , length of roots, leave area, and plant dry/wet weight) were evaluated with hydroponic lima bean (Phaseolus vulgaris L.). The lower concentration of NAA (10 ppm), 6-BA (5 ppm) and GA (5 ppm) with shortest soaking duration (0.5 hour) showed a higher survival of bean plant and higher growth rate and biomass at 5-day and 12-day hydroponic seedlings. Foliar application of a combination of humic acid, seaweed extract, urea and Hyporex II® was the best to have lima bean vigorousness and gained the highest density of Tetranychus urticae and egg production among all tested combination and solo fertilizers. Initial pH 6.0 of hydroponic water without any additives, but daily foliar spray of combined 4 fertilizers gained a higher and steady production of Tetranychus urticae eggs. Lima bean plant increase its degree of PPO and POD activities with intensity of injury of Tetranychus urticae. Degree of PPO and POD activities were verified as two mechanisms of lima bean plant to Tetranychus urticae injury. Suitable duration of lima bean plant for T. urticae rearing can be therefore determined by the activities of PPO and POD.