Dictyoglomus turgidum DSM 6724 α-Glucan Phosphorylase: Characterization and Its Application in Multi-enzyme Cascade Reaction for D-Tagatose Production.

Phosphorylase is a type of enzyme-producing sugar phosphates through the reversible phosphorolysis reactions of glycosides, which makes it an important starting enzyme in multi-enzyme systems for rare sugar biomanufacturing. To investigate its application in D-tagatose biosynthesis from maltodextrin using in vitro multi-enzyme cascade biosystem, the α-glucan phosphorylase (αGP; EC 2.4.1.1) from the thermophile D. turgidum DSM 6724 was prepared and characterized. It exhibited the specific activity of 30.28 U/mg at its optimal temperature of 70 °C. Thermostability results revealed that DituαGP could maintain more than 25% of initial activity for 4 h, even at 90 °C. The highest activity was observed at pH 5.5, and most divalent metal ions deactivated the enzyme. DituαGP exhibited great application potential in the multi-enzyme system that about 3.919 g/L of D-tagatose was produced from 150 g/L of maltodextrin within 36 h. DituαGP has played an important role in this biosystem and will also be applied in the synthesis of other rare sugars from maltodextrin.

Genetic and biochemical characterization of thermophilic ß-cyclodextrin glucanotransferase from Gracilibacillus alcaliphilus SK51.001.

BACKGROUND: Gracilibacillus alcaliphilus SK51.001, a strain that produces ß-CGTase (ß-cyclodextrin glucanotransferase) (EC 2.4.1.19), was screened and isolated from Sudanese soil. The objective of this study was to sequence and characterize the ß-CGTase gene from G. alcaliphilus SK51.001. RESULTS: According to 16S rRNA analysis of the strain and its morphological shape, it was identified as G. alcaliphilus. The ß-CGTase gene was successfully cloned, sequenced, and expressed in Escherichia coli BL21. This gene showed 706 amino acid residues including 33 amino acids as a signal peptide. The active site residues of G. alcaliphilus SK51.001CGTase were described using enzyme modeling and docking with the products. The estimated molecular mass of G. alcaliphilus SK51.001CGTase was approximately 74 kDa as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the evaluation of the gel filtration showed approximately 85 kDa, which means G. alcaliphilus SK51.001CGTase is a monomer. The optimum temperature and pH of G. alcaliphilus SK51.001CGTase were 60 °C and 7.0 respectively. Gracilibacillus alcaliphilus SK51.001CGTase was comparatively stable at a pH levels between 6.0 and 9.0 and temperatures of 30-50 °C. The activity of G. alcaliphilus SK51.001CGTase was increased by Ni2+ , and Co2+ but inhibited by Al3+ and Fe3+ . The kinetic parameters of Km and Vmax were 2068.52 µg mL-1 and 0.13 µmol mL-1  min-1 , respectively. CONCLUSION: Gracilibacillus alcaliphilus SK51.001CGTase could hydrolyze soluble starch into α-, ß-, and γ-cyclodextrin in a ratio of 2: 83: 15% respectively. This high ratio production of ß-CD could allow the enzyme to be used in ß-CD production. © 2020 Society of Chemical Industry.

Characteristics of a fructose 6-phosphate 4-epimerase from Caldilinea aerophila DSM 14535 and its application for biosynthesis of tagatose.

Tagatose is a rare hexoketose with potential health benefits. Here, an enzyme, GatZ subunit ofd-tagatose-1,6-bisphosphate aldolase, was characterized. GatZ is involved in a multi-enzyme cascade reaction system that can produce tagatose from maltodextrin. It showed maximum activity at 70 °C and a pH 8.0, and required supplementation with 5 mM Mg2+ to achieve the highest catalytic activity. The Km and Vmax values of GatZ using fructose 6-phosphate as substrate were 5.66 mM and 0.0329 mmol/L min, respectively. An in vitro multi-enzyme system containing GatZ was constructed, and 1.75 g/L tagatose was produced from 5 g/L maltodextrin after 10 h. This biosystem could potentially enrich the application of C4 epimerases in rare sugar bioproduction.

Lactulose production by a thermostable glycoside hydrolase from the hyperthermophilic archaeon Caldivirga maquilingensis IC-167.

