-
[show abstract]
[hide abstract]
ABSTRACT: Difructosan anhydrides III (DFA III) are usually obtained by inulin conversion with inulin fructotransferase (IFTase). IFTase liquor is difficult to store for a long time, which could greatly restrict its application and DFA III production. To meet DFA III scale-up preparation, this work was explored to research dry powder preparation of IFTase from Arthrobacter aurescens SK 8.001 fermented liquor by ultrafiltration concentration, ammonium sulfate precipitation and freeze drying. IFTase powder (10.2g) was obtained from IFTase precipitation (126.4g) and its specific activity determined was 16.4U/mg. Dry powder of IFTase could maintain over 120days at different temperatures. These results showed that it is easy to scale up DFA III preparation for industrial capacity.
Carbohydrate polymers. 06/2013; 95(2):654-6.
-
[show abstract]
[hide abstract]
ABSTRACT: The gene coding for D-psicose 3-epimerase (DPEase) from Clostridium sp. BNL1100 was cloned and expressed in Escherichia coli. The recombinant enzyme was purified by Ni-affinity chromatography. It was a metal-dependent enzyme and required Co(2+) as optimum cofactor. It displayed catalytic activity maximally at pH 8.0 and 65 °C (as measured over 5 min). The optimum substrate was D-psicose, and the K m, turnover number (k cat), and catalytic efficiency (k cat/K m) for D-psicose were 227 mM, 32,185 min(-1), and 141 min(-1 )mM(-1), respectively. At pH 8.0 and 55 °C, 120 g D-psicose l(-1) was produced from 500 g D-fructose l(-1) after 5 h.
Biotechnology Letters 05/2013; · 1.68 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: D-Tagatose 3-epimerase family enzymes can efficiently catalyze the epimerization of free keto-sugars, which could be used for D-psicose production from D-fructose. In previous studies, all optimum pH values of these enzymes were found to be alkaline. In this study, a D-psicose 3-epimerase (DPEase) with neutral pH optimum from Clostridium bolteae (ATCC BAA-613) was identified and characterized. The gene encoding the recombinant DPEase was cloned and expressed in Escherichia coli. In order to characterize the catalytic properties, the recombinant DPEase was purified to electrophoretic homogeneity using nickel-affinity chromatography. Ethylenediaminetetraacetic acid was shown to inhibit the enzyme activity completely; therefore, the enzyme was identified as a metalloprotein that exhibited the highest activity in the presence of Co(2+). Although the DPEase demonstrated the most activity at a pH ranging from 6.5 to 7.5, it exhibited optimal activity at pH 7.0. The optimal temperature for the recombinant DPEase was 55 °C, and the half-life was 156 min at 55 °C. Using D-psicose as the substrate, the apparent K m, k cat, and catalytic efficiency (k cat/K m) were 27.4 mM, 49 s(-1), and 1.78 s(-1) mM(-1), respectively. Under the optimal conditions, the equilibrium ratio of D-fructose to D-psicose was 69:31. For high production of D-psicose, 216 g/L D-psicose could be produced with 28.8 % turnover yield at pH 6.5 and 55 °C. The recombinant DPEase exhibited weak-acid stability and thermostability and had a high affinity and turnover for the substrate D-fructose, indicating that the enzyme was a potential D-psicose producer for industrial production.
Applied Microbiology and Biotechnology 05/2013; · 3.42 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Difructosan anhydrides III (DFA III) preparation was usually obtained by inulin hydrolysis with inulin fructotransferase (IFTase). The fructofuranosidic linkages of inulin were the same as fructooligosaccharides (FOS), which was synthesized by sucrose with fructosyltransferase (FTase). FOS was mainly composed of 1-kestose (GF(2)), nystose (GF(3)) and fructofuranosylnystose (GF(4)), and nystose was observed to be the smallest substrate for IFTase to synthesize DFA III. So sucrose, much cheaper than inulin, was considered to produce DFA III by coupled FTase and IFTase reaction. DFA III yield was obtained about 100mg/g (DFA III weight/sucrose weight) through this method. The results demonstrated the high potential of the coupled enzyme reaction as a novel DFA III producing method.
