scholarly journals Galactose to tagatose isomerization at moderate temperatures with high conversion and productivity

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Josef R. Bober ◽  
Nikhil U. Nair
Keyword(s):  
Lactobacillus Plantarum ◽  
Elevated Temperatures ◽  
Catalytic Efficiency ◽  
Batch Process ◽  
High Conversion ◽  
Lactobacillus Sakei ◽  
Reaction Parameters ◽  
Equilibrium Conversion ◽  
Stability Issues ◽  
Arabinose Isomerase

Abstract There are many industrially-relevant enzymes that while active, are severely limited by thermodynamic, kinetic, or stability issues (isomerases, lyases, transglycosidases). In this work, we study Lactobacillus sakeil-arabinose isomerase (LsLAI) for d-galactose to d-tagatose isomerization—that is limited by all three reaction parameters. The enzyme demonstrates low catalytic efficiency, low thermostability at temperatures > 40 °C, and equilibrium conversion < 50%. After exploring several strategies to overcome these limitations, we show that encapsulating LsLAI in gram-positive Lactobacillus plantarum that is chemically permeabilized enables reactions at high rates, high conversions, and elevated temperatures. In a batch process, this system enables ~ 50% conversion in 4 h starting with 300 mM galactose (an average productivity of 37 mM h−1), and 85% conversion in 48 h. We suggest that such an approach may be invaluable for other enzymatic processes that are similarly kinetically-, thermodynamically-, and/or stability-limited.

scholarly journals Surpassing thermodynamic, kinetic, and stability barriers to isomerization catalysis for tagatose biosynthesis

2019 ◽  
Author(s):  
Josef R Bober ◽  
Nikhil Nair
Keyword(s):  
Enzymatic Reaction ◽  
Reaction System ◽  
Catalytic Efficiency ◽  
Batch Process ◽  
Product Formation ◽  
Cell System ◽  
Fast Kinetics ◽  
Equilibrium Conversion ◽  
Stability Issues ◽  
Arabinose Isomerase

AbstractThere are many enzymes that are relevant for making rare and valuable chemicals that while active, are severely limited by thermodynamic, kinetic, or stability issues (e.g. isomerases, lyases, transglycosidase etc.). In this work, we study an enzymatic reaction system −Lactobacillus sakeiL-arabinose isomerase (LsLAI) for D-galactose to D-tagatose isomerization – that is limited by all three reaction parameters. The enzyme has a low catalytic efficiency for non-natural substrate galactose, has low thermal stability at temperatures > 40 °C, and equilibrium conversion < 50%. After exploring several strategies to overcome these limitations, we finally show that encapsulating the enzyme in a gram-positive bacterium (Lactobacillus plantarum) that is chemically permeabilized can enable reactions at high rates, high conversion, and at high temperatures. The modified whole cell system stabilizes the enzyme, differentially partitions substrate and product across the membrane to shift the equilibrium toward product formation enables rapid transport of substrate and product for fast kinetics. In a batch process, this system enables approximately 50 % conversion in 4 h starting with 300 mM galactose (an average productivity of 37 mM/h), and 85 % conversion in 48 h, which are the highest reported for food-safe mesophilic tagatose synthesis. We suggest that such an approach may be invaluable for other enzymatic processes that are similarly kinetically-, thermodynamically-, and/or stability-limited.

scholarly journals Probing the Molecular Determinant for the Catalytic Efficiency of l-Arabinose Isomerase from Bacillus licheniformis

2010 ◽  
Vol 76 (5) ◽  
pp. 1653-1660 ◽  
Author(s):  
Ponnandy Prabhu ◽  
Marimuthu Jeya ◽  
Jung-Kul Lee
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Bacillus Licheniformis ◽  
Catalytic Efficiency ◽  
Homology Model ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Dynamics Simulation ◽  
High Turnover ◽  
Arabinose Isomerase