BACKGROUND: Lactulose has various uses in the food and pharmaceutical fields. Thermostable enzymes have many advantages for industrial exploitation, including high substrate solubilities as well as reduced risk of process contamination. RESULTS: Enzymatic synthesis of lactulose employing a transgalactosylation reaction by a recombinant thermostable glycoside hydrolase (GH1) from the hyperthermophilic archaeon Caldivirga maquilingensis IC-167 was investigated. The optimal pH for lactulose production was found to be 4.5, while the optimal temperature was 85 °C, before it dropped moderately to 83% at 90 °C. However, the relative activity for lactulose synthesis dropped sharply to 35% at 95 °C. At optimal reaction conditions of 70% (w/w) initial sugar substrates with molar ratio of lactose to fructose of 1:4, 15 U mL-1 enzyme concentration and 85 °C, the time course reaction produced a maximum lactulose concentration of 108 g L-1 at 4 h, corresponding to a lactulose yield of 14% and 27 g L-1 h-1 productivity with 84% lactose conversion. The transgalactosylation reaction for lactulose synthesis was greatly influenced by the ratio of galactose donor to acceptor. CONCLUSION: This novel GH1 may be useful for process applications owing to its high activity in very concentrated substrate reaction media and promising thermostability. © 2017 Society of Chemical Industry.

Characterization of a thermostable glycoside hydrolase (CMbg0408) from the hyperthermophilic archaeon Caldivirga maquilingensis IC-167.

BACKGROUND: Hyperthermophilic archaea capable of functioning optimally at very high temperatures are a good source of unique and industrially important thermostable enzymes. RESULTS: A glycoside hydrolase family 1 ß-galactosidase gene (BglB) from a hyperthermophilic archaeon Caldivirga maquilingensis IC-167 was cloned and expressed in Escherichia coli. The recombinant enzyme (CMbg0408) displayed optimum activity at 110 °C and pH 5.0. It also retained 92% and 70% of its maximal activity at 115 and 120 °C, respectively. The enzyme was completely thermostable and active after 120 min of incubation at 80 and 90 °C. It also showed broad substrate specificity with activities of 8876 ± 185 U mg-1 for p-nitrophenyl-ß-d-galactopyranoside, 4464 ± 172 U mg-1 for p-nitrophenyl-ß-d-glucopyranoside, 1486 ± 68 U mg-1 for o-nitrophenyl-ß-d-galactopyranoside, 2250 ± 86 U mg-1 for o-nitrophenyl-ß-d-xylopyranoside and 175 ± 4 U mg-1 for lactose. A catalytic efficiency (kcat /Km ) of 3059 ± 122 mmol L-1 s-1 and Km value of 8.1 ± 0.08 mmol L-1 were displayed towards p-nitrophenyl-ß-d-galactopyranoside. CONCLUSION: As a result of its remarkable thermostability and high activity at high temperatures, this novel ß-galactosidase may be useful for food and pharmaceutical applications. © 2016 Society of Chemical Industry.

Cloning and characterization of a new ribitol dehydrogenase from Providencia alcalifaciens RIMD 1656011.

BACKGROUND: A new ribitol dehydrogenase gene was cloned from Providencia alcalifaciens RIMD 1656011 and expressed in Escherichia coli BL21. This study aimed to purify and characterize the ribitol dehydrogenase from P. alcalifaciens RIMD 1656011 and investigate its substrate specificity for potential use as an industrial enzyme. RESULTS: The protein was purified by nickel affinity chromatography. The molecular mass of the purified enzyme was determined as ∼25 000 and 26 650 Da through sodium dodecyl sulfate polyacrylamide gel electrophoresis and liquid chromatography/mass spectrometry respectively. The result for native molecular mass (104 kDa) suggested that the enzyme functions as a tetramer. Optimum activity of the enzyme was determined at pH 10.0 and a temperature of 35 °C. Regarding its thermal stability, the enzyme retained 72, 72, 48 and 0% of its initial activity after 4 h at 25, 30, 40 and 50 °C respectively. The Km , kcat and kcat /Km values of the enzyme for the substrate ribitol were determined as 13.9 mmol L(-1) , 10.0 s(-1) and 0.71 L mmol(-1) s(-1) respectively. The Km of NAD(+) was 0.042 mmol L(-1) . CONCLUSION: The substrate specificity indicated that the ribitol dehydrogenase from P. alcalifaciens RIMD 1656011 can be used for direct production of allitol from d-fructose without any by-product formation. © 2015 Society of Chemical Industry.