Lipase B from Pseudozyma antarctica (PalB), had been expressed in several recombinant protein hosts and showed very good transesterification activity for biodiesel production. However, the functional expression could not be demonstrated until recently in the most popular recombinant protein expression system, e.g. Escherichia coli, and the expression performance stands improvement. The probable reason is that PalB is a lipase with more hydrophobic surface and three disulfide bonds and thus may not be easily expressed in E. coli functionally. This thesis focuses on the identification of factors limiting heterologous expression of PalB in E. coli through a systematic study by using several strategies, including the different expression compartments, fusion tags, folding factors, and host strains. Functional expression of PalB in the cytoplasm of E. coli was explored using BL21(DE3) and its mutant derivative Origami B(DE3) as the host. Bioactive PalB was obtained in the reduced cytoplasm of BL21(DE3), implying that the formation of disulfide bond was not strictly required for functional expression. However, the expression was ineffective and was primarily limited by formation of PalB inclusion bodies and growth arrest, both of which were associated with PalB misfolding and deteriorated physiology. The culture performance in terms of cell growth and PalB expression level could be significantly improved by simultaneous coexpression of multiple chaperones of trigger factor and GroEL/ES, but not individual coexpression of either one of them. It was proposed that the two chaperones mediate the early stage and late stage of cytoplasmic PalB folding and would be required simultaneously for boosting both the overall PalB synthesis rate and the cytoplasmic folding efficiency. On the other hand, a much higher bioactive PalB was produced in Origami B(DE3) harboring the same PalB expression vector. Furthermore, the significant high bioactive PalB was produced by coexpression of periplasmic folding factor without a signal peptide (e.g., coexpression of DsbA, and DsbC). Coexpression of DsbA was found to be effective in enhancing PalB expression and such an improvement was more pronounced in Origami B(DE3), suggesting that both folding and disulfide bond formation could be the major factors limiting PalB expression. The fusion tag technique was also explored by constructing several PalB fusions for the evaluation of their expression performance. While the solubility was enhanced for most PalB fusions, only the DsbA tag was effective in boosting PalB activity possibly via both enhanced solubility and correct disulfide bond formation. Our results suggest that solubilization of PalB fusions did not necessarily result in the development of PalB activity which could be closely associated with correct disulfide bond formation. While PalB was stably expressed in the cytoplasm, most of the expressed gene product aggregated in cells as inactive inclusion bodies. In contrast, PalB was extremely unstable when expressed in the periplasm, also leading to poor expression performance. Such unstable PalB can be rescued by coexpression of several periplasmic folding factors, such as DegP, FkpA, DsbA, and DsbC, but not cytoplasmic chaperones. As a result, the performance for functional PalB expression in the periplasm was significantly improved. This is the first report demonstrating the use of folding factors to rescue the extremely unstable gene product that is otherwise completely degradable. On the other hand, functional expression of PalB in the periplasm was explored using four fusion tags, e.g., DsbC, DsbA, maltose binding protein (MBP), and FLAG in the sequence of increasing expression efficacy. Amongst these fusion tags for functional expression of PalB, FLAG and MBP appear to be the most effective ones in terms of boosting enzyme activity and enhancing solubility of gene products, respectively. Overexpression of these PalB fusions often resulted in concomitant formation of insoluble inclusion bodies. Coexpression of a selection of periplasmic folding factors, including DegP (and its mutant variant of DegPS210A), FkpA, DsbA, DsbC, and a cocktail of SurA, FkpA, DsbA, and DsbC, could improve the expression performance. Coexpression of DsbA appeared to be the most effective in reducing the formation of inclusion bodies for the four PalB fusions, implying that functional expression of PalB could be limited by initial bridging of disulfide bonds. Culture performance for functional expression of PalB was optimized by overexpressing FLAG-PalB with DsbA coexpression, resulting in a high volumetric PalB activity of 360 U/liter. Without extracting protein from cells the whole cell can be directly used as a platform for the immobilized enzyme. Proof-of-concept experimentation was conducted by PalB display on the E. coli cell surface. By fusing the palB gene in between the signal peptide phoA and an autotransporter Protein EstA’s gene under the lac promoter, PalB was successfully displayed on the E. coli cell surface. However, cells encountered a severe physiological stress. Coexpression of various periplasmic folding factors, e.g., DegP, SurA, DsbA and DsbC could erease the physiological stress, but only DsbA was demonstrated to be effective to restore cell physiology and increase PalB expression level. Key words: enzyme, Escherichia coli, chaperone, folding factor, fusion tag, gene expression, Pseudozyma antarctica, lipase B, recombinant protein production