ABSTRACT Bacillus licheniformis l-arabinose isomerase (l-AI) is distinguished from other l-AIs by its high degree of substrate specificity for l-arabinose and its high turnover rate. A systematic strategy that included a sequence alignment-based first screening of residues and a homology model-based second screening, followed by site-directed mutagenesis to alter individual screened residues, was used to study the molecular determinants for the catalytic efficiency of B. licheniformis l-AI. One conserved amino acid, Y333, in the substrate binding pocket of the wild-type B. licheniformis l-AI was identified as an important residue affecting the catalytic efficiency of B. licheniformis l-AI. Further insights into the function of residue Y333 were obtained by replacing it with other aromatic, nonpolar hydrophobic amino acids or polar amino acids. Replacing Y333 with the aromatic amino acid Phe did not alter catalytic efficiency toward l-arabinose. In contrast, the activities of mutants containing a hydrophobic amino acid (Ala, Val, or Leu) at position 333 decreased as the size of the hydrophobic side chain of the amino acid decreased. However, mutants containing hydrophilic and charged amino acids, such as Asp, Glu, and Lys, showed almost no activity with l-arabinose. These data and a molecular dynamics simulation suggest that Y333 is involved in the catalytic efficiency of B. licheniformis l-AI.

scholarly journals D-Tagatose production by Lactococcus lactis NZ9000 Cells Harboring Lactobacillus plantarum L-arabinose Isomerase

2017 ◽  
Vol 51 (2) ◽  
pp. 288-294 ◽  
Author(s):  
Yao Zhang ◽  
Yanli Fan ◽  
Haijie Hu ◽  
Haixu Yang ◽  
Xuegang Luo ◽  
...  
Keyword(s):  
Lactobacillus Plantarum ◽  
Lactococcus Lactis ◽  
Arabinose Isomerase

scholarly journals Unravelling the diversity of glycoside hydrolase family 13 α-amylases from Lactobacillus plantarum WCFS1

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Laura Plaza-Vinuesa ◽  
Oswaldo Hernandez-Hernandez ◽  
F. Javier Moreno ◽  
Blanca de las Rivas ◽  
Rosario Muñoz
Keyword(s):  
Lactobacillus Plantarum ◽  
Glycoside Hydrolase ◽  
Catalytic Efficiency ◽  
Hydrolytic Activity ◽  
Structural Features ◽  
Biochemical Properties ◽  
Glycoside Hydrolase Family ◽  
Reaction Conditions ◽  
Genes Encoding ◽  
Hydrolysis Of

Abstract Background α-Amylases specifically catalyse the hydrolysis of the internal α-1, 4-glucosidic linkages of starch. Glycoside hydrolase (GH) family 13 is the main α-amylase family in the carbohydrate-active database. Lactobacillus plantarum WCFS1 possesses eleven proteins included in GH13 family. Among these, proteins annotated as maltose-forming α-amylase (Lp_0179) and maltogenic α-amylase (Lp_2757) were included. Results In this study, Lp_0179 and Lp_2757 L. plantarum α-amylases were structurally and biochemically characterized. Lp_2757 displayed structural features typical of GH13_20 subfamily which were absent in Lp_0179. Genes encoding Lp_0179 (Amy2) and Lp_2757 were cloned and overexpressed in Escherichia coli BL21(DE3). Purified proteins showed high hydrolytic activity on pNP-α-D-maltopyranoside, being the catalytic efficiency of Lp_0179 remarkably higher. In relation to the hydrolysis of starch-related carbohydrates, Lp_0179 only hydrolysed maltopentaose and dextrin, demonstrating that is an exotype glucan hydrolase. However, Lp_2757 was also able to hydrolyze cyclodextrins and other non-cyclic oligo- and polysaccharides, revealing a great preference towards α-1,4-linkages typical of maltogenic amylases. Conclusions The substrate range as well as the biochemical properties exhibited by Lp_2757 maltogenic α-amylase suggest that this enzyme could be a very promising enzyme for the hydrolysis of α-1,4 glycosidic linkages present in a broad number of starch-carbohydrates, as well as for the investigation of an hypothetical transglucosylation activity under appropriate reaction conditions.

