Function of a cloned polyphenolase in organic synthesis
- Authors: Naidoo, Michael Joseph
- Date: 1995
- Subjects: Polyphenols , Catechol , Streptomacyes , Organic compounds -- Synthesis , Mutagenesis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4042 , http://hdl.handle.net/10962/d1004103 , Polyphenols , Catechol , Streptomacyes , Organic compounds -- Synthesis , Mutagenesis
- Description: The enzyme polyphenolase, which catalyses the oxidation of phenols to catechols and subsequently dehydrogenates these to o-quinones, is widely distributed in nature. The multicopy plasmid vector pIJ702 contains a mel gene from Streptomyces antibioticus, that codes for the production of a polyphenol oxidase. The plasmid was isolated from Streptomyces lividans 66pIJ702 and subjected to a variety of mutagenic treatments in order to establish a structurefunction relationship for the polyphenolase enzymes. An attempt was made to engineer the polyphenolase enzyme by localized random mutagenesis in vitro of the mel gene on pIJ702, in order to alter properties like productivity, activity and substrate specificity. It was hoped to alter the amino acid sequence of the active site of the enzyme in order to facilitate catalysis in an organic environment. The plasmid was subsequently transformed into a plasmid-free Streptomyces strain, and enzyme production was carried out in batch culture systems, in order to determine the effect of the height treatment, and to isolate and propagate functional polyphenolase mutants for organic synthesis.
- Full Text:
- Date Issued: 1995
- Authors: Naidoo, Michael Joseph
- Date: 1995
- Subjects: Polyphenols , Catechol , Streptomacyes , Organic compounds -- Synthesis , Mutagenesis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4042 , http://hdl.handle.net/10962/d1004103 , Polyphenols , Catechol , Streptomacyes , Organic compounds -- Synthesis , Mutagenesis
- Description: The enzyme polyphenolase, which catalyses the oxidation of phenols to catechols and subsequently dehydrogenates these to o-quinones, is widely distributed in nature. The multicopy plasmid vector pIJ702 contains a mel gene from Streptomyces antibioticus, that codes for the production of a polyphenol oxidase. The plasmid was isolated from Streptomyces lividans 66pIJ702 and subjected to a variety of mutagenic treatments in order to establish a structurefunction relationship for the polyphenolase enzymes. An attempt was made to engineer the polyphenolase enzyme by localized random mutagenesis in vitro of the mel gene on pIJ702, in order to alter properties like productivity, activity and substrate specificity. It was hoped to alter the amino acid sequence of the active site of the enzyme in order to facilitate catalysis in an organic environment. The plasmid was subsequently transformed into a plasmid-free Streptomyces strain, and enzyme production was carried out in batch culture systems, in order to determine the effect of the height treatment, and to isolate and propagate functional polyphenolase mutants for organic synthesis.
- Full Text:
- Date Issued: 1995
The conflict between adaptation and constraint : the case of the Siphonariid limpets
- Authors: Chambers, Richard Jonathan
- Date: 1995
- Subjects: Siphonaria -- Adaptation Limpets -- Adaptation Pulmonata
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:5657 , http://hdl.handle.net/10962/d1005340
- Description: The reproductive strategies of marine invertebrates have been related to various aspects of both their ecology, and their phylogenetic history. It has been the purpose of this thesis to try and separate these components among Siphonaria, a group of marine pulmonates. The taxonomy of these species is revised and I conclude that nine species are valid. All species deposit benthic egg masses and development may be either direct (S. anneae, S. compressa, S. dayi, S. nigerrima, S. serrata and S. tenuicostulata) or planktonic (S. capensis, S.concinna and S. oculus). Data on distribution and life-history relating to mode of larval development is then presented for 26 species of Siphonaria worldwide. Fifteen species are direct developers, nine are planktonic developers and a further two appear to have a dual developmental capacity, retaining both the velar swimming apparatus of a planktonic developer and the crawling foot of a direct developer. Direct developing species hatch from larger egg capsules, and generally occur higher on the shore than planktonic developers. Worldwide, planktonic developers are more widespread than direct developers, and individual planktonic species may have a greater latitudinal range. In most S~honaria subgenera, mode of larval development appears to be constant, although two subgenera (Patel/opsis and Sacculosiphonaria) include both developmental types. Locally, the intertidal zonation of three sympatric species (S. capensis, S. concinna and S. serrata) does not support a model which predicts direct development on the high shore and planktonic development on the low shore. However, distributions do correspond to particular intertidal microhabitats, and while there may be no, direct relationship between mode of larval