Lipase B from Pseudozyma antarctica (PalB), had been expressed in several recombinant protein hosts and showed very good transesterification activity for biodiesel production. However, the functional expression could not be demonstrated until recently in the most popular recombinant protein expression system, e.g. Escherichia coli, and the expression performance stands improvement. The probable reason is that PalB is a lipase with more hydrophobic surface and three disulfide bonds and thus may not be easily expressed in E. coli functionally. This thesis focuses on the identification of factors limiting heterologous expression of PalB in E. coli through a systematic study by using several strategies, including the different expression compartments, fusion tags, folding factors, and host strains. Functional expression of PalB in the cytoplasm of E. coli was explored using BL21(DE3) and its mutant derivative Origami B(DE3) as the host. Bioactive PalB was obtained in the reduced cytoplasm of BL21(DE3), implying that the formation of disulfide bond was not strictly required for functional expression. However, the expression was ineffective and was primarily limited by formation of PalB inclusion bodies and growth arrest, both of which were associated with PalB misfolding and deteriorated physiology. The culture performance in terms of cell growth and PalB expression level could be significantly improved by simultaneous coexpression of multiple chaperones of trigger factor and GroEL/ES, but not individual coexpression of either one of them. It was proposed that the two chaperones mediate the early stage and late stage of cytoplasmic PalB folding and would be required simultaneously for boosting both the overall PalB synthesis rate and the cytoplasmic folding efficiency. On the other hand, a much higher bioactive PalB was produced in Origami B(DE3) harboring the same PalB expression vector. Furthermore, the significant high bioactive PalB was produced by coexpression of periplasmic folding factor without a signal peptide (e.g., coexpression of DsbA, and DsbC). Coexpression of DsbA was found to be effective in enhancing PalB expression and such an improvement was more pronounced in Origami B(DE3), suggesting that both folding and disulfide bond formation could be the major factors limiting PalB expression. The fusion tag technique was also explored by constructing several PalB fusions for the evaluation of their expression performance. While the solubility was enhanced for most PalB fusions, only the DsbA tag was effective in boosting PalB activity possibly via both enhanced solubility and correct disulfide bond formation. Our results suggest that solubilization of PalB fusions did not necessarily result in the development of PalB activity which could be closely associated with correct disulfide bond formation. While PalB was stably expressed in the cytoplasm, most of the expressed gene product aggregated in cells as inactive inclusion bodies. In contrast, PalB was extremely unstable when expressed in the periplasm, also leading to poor expression performance. Such unstable PalB can be rescued by coexpression of several periplasmic folding factors, such as DegP, FkpA, DsbA, and DsbC, but not cytoplasmic chaperones. As a result, the performance for functional PalB expression in the periplasm was significantly improved. This is the first report demonstrating the use of folding factors to rescue the extremely unstable gene product that is otherwise completely degradable. On the other hand, functional expression of PalB in the periplasm was explored using four fusion tags, e.g., DsbC, DsbA, maltose binding protein (MBP), and FLAG in the sequence of increasing expression efficacy. Amongst these fusion tags for functional expression of PalB, FLAG and MBP appear to be the most effective ones in terms of boosting enzyme activity and enhancing solubility of gene products, respectively. Overexpression of these PalB fusions often resulted in concomitant formation of insoluble inclusion bodies. Coexpression of a selection of periplasmic folding factors, including DegP (and its mutant variant of DegPS210A), FkpA, DsbA, DsbC, and a cocktail of SurA, FkpA, DsbA, and DsbC, could improve the expression performance. Coexpression of DsbA appeared to be the most effective in reducing the formation of inclusion bodies for the four PalB fusions, implying that functional expression of PalB could be limited by initial bridging of disulfide bonds. Culture performance for functional expression of PalB was optimized by overexpressing FLAG-PalB with DsbA coexpression, resulting in a high volumetric PalB activity of 360 U/liter. Without extracting protein from cells the whole cell can be directly used as a platform for the immobilized enzyme. Proof-of-concept experimentation was conducted by PalB display on the E. coli cell surface. By fusing the palB gene in between the signal peptide phoA and an autotransporter Protein EstA’s gene under the lac promoter, PalB was successfully displayed on the E. coli cell surface. However, cells encountered a severe physiological stress. Coexpression of various periplasmic folding factors, e.g., DegP, SurA, DsbA and DsbC could erease the physiological stress, but only DsbA was demonstrated to be effective to restore cell physiology and increase PalB expression level. Key words: enzyme, Escherichia coli, chaperone, folding factor, fusion tag, gene expression, Pseudozyma antarctica, lipase B, recombinant protein production

