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1.
Comput Biol Chem ; 48: 64-70, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24378653

RESUMO

Sequence subgrouping for a given sequence set can enable various informative tasks such as the functional discrimination of sequence subsets and the functional inference of unknown sequences. Because an identity threshold for sequence subgrouping may vary according to the given sequence set, it is highly desirable to construct a robust subgrouping algorithm which automatically identifies an optimal identity threshold and generates subgroups for a given sequence set. To meet this end, an automatic sequence subgrouping method, named 'Subgrouping Automata' was constructed. Firstly, tree analysis module analyzes the structure of tree and calculates the all possible subgroups in each node. Sequence similarity analysis module calculates average sequence similarity for all subgroups in each node. Representative sequence generation module finds a representative sequence using profile analysis and self-scoring for each subgroup. For all nodes, average sequence similarities are calculated and 'Subgrouping Automata' searches a node showing statistically maximum sequence similarity increase using Student's t-value. A node showing the maximum t-value, which gives the most significant differences in average sequence similarity between two adjacent nodes, is determined as an optimum subgrouping node in the phylogenetic tree. Further analysis showed that the optimum subgrouping node from SA prevents under-subgrouping and over-subgrouping.


Assuntos
Algoritmos , Análise de Sequência de Proteína , 4-Aminobutirato Transaminase/química , Carboxiliases/química , Ornitina-Oxo-Ácido Transaminase/química , Filogenia , Alinhamento de Sequência , beta-Alanina-Piruvato Transaminase/química
2.
Biotechnol Bioeng ; 96(3): 559-69, 2007 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-16902948

RESUMO

Biocatalysis continues to emerge as a powerful technique for the efficient synthesis of optically pure pharmaceuticals that are difficult to access via conventional chemistry. The power of biocatalysis can be enhanced if two or more reactions can be achieved by a single whole cell biocatalyst containing a pathway designed de-novo to facilitate a required synthetic sequence. The enzymes transketolase (TK) and transaminase (TAm) respectively catalyze asymmetric carbon--carbon bond formation and amine group addition to suitable substrate molecules. The ability of a transaminase to accept the product of the transketolase reaction can allow the two catalysts to be employed in series to create chiral amino-alcohols from achiral substrates. As proof of principle, the beta-alanine: pyruvate aminotransferase (beta-A:P TAm) from Pseudomonas aeruginosa has been cloned, to create plasmid pQR428, for overexpression in E.coli strain BL21gold(DE3). Production of the beta-A:P TAm alongside the native transketolase (overexpressed from plasmid pQR411), in a single E.coli host, has created a novel biocatalyst capable of the synthesis of chiral amino alcohols via a synthetic two-step pathway. The feasibility of using the biocatalyst has been demonstrated by the formation of a single diastereoisomer of 2-amino-1,3,4-butanetriol (ABT) product, in up to 21% mol/mol yield, by the beta-A:P TAm, via transamination of L-erythrulose synthesized by TK, from achiral substrates glycolaldehyde (GA) and beta-hydroxypyruvate (beta-HPA). ABT synthesis was achieved in a one-pot process, using either whole cells of the dual plasmid strain or cell lysate, while the dual alcohol-amine functionality of ABT makes it an excellent synthon for many pharmaceutical syntheses.


Assuntos
Amino Álcoois/síntese química , Proteínas de Bactérias/química , Escherichia coli/enzimologia , Pseudomonas aeruginosa/enzimologia , Transcetolase/química , beta-Alanina-Piruvato Transaminase/química , Proteínas de Bactérias/genética , Catálise , Sistema Livre de Células/enzimologia , Escherichia coli/genética , Pseudomonas aeruginosa/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Estereoisomerismo , Transcetolase/genética , beta-Alanina-Piruvato Transaminase/genética
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