Carbohydrate polymers. 02/2013; 92(2):1608-11.
-
[show abstract]
[hide abstract]
ABSTRACT: The gene coding for ribose-5-phosphate isomerase (Rpi) from Thermotoga lettingae TMO was cloned and expressed in E. coli. The recombinant enzyme was purified by Ni-affinity chromatography. It converted D-psicose to D-allose maximally at 75 °C and pH 8.0 with a 32 % conversion yield. The k (m), turnover number (k (cat)), and catalytic efficiency (k (cat) k (m) (-1) ) for substrate D-psicose were 64 mM, 6.98 min(-1) and 0.11 mM(-1) min(-1) respectively.
Biotechnology Letters 02/2013; · 1.68 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Epilactose (4-O-β-D-galactopyranosyl-D-mannose), an epimer of lactose, is a rare disaccharide existing extremely small quantities in heat-treated milk, in which epilactose is produced by non-enzymatic catalysis from lactose. This disaccharide is a kind of non-digestible carbohydrate, has a good prebiotic effect, and promotes intestinal mineral absorption. This article presents a review of recent studies on epilactose formation in food system, qualitative and quantitative analysis, and its physiological functions. In addition, the biochemical properties and kinetic parameters of the epilactose-producing enzyme, cellobiose 2-epimerase, are compared, and the biotechnological production of epilactose from lactose is reviewed.
Applied Microbiology and Biotechnology 01/2013; · 3.42 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The noncharacterized protein CLOSCI_02528 from Clostridium scindens ATCC 35704 was characterized as D-psicose 3-epimerase. The enzyme showed maximum activity at pH 7.5 and 60°C. The half-life of the enzyme at 50°C was 108 min, suggesting the enzyme was relatively thermostable. It was strictly metal-dependent and required Mn(2+) as optimum cofactor for activity. In addition, Mn(2+) improved the structural stability during both heat- and urea-induced unfolding. Using circular dichroism measurements, the apparent melting temperature (T m) and the urea midtransition concentration (C m) of metal-free enzyme were 64.4°C and 2.68 M. By comparison, the Mn(2+)-bound enzyme showed higher T m and C m with 67.3°C and 5.09 M. The Michaelis-Menten constant (K m), turnover number (k cat), and catalytic efficiency (k cat/K m) values for substrate D-psicose were estimated to be 28.3 mM, 1826.8 s(-1), and 64.5 mM(-1) s(-1), respectively. The enzyme could effectively produce D-psicose from D-fructose with the turnover ratio of 28%.
PLoS ONE 01/2013; 8(4):e62987. · 4.09 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A chickpea protein isolate (CPI) was pretreated before hydrolysis under a pressure that varied between 100 and 600MPa. The hydrolysis rate increased significantly with pressure above 300MPa. At 40min, the DH of the control was 15.3%, while the DH of the CPI treated at 300MPa was 18.5%, which reached 23.74% post treatment at 400MPa. The pretreatment of CPI above 300MPa enhanced the superoxide anion capturing rate of enzymatic hydrolysis. Pretreatment at 400MPa significantly reduced the hydrolysis time with the release of antioxidant peptides. While hydrolysis by Alcalase during treatment at high pressure (100-300MPa) significantly increased the degree of hydrolysis (DH), its maximum value peaked after hydrolysis at 200MPa for 30min. In addition, hydrolysates obtained at high pressure (100-300MPa) had a higher superoxide anion capturing rate. High-pressure treatment at 200MPa for 20min resulted in products with high antioxidative activity. The molecular-weight (MW) determination of the enzymatic hydrolysates indicated that hydrolysis at high pressure could significantly increase the amount of low-molecular-weight peptides.