scholarly journals Characterization of a Thermostable l-Arabinose (d-Galactose) Isomerase from the Hyperthermophilic Eubacterium Thermotoga maritima

2004 ◽  
Vol 70 (3) ◽  
pp. 1397-1404 ◽  
Author(s):  
Dong-Woo Lee ◽  
Hyeung-Jin Jang ◽  
Eun-Ah Choe ◽  
Byoung-Chan Kim ◽  
Sang-Jae Lee ◽  
...  
Keyword(s):  
Amino Acid ◽  
Elevated Temperatures ◽  
Thermotoga Maritima ◽  
Sequence Similarity ◽  
Gel Filtration ◽  
Catalytic Efficiency ◽  
Maximal Activity ◽  
Recombinant Enzyme ◽  
Amino Acid Sequence Similarity ◽  
Calculated Molecular Weight

ABSTRACT The araA gene encoding l-arabinose isomerase (AI) from the hyperthermophilic bacterium Thermotoga maritima was cloned and overexpressed in Escherichia coli as a fusion protein containing a C-terminal hexahistidine sequence. This gene encodes a 497-amino-acid protein with a calculated molecular weight of 56,658. The recombinant enzyme was purified to homogeneity by heat precipitation followed by Ni2+ affinity chromatography. The native enzyme was estimated by gel filtration chromatography to be a homotetramer with a molecular mass of 232 kDa. The purified recombinant enzyme had an isoelectric point of 5.7 and exhibited maximal activity at 90�C and pH 7.5 under the assay conditions used. Its apparent Km values for l-arabinose and d-galactose were 31 and 60 mM, respectively; the apparent V max values (at 90�C) were 41.3 U/mg (l-arabinose) and 8.9 U/mg (d-galactose), and the catalytic efficiencies (k cat/Km ) of the enzyme were 74.8 mM−1 � min−1 (l-arabinose) and 8.5 mM−1 � min−1 (d-galactose). Although the T. maritima AI exhibited high levels of amino acid sequence similarity (>70%) to other heat-labile mesophilic AIs, it had greater thermostability and higher catalytic efficiency than its mesophilic counterparts at elevated temperatures. In addition, it was more thermostable in the presence of Mn2+ and/or Co2+ than in the absence of these ions. The enzyme carried out the isomerization of d-galactose to d-tagatose with a conversion yield of 56% for 6 h at 80�C.

scholarly journals Thermal Inactivation of a Cold-Active Esterase PMGL3 Isolated from the Permafrost Metagenomic Library

Biomolecules ◽  
2019 ◽  
Vol 9 (12) ◽  
pp. 880 ◽  
Author(s):  
M.V. Kryukova ◽  
L.E. Petrovskaya ◽  
E.A. Kryukova ◽  
G.Yu. Lomakina ◽  
S.A. Yakimov ◽  
...  
Keyword(s):  
Thermal Inactivation ◽  
Elevated Temperatures ◽  
Metagenomic Library ◽  
Catalytic Efficiency ◽  
Surface Hydrophobicity ◽  
Maximum Activity ◽  
Hormone Sensitive Lipase ◽  
Hydrophobic Residues ◽  
Improved Stability ◽  
Hormone Sensitive

PMGL3 is a cold-adapted esterase which was recently isolated from the permafrost metagenomic library. It exhibits maximum activity at 30 °C and low stability at elevated temperatures (40 °C and higher). Sequence alignment has revealed that PMGL3 is a member of the hormone-sensitive lipase (HSL) family. In this work, we demonstrated that incubation at 40 °C led to the inactivation of the enzyme (t1/2 = 36 min), which was accompanied by the formation of tetramers and higher molecular weight aggregates. In order to increase the thermal stability of PMGL3, its two cysteines Cys49 and Cys207 were substituted by the hydrophobic residues, which are found at the corresponding positions of thermostable esterases from the HSL family. One of the obtained mutants, C207F, possessed improved stability at 40 °C (t1/2 = 169 min) and increased surface hydrophobicity, whereas C49V was less stable in comparison with the wild type PMGL3. Both mutants exhibited reduced values of Vmax and kcat, while C207F demonstrated increased affinity to the substrate, and improved catalytic efficiency.

