Fumarase From Cyanidioschyzon merolae Stably Shows High Catalytic Activity for Fumarate Hydration Under High Temperature Conditions

Abstract

Fumarases (Fums) catalyze the reversible reaction converting fumarate to l-malate. There are two kinds of Fums: Class І and ІІ. Thermostable Class ІІ Fums, from mesophilic microorganisms, are utilized for industrial l-malate production. However, the low thermostability of these Fums is a limitation in industrial l-malate production. Therefore, an alternative Class ІІ Fum that shows high activity and thermostability is required to overcome this drawback. Thermophilic microalgae and cyanobacteria can use carbon dioxide as a carbon source and are easy to cultivate. Among them, Cyanidioschyzon merolae and Thermosynechococcus elongatus are model organisms to study cell biology and structural biology, respectively. We biochemically analyzed Class ІІ Fums from C. merolae (CmFUM) and T. elongatus (TeFum). Both CmFUM and TeFum preferentially catalyzed fumarate hydration. The catalytic activity of CmFUM for fumarate hydration in the optimum conditions (52°C and pH 7.5) is higher compared to those of Class ІІ Fums from other organisms and TeFum. Thermostability tests of CmFUM revealed that CmFUM showed higher thermostability than those of Class ІІ Fums from other microorganisms. The yield of l-malate obtained from fumarate hydration catalyzed by CmFUM was 75-81%. In summary, CmFum has suitable properties for efficient l-malate production.

Biochemical Properties of β-Amylase from Red Algae and Improvement of Its Thermostability through Immobilization

“…coli BL21 (DE3) was cultured with several liters of the LB medium (Becton, Dickinson and Company, Franklin Lakes, NJ) at 30 °C overnight with shaking at 150 rpm, and 0.01 mM isopropyl β- d -1-thiogalactopyranoside (Wako Chemical, Osaka, Japan) was added to the culture medium to induce the expression of GST-tagged CmBAM. The expressed GST-tagged CmBAM protein was purified by affinity chromatography, with some modifications . The E.…”

“…The expressed GST-tagged CmBAM protein was purified by affinity chromatography, with some modifications. 22 The E. coli culture medium (4 L) was collected by repeated centrifugation (5800 g , 2 min, 25 °C) and added to approximately 35 mL of 1× PBS-T (0.14 M NaCl, 2.7 mM KCl, 8.1 mM Na 2 HPO 4 ·12H 2 O, 1.5 mM KH 2 PO 4 , 0.05% Tween-20) with cOmplete Mini protease inhibitor cocktail 1/5 tablets. The collected E. coli cells were sonicated 10 times for 20 s each time with 20% amplitude to disrupt the cells and elute GST-tagged CmBAM into the soluble fraction (VC-750, EYELA, Tokyo, Japan).…”

“…merolae has been sequenced. , As a model organism, the cell cycle, cell and organelle division, − genes related to oil and fat production, and environmental response mechanisms have been studied biochemically. − A few proteins that have been studied thus far in C. merolae are thermostable, , while more enzymes are expected to be thermostable. C.…”

“… 15 , 16 As a model organism, the cell cycle, cell and organelle division, 16 − 18 genes related to oil and fat production, and environmental response mechanisms have been studied biochemically. 19 − 21 A few proteins that have been studied thus far in C. merolae are thermostable, 22 , 23 while more enzymes are expected to be thermostable. C. merolae has no cell wall, and intracellular components can be easily extracted using the osmotic shock method.…”

Immobilization of fumarase from thermophilic eukaryotic red alga <i>Cyanidioschyzon merolae</i> on ceramic carrier

“…This fumarase exhibited an optimal reaction temperature of 52°C and a relatively high activity of approximately 110 U/mg for fumarate (Ito et al, 2020). Furthermore, the duration of heat treatment that the enzyme could withstand before activity dropped by 50% (t1/2) was 507 min at 50°C.…”