Food Chemistry 12/2012; 135(3):904-12. · 3.65 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 3-Phenyllactic acid (PLA), which is an organic acid widely existing in honey and lactic acid bacteria fermented food, can be produced by many microorganisms, especially lactic acid bacteria. It was proved as an ideal antimicrobial compound with broad and effective antimicrobial activity against both bacteria and fungi. In addition, it could be used as feed additives to replace antibiotics in livestock feeds. This article presented a review of recent studies on the existing resource, antimicrobial activity, and measurement of PLA. In addition, microorganism strains and dehydrogenases producing PLA were reviewed in detail, the metabolic pathway and regulation of PLA synthesis in LAB strains were discussed, and high-level bioproduction of PLA by microorganism fermentation was also summarized.
Applied Microbiology and Biotechnology 07/2012; 95(5):1155-63. · 3.42 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: D-Psicose is a hexoketose monosaccharide sweetener, which is a C-3 epimer of D-fructose and is rarely found in nature. It has 70 % relative sweetness but 0.3 % energy of sucrose, and is suggested as an ideal sucrose substitute for food products. It shows important physiological functions, such as blood glucose suppressive effect, reactive oxygen species scavenging activity, and neuroprotective effect. It also improves the gelling behavior and produces good flavor during food process. This article presents a review of recent studies on the properties, physiological functions, and food application of D-psicose. In addition, the biochemical properties of D-tagatose 3-epimerase family enzymes and the D-psicose-producing enzyme are compared, and the biotechnological production of D-psicose from D-fructose is reviewed.
Applied Microbiology and Biotechnology 05/2012; 94(6):1461-7. · 3.42 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: D-psicose exits in an extremely small amount in nature and is difficult to be chemically synthesized. Only three bacteria have
been used in the biotransformation of D-psicose from D-fructose. In this paper, another bacterium which could convert D-fructose to D-psicose was isolated and identified as Rhodobacter sphaeroides. The process parameters of D-psicose production using permeabilized cells of Rhodobacter sphaeroides SK011 were optimized, including the permeabilization procedure: 0.1% (w/v) CTAB, 10 min, and reaction conditions: cell concentration,
30 g dry cell wt/L; concentration of substrate, 50 g/L; 40°C, pH 9.0; reaction time, 8 h. Under the optimized conditions,
the permeabilized cells produced approximately 6.5 g/L D-psicose with a D-psicose productivity of 0.82 g·L−1·h−1. This is the first report of bioproduction of D-psicose using permeabilized cells of Rhodobacter sphaeroides.
Frontiers of Chemical Engineering in China 04/2012; 3(4):393-398.
-
[show abstract]
[hide abstract]
ABSTRACT: D-Lactate dehydrogenase (D-LDH) from Pediococcus pentosaceus ATCC 25745 was found to produce D-3-phenyllactic acid from phenylpyruvate. The optimum pH and temperature for enzyme activity were pH 5.5 and 45 °C. The Michaelis-Menten constant (K(m)), turnover number (k(cat)), and catalytic efficiency (k(cat)/K(m)) values for the substrate phenylpyruvate were estimated to be 1.73 mmol/L, 173 s(-1), and 100 (mmol/L)(-1) s(-1) respectively.
Bioscience Biotechnology and Biochemistry 04/2012; 76(4):853-5. · 1.28 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The gene coding for D-lactate dehydrogenase (D-LDH) from Pediococcus acidilactici DSM 20284 was cloned and expressed in E. coli. The recombinant enzyme was purified by nickel-affinity chromatography. It converted phenylpyruvic acid (PPA) to 3-phenyllactic acid maximally at 30°C and pH 5.5 with a specific activity of 140 and 422 U/mg for PPA and pyruvate, respectively. The K(m), turnover number (k(cat)), and catalytic efficiency (k(cat)/K(m)) for PPA were 2.9 mM, 305 s(-1), and 105 mM(-1) s(-1), respectively.