Le biodiesel, consid??r?? comme une solution pour remplacer le p??trodiesel est un sujet de recherche mondial. Un des principaux probl??mes associ??s au d??veloppement industriel du biodiesel est la source de mati??re premi??re ainsi que le proc??d?? de transformation. Ainsi, la pr??sente ??tude a pour but de trouver une source de mati??re premi??re durable pour la production industrielle de biodiesel et de d??terminer le proc??d?? de transformation de la mati??re premi??re, le plus appropri??, ainsi que les meilleures conditions op??ratoires. Durant la premi??re ??tape de ce projet, une source de mati??re premi??re durable a ??t?? s??lectionn??e : les microalgues. Le proc??d?? de transformation ??tudi?? est la transest??rification enzymatique. L???huile d???olive, huile ayant une composition en acides gras similaires ?? celle de l???huile de la microalgue Chlorella protothecoides, a ??t?? choisie pour effectuer les r??actions. Pendant la deuxi??me ??tape, le proc??d?? de standardisation de la r??action (bior??acteur de 5 mL) consistait ?? faire varier : le type de catalyseur (lipases de Candida antarctica (Novozym?? 435) et de Thermomyces lanuginosus (TL I150)), la concentration du catalyseur (7 ?? 14 % m/mhuile), la temp??rature de r??action (25 ?? 50 ??C) et le ratio molaire alcool:huile (3:1 ?? 4:1) ; la vitesse d???agitation ??tant de 150 rpm pour toutes les r??actions. Des techniques d???optimisation telle que la preincubation de l???enzyme ont ??t?? ??galement essay??es. Le rendement en esters alkyliques de la r??action de transest??rification enzymatique de l???huile en fonction du temps est la variable de contr??le pour toutes les r??actions. La standardisation des variables du proc??d?? a ??t?? faite en fonction de la r??duction du temps de r??action et du rendement en esters alkyliques. Un rendement ??lev?? en esters alkyliques de 92 % (m/m) a ??t?? obtenu sous les conditions op??ratoires suivantes : une concentration de catalyseur (TL I150) de 7 % (m/mhuile), une temp??rature de r??action de 25 ??C, un ratio molaire alcool:huile de 3:1 et un temps de r??action de 4 h ; la lipase a ??t?? preincub??e pendant 6 h avant la r??action de transest??rification. Le temps de r??action, un des param??tres importants lors du proc??d?? de standardisation des variables, a ??t?? r??duit de 24 ?? 4 h. Un autre param??tre significatif de la r??action est la temp??rature : une temp??rature de 25 ??C a ??t?? utilis??e; cette temp??rature de r??action est faible et rend le proc??d?? au niveau industriel plus attrayant.

The objective of the research was to carry out the biogeneration of lipophenols by enzymatic esterification of tricaprylin and caprylic acid with catechin and catechol in a model hexane system. Commercial lipases, including Lipase N from Rhizopus niveus, Lipozyme IM from Mucor miehei and Novozym 435 from Candida antarctica were used throughout this study. The effects of reaction time, incubation temperatures and agitation speeds on enzymatic hydrolytic activity were investigated to determine the optimal conditions for biocatalysis. The optimal temperatures for biocatalysis were determined to be 37.5°C for Lipase N, and 55°C for Lipozyme IM and Novozym 435; the optimum agitation speed was 100 rpm. Using Lipase N, maximum hydrolysis of 1.66 mumol free fatty acids/mL was obtained after 1.5 days of incubation, while with Lipozyme IM, maximum hydrolysis of 8.1 and 8.5 mumol free fatty acids/mL was obtained after 1 and 4 days, respectively. With Novozym 435, the highest hydrolysis of 4.0 and 6.1 mumol free fatty acids/mL were found after 2 and 9 days, respectively.

No description available.

