Levansucrase optimization using solid state fermentation and levan biological activities studies
Mona A. EsawyAzza M. Abdel‐FattahMamdouh M. AliWafaa A. HelmyBassem M. SalamaHanan A. A. TaieAmal M. HashemGhada E. A. Awad
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Levansucrase
Yeast extract
Solid-State Fermentation
Levansucrase
Fructan
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Levansucrase
Fructan
Molar mass distribution
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SUMMARY: Levansucrase in the corynebacterium studied is apparently an adaptive enzyme. Its optimal formation is dependent on the presence of an inducer (sucrose) and an organic nitrogen source. High nitrogen concentrations increase growth rate and sucrose hydrolysis and significantly decrease levansucrase formation. The pH activity curve of the levansucrase studied is asymmetric and the optimal pH value for its activity is 7·0. These findings suggest that this levansucrase differs from other levansucrases so far described.
Levansucrase
Inducer
Corynebacterium
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Optimized conditions for the levan production are necessary to increase its industrial application. This study aimed to optimize the production of levan synthesized by Bacillus licheniformis by factorial design and response surface methodology. The variables involved in this study were sucrose concentration (X1), temperature (X2), agitation (X3) and yeast extract concentration (X4). In view of the independent variables studied, the experiment showed the best results at: sucrose concentration (300 g/l), temperature 40°C, agitation 150 rpm and yeast extract concentration 3 g/l when an average production of 30.66 g/l was obtained.
Bacillus licheniformis
Yeast extract
Fractional factorial design
Central composite design
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Yeast extract
Plackett–Burman design
Fractional factorial design
Central composite design
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Abstract ObjectivesThe yield of levan extracted from microbial fermentation broth is low, so in vitro catalytic synthesis of levan by levansucrase is expected to be one of the industrial production approaches of levan. ResultsA recombinant plasmid Pet-28A-AcmA-Z constructed in the previous study was used to produce levansucrase. The recombinant levansucrase could be easily purified in one step and the purified enzyme had a single band clearly visible in SDS-PAGE. The conditions for enzymatic reactions was optimal at pH 5.2 and 40 ℃, and the activity of enzymes was stimulated by K + and Ca 2+ . The yield of levan biosynthesis from 10% (w/v) sucrose with 6.45 U/g sucrose of levansucrase was 30.6 g/L. The molecular weight of the levan was about 1.56×10 6 Da, as measured by GPC. HPIC analysis showed that the monosaccharide composition of the levan was fructose and glucose. The results of FTIR and NMR analysis indicated that the polymer produced by the recombinant levansucrase was β-(2, 6) levan.ConclusionsThe results of this study provide a basis for large-scale production of levan by enzymatic method.
Levansucrase
Monosaccharide
Zymomonas mobilis
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Levansucrases are bacterial enzymes which produce fructan polymers from sucrose via hydrolysis and transfructosylation activities.These polymers; levan and fructooligosaccharides are valuable for food and pharmaceutical industries.Levansucrases from Gram-positive bacteria such as Bacillus subtilis tend to produce levan, while those from Gram-negative bacteria preferentially produce fructooligosaccharides.Zymomonas mobilis is an efficient levansucrase producer and its extracellular levansucrase can produce both fructooligosaccharides and levan depending on the reaction parameters.In this study, the structure of Z. mobilis levansucrase was modeled in order to help to understand the structure-function relationship of the enzyme.Furthermore, amino acids previously reported to be important for levansucrase activity were mapped on the model.The structural model presents a five-bladed propeller with a deep, negatively charged central pocket, similar to other bacterial levansucrases.Mapping showed that amino acids which previously reported to affect fructan length are located on the periphery of the structure covering the active site central pocket.Thus it is showed that, for the first time, that hydrolysis and transfructosylation reactions are catalyzed on different parts of Z. mobilis levansucrase structure.The structural location of the critical amino acids will pave the way to identify other residues which control fructan length by site directed mutagenesis without altering the overall fold of the enzyme. Zymomonas mobilis Levansükrazının Yapısal Modellemesi ve Yapı-Fonksiyon AnaliziAnahtar Kelimeler Levansükraz Zymomonas mobilis, Protein yapı modelleme Yapısal biyoinformatik Özet: Bakteriyal enzimler olan levansükrazlar, hidroliz ve transfruktosilasyon aktiviteleri ile sakkarozdan fruktan polimer oluşumunu katalizlerler.Bu polimerler; levan ve fruktooligosakkaritler, gıda ve ilaç endüstrileri için değerlidir
Levansucrase
Zymomonas mobilis
Fructan
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1. Bacillus subtilis levansucrase was isolated from cultured cells grown on a medium containing sucrose at 37°C for 40 hr and purified as an electrophoretically pure state which is free from invertase.2. The enzyme produced levan predominantly toward sucrose alone. But heterofructooligosaccharides were synthesized under the co-existence of mono or oligosaccharide. The maximum efficiency (80%) of transfructosylation from sucrose to acceptor sugars by the enzyme obtained in the case of the acceptor/donor=l or more in molar ratio.3. Oligosaccharide formed by the levansucrase of Bacillus subtilis from sucrose and reducing oligosaccharide was non reducing sugar that C2 of fructosyl residue is linked to Cl of reducing end of acceptor sugars.4. Levansucrase linked to CH-sepharose 4B to produce immobilized enzyme was increased thermal stability 30°C to 40t and used repeatedly to make maltooligosaccharide-F without loss of the activity.5. The susceptibility of oligosaccharide-F to human amylases was tested. Heterooligosaccharides produced by levansucrase can be used as a substrate for screening of new carbohydrolases.
Levansucrase
Oligosaccharide
Acceptor
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Abstract Purpose: The yield of levan extracted from microbial fermentation broth is low, so in vitro catalytic synthesis of levan by levansucrase is expected to be one of the industrial production approaches of levan. Methods: A recombinant plasmid pET-28a-AcmA-Z constructed in the previous study was used to produce levansucrase. The effects of temperature, pH, and metal ions on the levan formation activity of the levansucrase were investigated. The polymer was analyzed by means of HPIC, FTIR, NMR techniques. Results: The recombinant levansucrase could be easily purified in one step and the purified enzyme had a single band clearly visible in SDS-PAGE. The conditions for enzymatic reactions was optimal at pH 5.2 and 40 ℃, and the activity of enzymes was stimulated by K + and Ca 2+ . The yield of levan biosynthesis from 10% (w/v) sucrose with 6.45 U/g sucrose of levansucrase was 30.6 g/L. The molecular weight of the levan was about 1.56×10 6 Da, as measured by GPC. HPIC analysis showed that the monosaccharide composition of the levan was fructose and glucose. The results of FTIR and NMR analysis indicated that the polymer produced by the recombinant levansucrase was β-(2, 6) levan. Conclusions: The results of this study provide a basis for large-scale production of levan by enzymatic method.
Levansucrase
Monosaccharide
Zymomonas mobilis
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