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A beta-phosphoglucomutase in carbohydrate metabolism of Lactococcus lactis

Qian, Ny LU (1997)
Abstract (Swedish)
Popular Abstract in Swedish

Ett beta-fosfoglukomutas i kolhydrat-metabolismen hos Lactococcus lactis
Abstract
Phosphoglucomutase (PGM) catalyzes the reversible conversion of glucose 1-phosphate to glucose 6-phosphate. Two distinct forms of PGM have been purifed and charactierized in Lactococcus lactis growing on maltose: one specific for the alpha-isomer, and the other specific for the beta-isomer of glucose 1-phosphate. Beta-PGM was purified and polyclonal antisera to the enzyme were generated. Beta-PGM was determined to be a monomeric polypeptide with a molecular weight of around 25 kDa, and with an isoelectric point of 4.8. The corresponding values for alpha-PGM were 65 kDa and 4.4, respectively. The gene encoding beta-PGM (bgmB) was cloned from a genomic library of L. lactis using the antisera. The nucleotide sequence of a 5695 bp DNA fragment... (More)
Phosphoglucomutase (PGM) catalyzes the reversible conversion of glucose 1-phosphate to glucose 6-phosphate. Two distinct forms of PGM have been purifed and charactierized in Lactococcus lactis growing on maltose: one specific for the alpha-isomer, and the other specific for the beta-isomer of glucose 1-phosphate. Beta-PGM was purified and polyclonal antisera to the enzyme were generated. Beta-PGM was determined to be a monomeric polypeptide with a molecular weight of around 25 kDa, and with an isoelectric point of 4.8. The corresponding values for alpha-PGM were 65 kDa and 4.4, respectively. The gene encoding beta-PGM (bgmB) was cloned from a genomic library of L. lactis using the antisera. The nucleotide sequence of a 5695 bp DNA fragment was determined, which contained six open reading frames, including pgmB. Amino acid sequence analysis revealed that the pgmB product had no significant similarity to any known proteins and did not contain the highly conserved catalytic site of alpha-PGM. Howerver, a short sequence at the N-terminal of beta-PGM was found to be similar to known metal-binding domains. ORF1 upstream of pgmB was found to be related to trehalose 6-phosphotase and phosphorylase. mRNA analysis and expression study of beta-PGM indicated that pgmB was transcribed not only alone but also together with other gene(s), probably ORF1. The enzyme assay, Western blot and Northern blot analysis revealed that beta-PGM was not under the control of allosteric regulation. Rather, the enzyme synthesis was induced by maltose, trehalose, and sucrose, and was repressed by glucose and lactose in the growth medium. The specific activity of beta-PGM during fermentation was found to be dependent on the maltose concentration in the medium. Alpha-PGM was found to be a constitutive enzyme, although its expression level was dependent on the carbohydrate source. A pathway for maltose metabolism was proposed, and its control and end-product formation were studied. It was suggested that the product formation was dependent on the initial degradation of maltose. The activaties of the first two enzymes in the degradation, maltose phosphorylase (MP) and beta-PGM, were higher in the strains producing more lactate and less other acids. Moreover, more lactate was producecd when maltose concentration was increased. pgmB was cloned and expressed in L. lactis 65.1, which exhibits homolactic formation on glucose, but produces mixed acids when grown on maltose. Two strains with different levels of beta-PGM activity were obtained. Although the product formation pattern were not changed by the overexpression of the gene, it is suggested that MP should also be overproduced in order to change the product formation of L. lactis. (Less)
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author
opponent
  • Valla, Svein
organization
publishing date
type
Thesis
publication status
published
subject
keywords
mykologi, virologi, bakteriologi, Mikrobiologi, mycology, gene regulation, beta-phosphoglucomutase, maltose metabolism, lactate, metabolic engineering, alpha-phosphoglucomutase, Lactococcus lactis, Microbiology, virology, bacteriology, Biochemistry, Metabolism, Biokemi
pages
102 pages
publisher
Department of Applied Microbiology, Lund University
defense location
Hall C, Chemical Center,
defense date
1997-05-29 10:15
external identifiers
  • other:ISRN: LUTKDH/TKMB-1025/1997-SE
ISBN
91-628-2515-1
language
English
LU publication?
yes
id
d9dd42bf-1037-4866-abdf-7165dba217cd (old id 29434)
date added to LUP
2007-06-14 15:38:35
date last changed
2016-09-19 08:45:03
@phdthesis{d9dd42bf-1037-4866-abdf-7165dba217cd,
  abstract     = {Phosphoglucomutase (PGM) catalyzes the reversible conversion of glucose 1-phosphate to glucose 6-phosphate. Two distinct forms of PGM have been purifed and charactierized in Lactococcus lactis growing on maltose: one specific for the alpha-isomer, and the other specific for the beta-isomer of glucose 1-phosphate. Beta-PGM was purified and polyclonal antisera to the enzyme were generated. Beta-PGM was determined to be a monomeric polypeptide with a molecular weight of around 25 kDa, and with an isoelectric point of 4.8. The corresponding values for alpha-PGM were 65 kDa and 4.4, respectively. The gene encoding beta-PGM (bgmB) was cloned from a genomic library of L. lactis using the antisera. The nucleotide sequence of a 5695 bp DNA fragment was determined, which contained six open reading frames, including pgmB. Amino acid sequence analysis revealed that the pgmB product had no significant similarity to any known proteins and did not contain the highly conserved catalytic site of alpha-PGM. Howerver, a short sequence at the N-terminal of beta-PGM was found to be similar to known metal-binding domains. ORF1 upstream of pgmB was found to be related to trehalose 6-phosphotase and phosphorylase. mRNA analysis and expression study of beta-PGM indicated that pgmB was transcribed not only alone but also together with other gene(s), probably ORF1. The enzyme assay, Western blot and Northern blot analysis revealed that beta-PGM was not under the control of allosteric regulation. Rather, the enzyme synthesis was induced by maltose, trehalose, and sucrose, and was repressed by glucose and lactose in the growth medium. The specific activity of beta-PGM during fermentation was found to be dependent on the maltose concentration in the medium. Alpha-PGM was found to be a constitutive enzyme, although its expression level was dependent on the carbohydrate source. A pathway for maltose metabolism was proposed, and its control and end-product formation were studied. It was suggested that the product formation was dependent on the initial degradation of maltose. The activaties of the first two enzymes in the degradation, maltose phosphorylase (MP) and beta-PGM, were higher in the strains producing more lactate and less other acids. Moreover, more lactate was producecd when maltose concentration was increased. pgmB was cloned and expressed in L. lactis 65.1, which exhibits homolactic formation on glucose, but produces mixed acids when grown on maltose. Two strains with different levels of beta-PGM activity were obtained. Although the product formation pattern were not changed by the overexpression of the gene, it is suggested that MP should also be overproduced in order to change the product formation of L. lactis.},
  author       = {Qian, Ny},
  isbn         = {91-628-2515-1},
  keyword      = {mykologi,virologi,bakteriologi,Mikrobiologi,mycology,gene regulation,beta-phosphoglucomutase,maltose metabolism,lactate,metabolic engineering,alpha-phosphoglucomutase,Lactococcus lactis,Microbiology,virology,bacteriology,Biochemistry,Metabolism,Biokemi},
  language     = {eng},
  pages        = {102},
  publisher    = {Department of Applied Microbiology, Lund University},
  school       = {Lund University},
  title        = {A beta-phosphoglucomutase in carbohydrate metabolism of Lactococcus lactis},
  year         = {1997},
}