Childhood acute lymphoblastic leukemia (ALL) is the most common malignancy in children. Several proto-oncogenes that encode nuclear proteins are activated by various chromosomal translocations in ALL including TALI, TAL2, and LMO1 and LMO2. Ectopic TALI expression is observed in about 50 % of T-ALL and is the most common genetic anomaly associated with this pathology. Of interest to the present work is the characterization of various multiprotein complexes and protein protein interactions that drive T-ALL progression (as it relates to TALI and LMO1) and over expression of TALI and LMO1 has been shown to have inhibitory effects on apoptosis. Recent data suggests possible interactions between these two oncoproteins and the protein product of the lipoprotein lipase (LPL) gene. Lipoprotein lipase has a complex pattern of regulation and can be regulated in different ways including down-regulation upon induction of TNF-a in 3T3-L1 cells. Thus, this study was undertaken to determine if LPL is expressed in cells over expressing TAL1 and LMO1. Results from this study demonstrated an increase in LPL expression at both transcriptional and translational level in cells engineered to express TAL1 alone and TAL1 and LMO1 together. This finding is a step forward to understanding mechanisms that result in apoptosis prevention in T-ALL. Therefore, the apoptosis preventive role seen in cells that over express TAL and LMO1 and the presence of LPL in the same cell line, theorizes an apoptosis preventive role for lipoprotein lipase as well. / Department of Physiology and Health Science

Hubbard Foods Limited of Auckland makes a variety of oats-based value-added products. In the preparation of a range of products at Hubbard Foods, technical staff has become aware of a bitterness problem that sporadically appears in toasted oats. Toasting involves dry heating to about 150°C resulting in the golden colour and flavour development necessary for range of products. Bitterness development has been described in the literature, but Hubbard staff is necessarily focussed on production issues, rather than on a sporadic problem seemingly outside the scope of production variables. The author of this thesis set out to identify the cause and suggest a remedy. Prior research with oats has shown that bitterness and associated off-flavours are linked to the accumulation of free fatty acids, their volatile oxidation products, and possibly amino acids and certain phenols. Oats are distinguished from related grains by their high relative fat content, about seven percent, and an associated very active lipase. The free fatty acids stem from the lipase activity that should be, but may not be, inactivated at source in Australia. This is achieved in the milling process by physical disruption and moist heating to a temperature at which the enzyme is denatured. However, residual lipase activity may adversely affect oats quality during time in storage and transit. A number of analytical methods for cereals were adapted to match the constraints of time and resources. These methods were for colour, moisture, peroxidase activity, p-anisidine, and fat and free fatty acids content, composition of fatty acids, total phenols, volatiles, and bitterness as perceived by an analytical sensory panel of four people. Determination of lipase activity is very expensive, so peroxidase activity is commercially used as an indicator. If the latter is inactive, the former will necessarily be also inactive. The designed methods were first applied to 17 oats lots passing through the Hubbard environment, where 14 were paired raw and toasted. The values of moisture, fat content, free fatty acids content and total phenols were within the normal limits expected for commercial lots of oats compared with the previous studies. Not much variation was observed among the 17 oats lots, with the exception of lot DWHE25. Lot DWHE25 was a faulty product, which had high moisture content, high free fatty acids content, and tasted very bitter. The results suggested that moisture content, free fatty acids and bitterness were usually correlated. In spite of the differences encountered and the clues provided by extremes, the data generated from Hubbard oats lots did not provide enough variation in quality to lead to a definitive chemical model of bitter flavour development. But perhaps crucially, it was found that most samples as received from Hubbard Foods were peroxidase-active which conflicted with the results reported on specification sheets prepared by the oats supplier. These specifications accompanied each lot delivered to Hubbard Foods. Therefore, the supplier’s method was examined and was found to be deficient in one critically important respect. Their method omitted the key reactant hydrogen peroxide. Therefore, it is possible that the lipase was active in many of the samples. Therefore, some experiments were conducted where raw oats, from Hubbard Foods and a supermarket, were treated with water additions and stored for a period to examine the effect of moisture content on the quality and flavour deterioration on subsequent storage. Water-treated oats were toasted to simulate a typical Hubbard process, yielding a total of 58 samples with carrying moisture contents. The data set was statistically analysed to identify the cause of bitterness and the means of its control. The free fatty acids content, volatile compounds particularly hexanal, and total phenols increased with moisture content and storage time. The correlations between chemical analysis and sensory test indicated that free fatty acids positively correlated with bitterness (r = 0.71), and hexanal was also positively correlated. Total phenols did not appear to correlate with bitterness. Oats lots with high peroxidase activity tended to have the poorest quality, strongly implicating residual lipase activity as the critical factor. There were no important interactions between water addition and toasting for most of the experiments. Therefore, it seems likely that the toasting procedures at Hubbard Foods are not responsible for bitterness formation. The cause(s) of bitterness is certainly at source, with a faulty peroxidase test strongly implicated.

