scholarly journals Extracellular expression and characterization of an α-glucosidase from Oryza sativa and its transglycosylation for synthesis of 2-O-α-D-glucopyranosyl-L-ascorbic acid

2021 ◽  
Author(s):  
Xuelian Qi ◽  
Junlan Shao ◽  
Yinchu Cheng ◽  
Xiaoying He ◽  
Yan Li ◽  
...  
Keyword(s):  
Ascorbic Acid ◽  
Oryza Sativa ◽  
Pichia Pastoris ◽  
Specific Activity ◽  
Maximal Activity ◽  
Batch Cultivation ◽  
Glycoside Hydrolases ◽  
Glycosylation Reaction ◽  
Fed Batch Cultivation

Abstract: 2-O-α-D-Glucopyranosyl-L-ascorbic acid (AA-2G) is an important industrial derivative of L-ascorbic acid (AA), which has the distinct advantages of non-reducibility, antioxidation, and reproducible decomposition into L-ascorbic acid and glucose. Enzymatic synthesis is a preferred method for AA-2G production over alternative chemical synthesis owing to the regioselective glycosylation reaction. α-Glucosidase, an enzyme classed into O- glycoside hydrolases, may be used in glycosylation reactions to synthesize AA-2G. Here, one α-glucosidase from Oryza sativa (rAGL) was recombinantly produced in Pichia pastoris GS115 and used for biosynthesis of AA-2G with few intermediates and byproducts. The extracellular rAGL reached 9.11 U/mL after fed-batch cultivation for 102 h in a 5-L fermenter. The specific activity of purified rAGL is 49.83 U/mg at 37 °C and pH 4.0. The optimal temperature of rAGL was 65 °C, and it was stable below 55 °C. rAGL was active over the range of pH 3.0–7.0, with the maximal activity at pH 4.0. Under the condition of 37 °C , pH 4.0, equimolar maltose and AA·Na, 8.7±0.4 g/L of AA-2G was synthesized by rAGL. These studies lay the basis for the industrial application of recombinant α-glucosidase. Keywords: α-Glucosidase; Oryza sativa; 2-O-α-D-glucopyranosyl-L-ascorbic acid; Transglycosylation; Pichia pastoris

Modeling and Model Based Feeding Control for Pichia pastoris Fed-Batch Cultivation

2004 ◽  
Vol 37 (1) ◽  
pp. 451-456
Author(s):  
H.T. Ren ◽  
J.H. Deng ◽  
B.K. He ◽  
L.M. Ren ◽  
J.Q. Yuan
Keyword(s):  
Pichia Pastoris ◽  
Batch Cultivation ◽  
Fed Batch ◽  
Model Based ◽  
Feeding Control ◽  
Fed Batch Cultivation

Isolation and Characterization of Wild-Type Lipoxygenase LOXPsa1 from Pleurotus sapidus

2014 ◽  
Vol 69 (3-4) ◽  
pp. 149-154 ◽  
Author(s):  
Ina Plagemann ◽  
Ulrich Krings ◽  
Ralf G. Berger
Keyword(s):  
De Novo ◽  
Specific Activity ◽  
Maximal Activity ◽  
Wild Type ◽  
Isolation And Characterization ◽  
Peptide Mass ◽  
Purification Scheme ◽  
Pleurotus Sapidus ◽  
Nano Liquid Chromatography

The lipoxygenase LOXPsa1 of Pleurotus sapidus, originally investigated because of its ability to oxidize (+)-valencene to the valuable grapefruit aroma (+)-nootkatone, was isolated from the peptidase-rich lyophilisate using a three-step purification scheme including preparative isoelectric focusing and chromatographic techniques. Nano-liquid chromatography electrospray ionization tandem mass spectrometry (nLC-ESI-MS=MS) of the purified enzyme and peptide mass fingerprint analysis gave 38 peptides of the lipoxygenase from P. sapidus. Nearly 50% of the 643 amino acids long sequence encoded by the cDNA was covered. Both terminal peptides of the native LOXPsa1 were identified by de novo sequencing, and the postulated molecular mass of 72:5 kDa was confirmed. With linoleic acid as the substrate, the LOXPsa1 showed a specific activity of 113 U mg-1 and maximal activity at pH 7.0 and 30 °C, respectively.

scholarly journals Expression and characterization of Trichoderma reesei endoglucanase II in Pichia pastoris under the regulation of the GAP promoter

2020 ◽  
Vol 25 (2) ◽  
pp. 127
Author(s):  
Kezia Abib Yerah Tjandra ◽  
Kartika Sari Dewi ◽  
Asrul Muhamad Fuad ◽  
Trisanti Anindyawati
Keyword(s):  
Pichia Pastoris ◽  
Trichoderma Reesei ◽  
Specific Activity ◽  
Catalytic Efficiency ◽  
Endoglucanase Activity ◽  
Gap Promoter ◽  
Sds Page ◽  
Optimum Ph ◽  
High Concentrations

