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- Hansson, Therese, et al.
(författare)
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Improved oligosaccharide synthesis by protein engineering of β-glucosidase CelB from hyperthermophilic Pyrococcus furiosus
- 2001
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Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 73:3, s. 203-210
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Tidskriftsartikel (refereegranskat)abstract
- Enzymatic transglycosylation of lactose into oligosaccharides was studied using wild-type β-glucosidase (CelB) and active site mutants thereof (M424K, F426Y, M424K/F426Y) and wild-type β-mannosidase (BmnA) of the hyperthermophilic Pyrococcus furiosus. The effects of the mutations on kinetics, enzyme activity, and substrate specificity were determined. The oligosaccharide synthesis was carried out in aqueous solution at 95°C at different lactose concentrations and pH values. The results showed enhanced synthetic properties of the CelB mutant enzymes. An exchange of one phenylalanine to tyrosine (F426Y) increased the oligosaccharide yield (45%) compared with the wild-type CelB (40%). Incorporation of a positively charged group in the active site (M424K) increased the pH optimum of transglycosylation reaction of CelB. The double mutant, M424K/ F426Y, showed much better transglycosylation properties at low (10-20%) lactose concentrations compared to the wild-type. At a lactose concentration of 10%, the oligosaccharide yield for the mutant was 40% compared to 18% for the wild-type. At optimal reaction conditions, a higher ratio of tetrasaccharides to trisaccharides was obtained with the double mutant (0.42, 10% lactose) compared to the wild-type (0.19, 70% lactose). At a lactose concentration as Iow as 10%, only trisaccharides were synthesized by CelB wild-type. The β-mannosidase BmnA from P. furiosus showed both β-glucosidase and β-galactosidase activity and in the transglycosylation of lactose the maximal oligosaccharide yield of BmnA was 44%. The oligosaccharide yields obtained in this study are high compared to those reported with other transglycosylating β-glycosidases in oligosaccharide synthesis from lactose.
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- Logan, NA, et al.
(författare)
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Polyphasic identification of Bacillus and Brevibacillus strains from clinical, dairy and industrial specimens and proposal of Brevibacillus invocatus sp nov.
- 2002
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Ingår i: International Journal of Systematic and Evolutionary Microbiology. - : Microbiology Society. - 1466-5026 .- 1466-5034. ; 52, s. 953-966
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Tidskriftsartikel (refereegranskat)abstract
- Thirty-three clinical, dairy and industrial isolates of aerobic endospore-forming bacteria which were unreactive in routine identification tests were characterized genotypically by using amplified rDNA restriction analysis (ARDRA), 16S rDNA sequencing and DNA-DNA reassociation, and phenotypically by using fatty acid methyll ester (FAME) analysis, SDS-PAGE of whole-cell proteins, API Biotype 100 assimilation tests and 16 other routine phenotypic tests. Three isolates were identified as strains of Bacillus badius, 12 as Brevibacillus agri, including 3 strains associated with an outbreak of water-borne illness, 4 as Brevibacillus centrosporus and 2 as Brevibacillus parabrevis; 12 strains contaminating an antibiotic production plant were recognized as members of a new species, for which the name Brevibacillus invocatus is proposed, with the type strain LMG 18962(T) (= B2156(T) = CIP 106911(T) = NCIMB 13772(T)).
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