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Zhang X.,Jiangnan University | Xiong Y.L.,University of Kentucky | Chen J.,Jiangnan University | Zhou L.,Roquette America
Journal of Agricultural and Food Chemistry | Year: 2013

Fourteen pea protein hydrolysates (PPHs) were prepared using different proteases and tested for antioxidant activity in a liposomal model system under oxidative stress (100 μM FeCl3/2 mM ascorbate). Almost all PPHs inhibited lipid oxidation, and those prepared from heated protein with Flavourzyme (Fla-PPH) or Protamex (Pro-PPH) were the most effective. Remarkable synergistic effects were observed on both Fla-PPH and Pro-PPH with licorice extract (LE). Electron microscopy revealed a self-assembled network that appeared to provide crucial protection of liposome against oxidation. The presence of LE enhanced the antioxidant potential by producing a more compact network apparently via PPH-LE complexation. Zeta-potential measurements suggested electrostatic interactions are important driving forces for the accumulation of active peptides at the liposome interface. Peptides rich in leucine, lysine, glutamic acid, glutamine, valine, or proline with a hydrophobic N-terminus, as identified by mass spectrometry, were implicated in the antioxidative protection. © 2013 American Chemical Society. Source

Zhang W.,Jiangnan University | Fang D.,Jiangnan University | Zhang T.,Jiangnan University | Zhou L.,Roquette America | And 2 more authors.
Journal of Agricultural and Food Chemistry | Year: 2013

The rare sugar d-psicose is an ideal sucrose substitute for food products, due to having 70% of the relative sweetness but 0.3% of the energy of sucrose. It also shows important physiological functions. d-Tagatose 3-epimerase (DTEase) family enzymes can produce d-psicose from d-fructose. In this paper, a new member of the DTEase family of enzymes was characterized from Desmospora sp. 8437 (GenBank accession no. WP-009711885) and was named Desmospora sp. d-psicose 3-epimerase (DPEase) due to its highest substrate specificity toward d-psicose. Desmospora sp. DPEase was strictly metal-dependent and displayed maximum activity in the presence of Co2+. The optimum pH and temperature were 7.5 and 60 C, respectively. The enzyme was relatively thermostable below 50 C, but easily lost initial activity when preincubated at 60 C. The thermostability property was almost not affected by the addition of Co2+. Desmospora sp. DPEase had relatively high catalysis efficiency for the substrates d-psicose and d-fructose, which were measured to be 327 and 116 mM-1 min -1, respectively. The equilibrium ratio between d-psicose and d-fructose of Desmospora sp. DPEase was 30:70. The enzyme could produce 142.5 g/L d-psicose from 500 g/L of d-fructose, suggesting that the enzyme is a potential d-psicose producer for industrial production. © 2013 American Chemical Society. Source

Fan C.,Jiangnan University | Xu W.,Jiangnan University | Zhang T.,Jiangnan University | Zhou L.,Roquette America | And 2 more authors.
Applied Biochemistry and Biotechnology | Year: 2015

A mutation, D478N, was obtained by an error-prone polymerase chain reaction using the l-arabinose isomerase (l-AI) gene from Alicyclobacillus hesperidum URH17-3-68 as the template. The mutated isomerase showed higher activity for d-galactose isomerization. The mutation site obtained from random mutagenesis was then introduced as a single-site mutation using site-directed mutagenesis. Single-site variants, D478N, D478Q, D478A, D478K, and D478R, were constructed. The optimum temperatures were all higher than 60 °C. D478A, D478N, and D478Q retained more than 80 % of the maximum relative activity of the wild-type l-AI at 75 °C. With the exception of the D478A variant, all variants showed decreased optimum pH values in the acidic range (6.0–6.5). All of the variant l-AIs could be significantly activated by the addition of Co2+ and Mn2+. D478N and D478Q showed higher catalytic efficiencies (kcat/Km) toward d-galactose than that of wild-type l-AI. In addition, the D478N and D478Q variants exhibited a much higher conversion ratio of d-galactose to d-tagatose at 6.0 than the wild-type l-AI. According to the molecular model, residue D478 was located on the surface of the enzyme and distant from the active site. It was supposed that the charged state of residue 478 may influence the optimum pH for substrate binding or isomerization. © 2015 Springer Science+Business Media New York Source

Zhang W.,Jiangnan University | Jia M.,Jiangnan University | Yu S.,Jiangnan University | Zhang T.,Jiangnan University | And 3 more authors.
Journal of Agricultural and Food Chemistry | Year: 2016

D-Psicose is a highly valuable rare sugar because of its excellent physiological properties and commercial potential. D-Psicose 3-epimerase (DPEase) is the key enzyme catalyzing the isomerization of D-fructose to D-psicose. However, the poor thermostability and low catalytic efficiency are serious constraints on industrial application. To address these issues, site-directed mutagenesis of Tyr68 and Gly109 of the Clostridium bolteae DPEase was performed. Compared with the wild-type enzyme, the Y68I variant displayed the highest substrate-binding affinity and catalytic efficiency, and the G109P variant showed the highest thermostability. Furthermore, the double-site Y68I/G109P variant was generated and exhibited excellent enzyme characteristics. The Km value decreased by 17.9%; the kcat/Km increased by 1.2-fold; the t1/2 increased from 156 to 260 min; and the melting temperature (Tm) increased by 2.4 °C. Moreover, Co2+ enhanced the thermostability significantly, including the t1/2 and Tm values. All of these indicated that the Y68I/G109P variant would be appropriate for the industrial production of D-psicose. © 2016 American Chemical Society. Source

Zhang W.,Jiangnan University | Li H.,Jiangnan University | Zhang T.,Jiangnan University | Jiang B.,Jiangnan University | And 2 more authors.
Journal of Molecular Catalysis B: Enzymatic | Year: 2015

Ketose 3-epimerase displayed an important role in not only the cyclic monosaccharides bioconversion strategy, named Izumoring, but also in the industrial biological production of D-psicose, a novel low-calorie rare sugar widely used in food and medical industries. Since the non-enzymatic side reactions could be reduced in acid conditions, slightly acidic pH optimum is one of the main issues for biological production of D-psicose. In this study, we first characterized an acidic ketose 3-epimerase, the recombinant D-psicose 3-epimerase (DPEase) from Dorea sp. CAG317. The protein exhibited high amino acid sequence identity with other reported DPEases, and was determined as a homotetramer with subunit molecular weight approximately 33 kDa, which was the same as other reported findings. The recombinant DPEase was a metal-dependent enzyme with the optimum metal cofactor as Co2+. In presence of 1 mM of Co2+, the enzyme displayed the maximal activity as 803 U/mg at pH 6.0 and 70 °C. The catalytic efficiency (kcat/Km) was measured to be 412 and 199 mM-1 min-1 toward D-psicose and D-fructose, respectively. The equilibrium ratio between D-fructose and D-psicose was approximately 30:70, and the elevated temperature did not significantly shift the equilibrium toward D-psicose. Compared to other reported DPEases, the recombinant Dorea sp. DPEase displayed significantly higher specific activity at acidic pHs and remarkably higher productivity of D-psicose at pH 6.0, indicating that it was appropriate for use as a new source of D-psicose producing enzyme. © 2015 Elsevier B.V. All rights reserved. Source

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