development and intertidal height, the physical structure of egg masses, and the microhabitats used for spawning appear adaptive with regards to desiccation in the intertidal. S. concinna (planktonic development) and S. serrata (direct development) occur in similar microhabitats and are likely to be under similar selection pressures. In having different modes of larval development, there appears to be more than one optimal solution in a particular selective regime. In addition, both species seem to apportion similar amounts of energy to reproduction for each spawning episode, and also annually suggesting an optimum allocation of resources to reproduction. Genetic investigations using PolyAcrylamide Gel Electrophoresis (PAGE) confirm the status of the southern African species initially described, and indicate greater genetic variability associated with planktonic developing species than direct developing species. The systematic relationships revealed by DNA fingerprinting support the current classification systems, and also have implications with regards the evolution of larval development: direct development may be the plesiomorphic condition in, and among, some Siphonaria groups. There are both phylogenetic and adaptive explanations for the distribution of reproductive mode among benthic marine invertebrates. An evolutionary question, however, is not just a matter of either adaptation or constraint, it is a combination of these. Both contribute to the distribution of developmental mode among Siphonaria.
- Full Text:
- Date Issued: 1995
- Authors: Chambers, Richard Jonathan
- Date: 1995
- Subjects: Siphonaria -- Adaptation Limpets -- Adaptation Pulmonata
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:5657 , http://hdl.handle.net/10962/d1005340
- Description: The reproductive strategies of marine invertebrates have been related to various aspects of both their ecology, and their phylogenetic history. It has been the purpose of this thesis to try and separate these components among Siphonaria, a group of marine pulmonates. The taxonomy of these species is revised and I conclude that nine species are valid. All species deposit benthic egg masses and development may be either direct (S. anneae, S. compressa, S. dayi, S. nigerrima, S. serrata and S. tenuicostulata) or planktonic (S. capensis, S.concinna and S. oculus). Data on distribution and life-history relating to mode of larval development is then presented for 26 species of Siphonaria worldwide. Fifteen species are direct developers, nine are planktonic developers and a further two appear to have a dual developmental capacity, retaining both the velar swimming apparatus of a planktonic developer and the crawling foot of a direct developer. Direct developing species hatch from larger egg capsules, and generally occur higher on the shore than planktonic developers. Worldwide, planktonic developers are more widespread than direct developers, and individual planktonic species may have a greater latitudinal range. In most S~honaria subgenera, mode of larval development appears to be constant, although two subgenera (Patel/opsis and Sacculosiphonaria) include both developmental types. Locally, the intertidal zonation of three sympatric species (S. capensis, S. concinna and S. serrata) does not support a model which predicts direct development on the high shore and planktonic development on the low shore. However, distributions do correspond to particular intertidal microhabitats, and while there may be no, direct relationship between mode of larval development and intertidal height, the physical structure of egg masses, and the microhabitats used for spawning appear adaptive with regards to desiccation in the intertidal. S. concinna (planktonic development) and S. serrata (direct development) occur in similar microhabitats and are likely to be under similar selection pressures. In having different modes of larval development, there appears to be more than one optimal solution in a particular selective regime. In addition, both species seem to apportion similar amounts of energy to reproduction for each spawning episode, and also annually suggesting an optimum allocation of resources to reproduction. Genetic investigations using PolyAcrylamide Gel Electrophoresis (PAGE) confirm the status of the southern African species initially described, and indicate greater genetic variability associated with planktonic developing species than direct developing species. The systematic relationships revealed by DNA fingerprinting support the current classification systems, and also have implications with regards the evolution of larval development: direct development may be the plesiomorphic condition in, and among, some Siphonaria groups. There are both phylogenetic and adaptive explanations for the distribution of reproductive mode among benthic marine invertebrates. An evolutionary question, however, is not just a matter of either adaptation or constraint, it is a combination of these. Both contribute to the distribution of developmental mode among Siphonaria.
- Full Text:
- Date Issued: 1995
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