scholarly journals DETECÇÃO DE BACTERIOCINAS PRODUZIDAS POR Lactobacillus plantarum BN EM MELAÇO DE CANA-DE-AÇÚCAR SOB FERMENTAÇÃO SUBMERSA

2002 ◽  
Vol 20 (1) ◽  
Author(s):  
JACIARA ZARPELLON MAZO ◽  
ERNANI S. SANT’ANNA ◽  
BERNADETTE D.G.M. FRANCO ◽  
ANNA C. S. PORTO ◽  
ÂNGELA M. FIORENTINI
Keyword(s):  
Staphylococcus Aureus ◽  
Listeria Monocytogenes ◽  
Lactobacillus Plantarum ◽  
Sugar Cane ◽  
Ammonium Citrate ◽  
Diffusion Method ◽  
Exponential Growth Phase ◽  
Lactobacillus Sakei ◽  
Fermentation Time ◽  
Cane Molasses

Verificou-se a presença de bacteriocinas produzidas por Lactobacillus plantarum BN (microrganismo teste) em caldo com 3% de melaço de cana-de-açúcar, centrifugado e enriquecido com extrato de leveduras, acetato de sódio e citrato de amônia. Os testes foram realizados em fermentador com volume de trabalho de 3,0 L, sob agitação contínua a 100 rpm, temperatura de 30 ± 0,1°C, aeração de 0,7 vvm, tempo de fermentação de 24 horas e inóculo aproximado de 6,0 Log10 UFC/mL, com tomada de amostras em intervalos de 2 horas. O maior número médio de células viáveis foi de 10 Log10 ciclos logarítmicos, nos intervalos de 12 a 18 horas de fermentação. O pH inicial de 6,49, após 24 horas diminuiu para 5,05. A detecção de bacteriocinas foi realizada no sobrenadante obtido por centrifugação do meio de cultivo, pelo método de difusão em orifícios, usando Lactobacillus sakei ATCC 15521 como microrganismo indicador. Verificouse a presença de bacteriocinas no meio de cultivo a partir de 8 horas de fermentação pela formação de halo inibitório, quando o microrganismo encontrava-se na fase exponencial de crescimento. Comprovou-se a natureza protéica da bacteriocina pelo uso da enzima a-quimotripsina. A bacteriocina produzida por L. plantarum BN apresentou efeito inibitório sobre Listeria monocytogenes ATCC 19112, mas não sobre Staphylococcus aureus ATCC 15489. DETECTION OF BACTERIOCIN PRODUCED BY Lactobacillus plantarum BN IN SUGAR-CANE MOLASSES BY SUBMERSE FERMENTATION Abstract The presence of bacteriocins produced by Lactobacillus plantarum BN (test microorganism) in 3% sugar cane molasses, centrifuged and enriched with yeast extract, sodium acetate and ammonium citrate, was verified. The tests were realized in a fermenter with 3.0 L work volume, under continuous agitation of 100 rpm, at a temperature of 30 ± 0.1 °C, 0.7 vvm aeration and fermentation time of 24 hours with an approximate inoculum of 6.0 Log10 CFU/mL with sample being taken at every 2 hours interval. The greatest number of viable cells observed was 10 Log10 logarithmic cycles at 12 and 18 hours fermentation intervals. Initial pH was 6,49 and after 24 hours, it decreased to 5,05. Bacteriocins detection was accomplished using the supernatant obtained by centrifugation of cultivation media in the well diffusion method using Lactobacillus sakei ATCC 15521 as the indicator microorganism. From 8 hours on of fermentation, presence of colonies surrounded by a clear zone of inhibition indicated bacteriocins production in cultivation media when the microorganism was in the exponential growth phase. The proteic nature of the bacteriocin was certified by using the a-chimotrypsin enzyme. The bacteriocin produced by Lactobacillus plantarum BN presented inhibiting effect over Listeria monocytogenes ATCC 19112 while this effect was not observed in Staphylococcus aureus ATCC 15489.

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