Biotechnology Letters 01/2012; 34(5):907-11. · 1.68 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: An ultrafiltration membrane bioreactor was used for the production of DFA III from enzymatic conversion of inulin. Compared with the traditional batch reactor, the productivity and purity of DFA III could be markedly enhanced and product inhibition was removed and IFTase could be continuously used for six runs in the UF membrane bioreactor. When the substrate concentration was 100 g/L, the concentration of DFA III was about 78.4 g/L, while the productivity and purity of DFA III could attain about 2385 and 92%, respectively.
Journal of Bioscience and Bioengineering 01/2012; 113(1):55-7. · 1.79 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Difructose anhydride (DFA) III is a natural and low-calorie sweetener. It stimulates the absorption of calcium and other minerals. Inulin fructotransferase (IFTase; EC 4.2.2.18), catalysing inulin hydrolysis to DFA III, is considered to be the most promising enzyme for the production of DFA III.
IFTase gene from Arthrobacter aurescens SK 8.001 was cloned and sequenced. Transformant with native IFTase signal peptide was a useful system for extracellular over-expression of IFTase, and its extracellular IFTase activity reached 81.0 U mL(-1) . This value was 4.1-fold of that obtained with A. aurescens SK 8.001 for IFTase production. The recombinant IFTase was purified to electrophoretical homogeneity and characterized. The enzyme showed maximum activity at pH 6.0 and 55 °C, and retained 81.3% of its initial activity after incubation at 60 °C for 4 h.
IFTase gene from A. aurescens SK 8.001 was cloned, sequenced and over-expressed in E. coli. IFTase was reported for the first time to be over-expressed extracellularly. The recombinant IFTase was purified and characterized, and shown to be a good candidate for potential application in DFA III production.
Journal of the Science of Food and Agriculture 12/2011; 91(15):2715-21. · 1.44 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: BACKGROUND: γ-Glutamyltranspeptidase (GGT; EC 2.3.2.2) is a widely distributed enzyme that is of interest in the food industry. In this study the effects of pH and dissolved oxygen (DO) on GGT synthesis from Bacillus subtilis SK 11.004 were investigated. RESULTS: GGT production increased to 0.5 U mL(-1) when the pH value was controlled at 6.5. The control of a single DO level revealed that the highest specific growth rate (3.42 h(-1) ) and GGT production rate (0.40 U g(-1) mL(-1) ) were obtained at DO levels of 40 and 10% respectively. To satisfy the different oxygen demands at different stages of cell growth and GGT synthesis, a stage DO level control strategy was designed as follows: 40% from 0 to 4 h, 30% from 4 to 6 h and 10% from 6 to 18 h. Furthermore, the maximum biomass (2.27 g L(-1) ) and GGT production (3.05 U mL(-1) ) could be obtained using a fermentation strategy combining a constant pH value with stage DO level control. CONCLUSION: The proposed fermentation strategy resulted in a 13.7-fold increase in GGT production. This finding should be of great importance for the industrial production of GGT. Copyright © 2011 Society of Chemical Industry.
Journal of the Science of Food and Agriculture 10/2011; · 1.44 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Difructose anhydride III (DFA III), the smallest cyclic disaccharide, consists of two fructose residues. DFA III is a hydrolysate of inulin and is rarely found in nature. Industrial interest in DFA III as a low-calorie sugar substitute is increasing. The present review describes the properties and physiological functions of DFA III as well as its commercial importance. Focus is also given on the biological production of DFA III from inulin, which contains enzyme resources, inulase II properties, and the capacity for mass DFA III production. Inulase II as an industrial enzyme and its molecular evolution are discussed as well. The aim is to better understand commercial-scale DFA III production as a food product.