Trichoderma reesei is known to be one of the organisms capable for producing various types of cellulase in high concentrations. Among these cellulases, the highest catalytic efficiency of endoglucanases II (EGII, EC 3.2.1.4) are considered important for industrial application. The characterization of the EGII is necessary since it is widely used in high-temperature reactions in the industries. In this study, the recombinant EGII protein was expressed in Pichia pastoris and it has a molecular mass of approximately 52 kDa. Recombinant EGII was purified using Ni-NTA affinity chromatography and characterized by SDS-PAGE and western blot analyses. The enzyme activity of recombinant EGII was measured using the Nelson Somogyi method to determine its optimum pH and temperature. The result showed that the maximum EGII expression was achieved after 72 h of culture incubation. The crude enzyme has optimum activity at pH 5.0, resulting in 16.3 U/mL and 14.6 U/mL activity at 40 °C and 50 °C, respectively. While the purified enzyme gave the specific activity of 115.7 U/mg under the optimum condition. Finally, our study demonstrated that recombinant EGII could retain the endoglucanase activity for 89% and 80% at 40 °C and 50 °C, respectively.

scholarly journals Characterization of a Mannose-6-Phosphate Isomerase fromThermus thermophilusand Increased l-Ribose Production by Its R142N Mutant

2010 ◽  
Vol 77 (3) ◽  
pp. 762-767 ◽  
Author(s):  
Soo-Jin Yeom ◽  
Eun-Sun Seo ◽  
Bi-Na Kim ◽  
Yeong-Su Kim ◽  
Deok-Kun Oh
Keyword(s):  
Specific Activity ◽  
Thermus Thermophilus ◽  
Catalytic Efficiency ◽  
Maximal Activity ◽  
Wild Type ◽  
Active Site Residues ◽  
Geobacillus Thermodenitrificans ◽  
Wild Type Enzyme ◽  
Mannose 6 Phosphate

ABSTRACTAn uncharacterized gene fromThermus thermophilus, thought to encode a mannose-6-phosphate isomerase, was cloned and expressed inEscherichia coli. The maximal activity of the recombinant enzyme forl-ribulose isomerization was observed at pH 7.0 and 75°C in the presence of 0.5 mM Cu2+. Among all of the pentoses and hexoses evaluated, the enzyme exhibited the highest activity for the conversion ofl-ribulose tol-ribose, a potential starting material for manyl-nucleoside-based pharmaceutical compounds. The active-site residues, predicted according to a homology-based model, were separately replaced with Ala. The residue at position 142 was correlated with an increase inl-ribulose isomerization activity. The R142N mutant showed the highest activity among mutants modified with Ala, Glu, Tyr, Lys, Asn, or Gln. The specific activity and catalytic efficiency (kcat/Km) forl-ribulose using the R142N mutant were 1.4- and 1.6-fold higher than those of the wild-type enzyme, respectively. Thekcat/Kmof the R142N mutant was 3.8-fold higher than that ofGeobacillus thermodenitrificansmannose-6-phosphate isomerase, which exhibited the highest activity to date for the previously reportedkcat/Km. The R142N mutant enzyme produced 213 g/literl-ribose from 300 g/literl-ribulose for 2 h, with a volumetric productivity of 107 g liter−1h−1, which was 1.5-fold higher than that of the wild-type enzyme.

scholarly journals Characterization of an Agrobacterium tumefaciensd-Psicose 3-Epimerase That Converts d-Fructose to d-Psicose

2006 ◽  
Vol 72 (2) ◽  
pp. 981-985 ◽  
Author(s):  
Hye-Jung Kim ◽  
Eun-Kyung Hyun ◽  
Yeong-Su Kim ◽  
Yong-Joo Lee ◽  
Deok-Kun Oh
Keyword(s):  
Escherichia Coli ◽  
Agrobacterium Tumefaciens ◽  
Molecular Mass ◽  
Specific Activity ◽  
Catalytic Efficiency ◽  
Maximal Activity ◽  
Turnover Number ◽  
Conversion Yield ◽  
Equilibrium Ratio

ABSTRACT The noncharacterized gene previously proposed as the d-tagatose 3-epimerase gene from Agrobacterium tumefaciens was cloned and expressed in Escherichia coli. The expressed enzyme was purified by three-step chromatography with a final specific activity of 8.89 U/mg. The molecular mass of the purified protein was estimated to be 132 kDa of four identical subunits. Mn2+ significantly increased the epimerization rate from d-fructose to d-psicose. The enzyme exhibited maximal activity at 50°C and pH 8.0 with Mn2+. The turnover number (k cat) and catalytic efficiency (k cat/Km ) of the enzyme for d-psicose were markedly higher than those for d-tagatose, suggesting that the enzyme is not d-tagatose 3-epimerase but d-psicose 3-epimerase. The equilibrium ratio between d-psicose and d-fructose was 32:68 at 30°C. d-Psicose was produced at 230 g/liter from 700-g/liter d-fructose at 50°C after 100 min, corresponding to a conversion yield of 32.9%.

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