Proteolytic processing of dextransucrase of Leuconostoc mesenteroides

SÃ¡nchez-GonzÃ¡lez, M

doi:10.1016/s0378-1097(99)00510-8

Cited by 8 publications

(13 citation statements)

References 12 publications

(14 reference statements)

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“…The presence of extracellular proteolytic activity in L. mesenteroides B512F and B512FMC, either grown in sucrose or glucose as carbon sources was previously reported (Sanchez-Gonzalez et al 1999). The protein pattern of L. mesenteroides B512F and B512FMC supernatants is shown ( Figure 1A, lanes 1 and 2, respectively).…”

Section: Protease Productionsupporting

confidence: 78%

“…The maximum proteolytic activity was produced earlier in the culture, after 2 h of fermentation, probably to allow for the uptake of protein present in the culture. This is also the case with the constitutive strain L. mesenteroides B512FMC (Sanchez-Gonzalez et al 1999). AS, CAS and TRP may induce stress conditions that result in ''de novo'' extracellular protease synthesis as already described by Rolla´n et al (1998) for Leuconostoc oenos cells.…”

Section: Discussionmentioning

confidence: 53%

“…In order to measure proteolytic activity, samples were concentrated at least 10-fold using an ultrafiltration system, equipped with an HP100-20, 100 kDa cut off hollow fibre cartridge (Amicon). As already described (Sanchez-Gonzalez et al 1999), the proteolytic activity is strongly associated with the DSRS so most of the proteolytic activity is concentrated with DSRS, including the low molecular weight 30 kDa protein.…”

Section: Culture Conditionsmentioning

confidence: 54%

“…In these reaction conditions, L. mesenteroides B512FMC did not release any other active forms from the native dextransucrase. As already shown, this is not the case when the pH is shifted to the alkaline region where extensive proteolysis takes place (Sanchez-Gonzalez et al 1999).…”

Section: Protease Productionmentioning

confidence: 77%

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Proteolytic modification of Leuconostoc mesenteroides B-512F dextransucrase

Argüello-Morales

Sánchez-González²,

Canedo

et al. 2005

Antonie Van Leeuwenhoek

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Multiple active lower molecular weight forms from Leuconostoc mesenteroides B512F dextransucrase have been reported. It has been suggested that they arise from proteolytic processing of a 170 kDa precursor. In this work, the simultaneous production of proteases and dextransucrase was studied in order to elucidate the dextransucrase proteolytic processing. The effect of the nitrogen source on protease and dextransucrase production was studied. Protease activity reaches a maximum early in the logarithmic phase of dextransucrase synthesis using the basal culture medium but the nitrogen source plays an important effect on growth: the highest protease concentration was obtained when ammonium sulfate, casaminoacids or tryptone were used. Two active forms of 155 and 129 kDa were systematically obtained from dextransucrase precursor by proteolysis. The amino termini of these forms were sequenced and the cleavage site deduced. Both forms of the enzyme obtained had the same cleavage site in the amino terminal region (F209-Y210). From dextransucrase analysis, various putative cleavage sites with the same sequence were found in the variable region and in the glucan binding domain. Although no structural differences were found in dextrans synthesized with both the precursor and the proteolyzed 155 kDa form under the same reaction conditions, their rheological behaviour was modified, with dextran of a lower viscosity yielded by the smaller form.

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Section: Protease Productionsupporting

confidence: 78%

Section: Discussionmentioning

confidence: 53%

Section: Culture Conditionsmentioning

confidence: 54%

Section: Protease Productionmentioning

confidence: 77%

See 2 more Smart Citations

Proteolytic modification of Leuconostoc mesenteroides B-512F dextransucrase

Argüello-Morales

Sánchez-González²,

Canedo

et al. 2005

Antonie Van Leeuwenhoek

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“…The enzyme was concentrated from 23.50 L to 1.65 L, giving 280 IUml -1 with only 7% loss in enzyme activity 66 . Purifi ed dextransucrase from L. mesenteroides B-512F was obtained by passing the culture supernatant through an ultra-fi ltration system, equipped with a 120 kDa cut off hollow fi ber cartridge 67 . Samples were lyophilized and re-suspended in buffer, containing CaCl 2 and sodium azide 67 .…”

Section: Ultrafi Ltrationmentioning

confidence: 99%

An overview of purification methods of glycoside hydrolase family 70 dextransucrase

2007

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The enzyme dextransucrase (sucrose:1, 6-α-D-glucan 6-α-glucosyltransferase, EC 2.4.1.5) catalyses the synthesis of exopolysaccharide, dextran from sucrose. This class of polysaccharide has been extensively exploited in pharmaceutical industry as blood volume expander, as stabiliser in food industry and as a chromatographic medium in fi ne chemical industry because of their nonionic nature and stability. Majority of the dextrans are synthesized from sucrose by dextransucrase secreted mainly by bacteria belonging to genera Leuconostoc, Streptococcus and Lactobacillus. Bulk of the information on purifi cation of extracellular dextransucrase has been generated from Leuconostoc species. Various methods such as precipitation by ammonium sulphate, ethanol or polyethylene glycol, phase partitioning, ultrafi ltration and chromatography have been used to purify the enzyme. Purifi cation of dextransucrase is rendered diffi cult by the presence of viscous dextran in the medium. However, processes like ultra-fi ltration, salt and PEG precipitation, chromatography and phase partitioning have been standardized and successfully used for higher scale purifi cation of the enzyme. A recombinant dextransucrase from Leuconostoc mesenteroides B-512F with a histidine tag has been expressed in E. coli cells and purifi ed by immobilized metal ion chromatography. This review reports the available information on purifi cation methods of dextransucrase from Leuconostoc mesenteroides strains.

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Maximization of dextransucrase activity expressed in E. coli by mutation and its functional characterization

Nam

Ko²,

Jang

et al. 2007

Biotechnol Lett

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A novel dextransucrase gene, DSRN, was obtained by ultrasoft X-ray treatment of the DSRB742 gene. The DSRN gene was further mutated via site-directed mutagenesis producing four mutants: DSRN1 (F196S), DSRN2 (Y346N), DSRN3 (K395T) and DSRN4 (P980T). Dextransucrases derived from DSRB742 and its mutants were expressed in E. coli and affinity-purified using dextran to give 80% purity. They had specific activities of 0.6-17 U/mg with Km values of 18-88 mM. DSRB742 had the lowest (0.02 s(-1) x mM(-1)) and DSRN1 had the highest (0.13 s(-1) x mM(-1)) Kcat/Km values. DSRN3 had the highest enzymatic transglycosylation efficiency with maltose (63% of theoretical), gentiobiose (39%), or salicine (40%).

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Proteolytic processing of dextransucrase of Leuconostoc mesenteroides

Cited by 8 publications

References 12 publications

Proteolytic modification of Leuconostoc mesenteroides B-512F dextransucrase

Proteolytic modification of Leuconostoc mesenteroides B-512F dextransucrase

An overview of purification methods of glycoside hydrolase family 70 dextransucrase

Maximization of dextransucrase activity expressed in E. coli by mutation and its functional characterization

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