Adsorption‐based immobilization of <i><scp>C</scp>aldicellulosiruptor saccharolyticus</i> cellobiose 2‐epimerase on <i><scp>B</scp>acillus subtilis</i> spores

Gu, Jun; Yang, Ruijin; Xiao, Hua; Zhang, Wenbin; Zhao, Wei

doi:10.1002/bab.1262

Cited by 24 publications

(9 citation statements)

References 30 publications

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“…The purification of CsCE with His-Trap affinity chromatography (Table 1) led to similar results as described previously . The enzyme activity produced in our study (3.32 µkat epilactose, 80°C /mg protein ) with lactose as the substrate was approximately 6.6 times higher than described before Gu et al, 2015). However, it has to be taken into account that the activity measurements were based on the formation of lactulose, the second product formed, whereas in our study, we defined the CsCE activity corresponding to epilactose generation, the first and only product formed during the assay period (4 to 10 min).…”

Section: Discussioncontrasting

confidence: 52%

Enzymatic production of lactulose and epilactose in milk

Rentschler

Schuh

Krewinkel

et al. 2015

Journal of Dairy Science

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The enzymatic production of lactulose was described recently through conversion of lactose by a thermophilic cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus (CsCE). In the current study, we examined the application of CsCE for lactulose and epilactose production in milk (1.5% fat). The bioconversions were carried out in stirred reaction vessels at 2 different temperatures (50 and 8°C) at a scale of 25 mL volume. At 50°C, 2 highly different CsCE amounts were investigated for the time course of formation of lactulose and epilactose. The conversion of milk lactose (initial lactose content of 48.5 ± 2.1 g/L) resulted in a final yield of 57.7% (28.0 g/L) lactulose and 15.5% (7.49 g/L) epilactose in the case of the approximately 9.5-fold higher CsCE amount (39.5 µkat epilactose, 50°C) after 24 h. Another enzymatic lactose conversion was carried out at low 8°C, an industrially relevant temperature for milk processing. Although the CsCE originated from a thermophilic microorganism, it was still applicable at 8°C. This enzymatic lactose conversion resulted in 56.7% (27.5 g/L) lactulose and 13.6% (6.57 g/L) epilactose from initial milk lactose after 72 h. The time courses of lactose conversion by CsCE suggested that first epilactose formed and afterward lactulose via epilactose. To the best of our knowledge, this is the first time that an enzyme has produced lactulose directly in milk in situ at industrially relevant temperatures.

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Section: Discussioncontrasting

confidence: 52%

Enzymatic production of lactulose and epilactose in milk

Rentschler

Schuh

Krewinkel

et al. 2015

Journal of Dairy Science

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“…The purification step of the enzyme was carried out using Q Sepharose Fast Flow and Sephadex G‐75 column chromatography (Gu et al ., ).…”

Section: Methodsmentioning

confidence: 97%

Lactulose production from lactose by recombinant cellobiose 2‐epimerase in permeabilised Escherichia coli cells

Wang

Yang

Xiao

et al. 2015

Int J of Food Sci Tech

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Caldicellulosiruptor saccharolyticus cellobiose 2-epimerase (CsCE) catalyses the single substrate lactose into lactulose and is the most efficient enzyme ever found for the enzymatic synthesis of lactulose. Ethanol-permeabilised Escherichia coli cells containing CsCE were used as biocatalysts for lactulose production. The reaction conditions for maximum lactulose production were optimised to be 600 g L À1 lactose, pH 7.5, 80°C and 12.5 U mL À1 of whole-cell biocatalyst. After incubated at Na 2 HPO 4 -NaH 2 PO 4 buffer for 2 h, approximately 390.59 g L À1 lactulose was obtained with a conversion yield of 65.1%. The lowest production and conversion yield of epilactose were also found in Na 2 HPO 4 -NaH 2 PO 4 buffer with a final concentration of 11.7 g L À1 and a conversion yield <2%. The results represent a promising technology to attain high production and conversion yield of lactulose with a high purity on industrial scale.

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“…Despite its entrenchment as a hypothesis, molecular state changes cannot account well for many commonly observed features of T opt . For example, T op t of reactions measured in vivo are often more than 20 °C below reported in vitro enzyme de-activation or denaturation temperatures, T den 4 , 5 , 10 , 11 and enzyme activities are often sustained well above reported in vitro T den in the presence of co-solvents or heat shock proteins 7 , 12 , 13 . T opt may depend on reaction characteristics as much or more than molecular state changes, as T opt often changes by 10–20 °C in response to changes in enzyme efficiency 14 , 15 or concentration 16 , 17 .…”

Section: Introductionmentioning

confidence: 98%

Reaction and diffusion thermodynamics explain optimal temperatures of biochemical reactions

Ritchie

2018

Sci Rep

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Ubiquitous declines in biochemical reaction rates above optimal temperatures (Topt) are normally attributed to enzyme state changes, but such mechanisms appear inadequate to explain pervasive Topt well below enzyme deactivation temperatures (Tden). Here, a meta-analysis of 92 experimental studies shows that product formation responds twice as strongly to increased temperature than diffusion or transport. This response difference has multiple consequences for biochemical reactions, such as potential shifts in the factors limiting reactions as temperature increases and reaction-diffusion dynamics that predict potential product inhibition and limitation of the reaction by entropy production at temperatures below Tden. Maximizing entropy production by the reaction predicts Topt that depend on enzyme concentration and efficiency as well as reaction favorability, which are patterns not predicted by mechanisms of enzyme state change. However, these predictions are strongly supported by patterns in a meta-analysis of 121 enzyme kinetic studies. Consequently, reaction-diffusion thermodynamics and entropy production may constrain organism performance at higher temperatures, yielding temperature optima of life that may depend on reaction characteristics and environmental features rather than just enzyme state changes.

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Adsorption‐based immobilization of Caldicellulosiruptor saccharolyticus cellobiose 2‐epimerase on Bacillus subtilis spores

Cited by 24 publications

References 30 publications

Enzymatic production of lactulose and epilactose in milk

Enzymatic production of lactulose and epilactose in milk

Lactulose production from lactose by recombinant cellobiose 2‐epimerase in permeabilised Escherichia coli cells

Reaction and diffusion thermodynamics explain optimal temperatures of biochemical reactions

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