Hubbard Foods Limited of Auckland makes a variety of oats-based value-added products. In the preparation of a range of products at Hubbard Foods, technical staff has become aware of a bitterness problem that sporadically appears in toasted oats. Toasting involves dry heating to about 150°C resulting in the golden colour and flavour development necessary for range of products. Bitterness development has been described in the literature, but Hubbard staff is necessarily focussed on production issues, rather than on a sporadic problem seemingly outside the scope of production variables. The author of this thesis set out to identify the cause and suggest a remedy. Prior research with oats has shown that bitterness and associated off-flavours are linked to the accumulation of free fatty acids, their volatile oxidation products, and possibly amino acids and certain phenols. Oats are distinguished from related grains by their high relative fat content, about seven percent, and an associated very active lipase. The free fatty acids stem from the lipase activity that should be, but may not be, inactivated at source in Australia. This is achieved in the milling process by physical disruption and moist heating to a temperature at which the enzyme is denatured. However, residual lipase activity may adversely affect oats quality during time in storage and transit. A number of analytical methods for cereals were adapted to match the constraints of time and resources. These methods were for colour, moisture, peroxidase activity, p-anisidine, and fat and free fatty acids content, composition of fatty acids, total phenols, volatiles, and bitterness as perceived by an analytical sensory panel of four people. Determination of lipase activity is very expensive, so peroxidase activity is commercially used as an indicator. If the latter is inactive, the former will necessarily be also inactive. The designed methods were first applied to 17 oats lots passing through the Hubbard environment, where 14 were paired raw and toasted. The values of moisture, fat content, free fatty acids content and total phenols were within the normal limits expected for commercial lots of oats compared with the previous studies. Not much variation was observed among the 17 oats lots, with the exception of lot DWHE25. Lot DWHE25 was a faulty product, which had high moisture content, high free fatty acids content, and tasted very bitter. The results suggested that moisture content, free fatty acids and bitterness were usually correlated. In spite of the differences encountered and the clues provided by extremes, the data generated from Hubbard oats lots did not provide enough variation in quality to lead to a definitive chemical model of bitter flavour development. But perhaps crucially, it was found that most samples as received from Hubbard Foods were peroxidase-active which conflicted with the results reported on specification sheets prepared by the oats supplier. These specifications accompanied each lot delivered to Hubbard Foods. Therefore, the supplier’s method was examined and was found to be deficient in one critically important respect. Their method omitted the key reactant hydrogen peroxide. Therefore, it is possible that the lipase was active in many of the samples. Therefore, some experiments were conducted where raw oats, from Hubbard Foods and a supermarket, were treated with water additions and stored for a period to examine the effect of moisture content on the quality and flavour deterioration on subsequent storage. Water-treated oats were toasted to simulate a typical Hubbard process, yielding a total of 58 samples with carrying moisture contents. The data set was statistically analysed to identify the cause of bitterness and the means of its control. The free fatty acids content, volatile compounds particularly hexanal, and total phenols increased with moisture content and storage time. The correlations between chemical analysis and sensory test indicated that free fatty acids positively correlated with bitterness (r = 0.71), and hexanal was also positively correlated. Total phenols did not appear to correlate with bitterness. Oats lots with high peroxidase activity tended to have the poorest quality, strongly implicating residual lipase activity as the critical factor. There were no important interactions between water addition and toasting for most of the experiments. Therefore, it seems likely that the toasting procedures at Hubbard Foods are not responsible for bitterness formation. The cause(s) of bitterness is certainly at source, with a faulty peroxidase test strongly implicated.

Diss. (sammanfattning) Umeå : Univ., 2004. / Härtill 5 uppsatser.

Diss. (sammanfattning) Umeå : Univ., 2007. / Härtill 4 uppsatser.