Applied Microbiology and Biotechnology 08/2011; 92(3):457-65. · 3.42 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The noncharacterized protein ACL75304 encoded by the gene Ccel_0941 from Clostridium cellulolyticum H10 (ATCC 35319), previously proposed as the xylose isomerase domain protein TIM barrel, was cloned and expressed in Escherichia coli . The expressed enzyme was purified by nickel-affinity chromatography with electrophoretic homogeneity and then characterized as d-psicose 3-epimerase. The enzyme was strictly metal-dependent and showed a maximal activity in the presence of Co(2+). The optimum pH and temperature for enzyme activity were 55 °C and pH 8.0. The half-lives for the enzyme at 60 °C were 6.8 h and 10 min when incubated with and without Co(2+), respectively, suggesting that this enzyme was extremely thermostable in the presence of Co(2+) but readily inactivated without metal ion. The Michaelis-Menten constant (K(m)), turnover number (k(cat)), and catalytic efficiency (k(cat)/K(m)) values of the enzyme for substrate d-psicose were estimated to be 17.4 mM, 3243.4 min(-1), and 186.4 mM min(-1), respectively. The enzyme carried out the epimerization of d-fructose to d-psicose with a conversion yield of 32% under optimal conditions, suggesting that the enzyme is a potential d-psicose producer.
Journal of Agricultural and Food Chemistry 06/2011; 59(14):7785-92. · 2.82 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: An extracellular γ-glutamyltranspeptidase (GGT) with a specific activity of 683.4 U/mg was purified to homogeneity from a culture filtrate of Bacillus subtilis SK11.004 in three steps and then characterized. The GGT is composed of one large subunit of 40 kDa and one small subunit of 21 kDa that was determined by SDS-PAGE and a molecular mass of 62 kDa that was determined by gel-filtration chromatography. The purified GGT had an optimal pH and temperature of 10 and 37 °C, respectively, and it was stable at pH 4.0-11.0 or <50 °C. The enzyme exhibited the highest affinity to imino acids (L-Pro) and then decreasing affinities for aromatic amino acids, ethylamine and basic amino acids. The K(m) values of hydrolysis and of transpeptidation for L-Gln were 3.16 mM and 0.83 mM, respectively, suggesting that the GGT likely synthesizes valuable γ-glutamyl peptides using L-Gln as γ-glutamyl donor. The effects of inhibitors on the enzyme suggested that the tryptophan residues and hydroxy groups of Ser or Thr are essential to enzyme activity. Based on the biochemical characteristics of the enzyme and lack of homology to previously identified proteins, it can be concluded that the GGT from B. subtilis SK11.004 is a novel enzyme.
Journal of Agricultural and Food Chemistry 05/2011; 59(11):6233-8. · 2.82 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Theanine, a unique amino acid found almost exclusively in tea plants, has various favourable physiological and pharmacological functions in humans. Gamma-glutamyltranspeptidase (GGT, EC 2.3.2.2) is considered to be the most effective enzyme for the production of theanine. In fact, GGT can catalyse the transfer of γ-glutamyl moieties from γ-glutamyl compounds to water (hydrolysis) or to amino acids and peptides (transpeptidation).
A novel strain, SK11.004, which produces GGT with high theanine-forming ability was isolated from fermented shrimp paste and identified as Bacillus subtilis through its physiological and biochemical properties as well as its 16S rDNA sequence analysis. Theanine (18.9 mmol L(-1)) was synthesised by GGT (0.06 U mL(-1)) through transfer reaction in the presence of glutamine (20 mmol L(-1)) as a donor and ethylamine HCl (50 mmol L(-1)) as an acceptor at pH 10 and 37 °C for 4 h, the conversion rate being up to 94%.
The enzymatic synthesis of theanine using GGT from a newly isolated strain Bacillus subtilis SK11.004 was found to be an efficient method. Moreover, compared with others, the GGT from B. subtilis SK11.004 exhibited the highest ratio of transferring activity to hydrolytic activity using glutamine, suggesting a high potential application in the production of theanine and other functional γ-glutamyl compounds.
Journal of the Science of Food and Agriculture 12/2010; 90(15):2563-7. · 1.44 Impact Factor
