Catalytically active inclusion bodies (CatIBs) induced by terminally attached self-assembling coiled-coil domains: To enhance the stability of (R)-hydroxynitrile lyase.

The catalytically-active inclusion bodies (CatIBs) represent a promising strategy for immobilizing enzyme without additional carriers and chemicals, which has aroused great attention in academic and industrial communities. In this work, we discovered two natural parallel right-handed coiled-coil tetramer peptides from PDB database by a structural mining strategy. The two self-assembling peptides, NSPdoT from rotavirus and HVdoT from human Vasodilator-stimulated phosphoprotein, efficiently induced the CatIBs formation of a (R)-Hydroxynitrile lyase from Arabidopsis thaliana (AtHNL) in Escherichia coli cells. This is convenient to simultaneously purify and immobilize the target proteins as biocatalysts. As expected, HVdoT-AtHNL and NSPdoT-AtHNL possessed drastically increased tolerance toward lower pH values, which will be very critical to synthesize cyanohydrins under acidic condition for suppressing the non-enzymatic side reaction. In addition. AtHNL-CatIBs are produced at high yield in host cells as bioactive microparticles, which exhibited high thermal and pH stabilities. Therefore, the CatIBs method represent a promising application for the immobilization of enzymes in the biocatalysis field.

Biomimetic mineralization of nitrile hydratase into a mesoporous cobalt-based metal-organic framework for efficient biocatalysis.

Nitrile hydratases (NHases) have attracted considerable attention owing to their application in the synthesis of valuable amides under mild conditions. However, the poor stability of NHases is still one of the main drawbacks for their industrial application. Recently, mesoporous metal-organic frameworks (MOFs) have been explored as an attractive support material for immobilizing enzymes. Here, we encapsulated a recombinant cobalt-type NHase from Aurantimonas manganoxydans into the cobalt-based MOF ZIF-67 by a biomimetic mineralization strategy. The nano-catalyst NHase1229@ZIF-67 shows high catalytic activity for the hydration of 3-cyanopyridine to nicotinamide, and its specific activity reached 29.5 U mg-1. The NHase1229@ZIF-67 nanoparticles show a significant improvement in the thermal stability of NHase1229. The optimum reaction temperature of NHase1229@ZIF-67 is at 50-55 °C, and it still retained 40% of the maximum activity at 70 °C. However, the free NHase1229 completely lost its catalytic activity at 70 °C. The half-lives of NHase1229@ZIF-67 at 30 and 40 °C were 102.0 h and 26.5 h, respectively. NHase1229@ZIF-67 nanoparticles exhibit an excellent cycling performance, and their catalytic efficiency did not significantly decrease in the initial 6 cycles using 0.9 M 3-cyanopyridine as the substrate. In a fed-batch reaction, NHase1229@ZIF-67 can efficiently hydrate 3-cyanopyridine to nicotinamide, and the space-time yield was calculated to be 110 g·L-1·h-1. Therefore, the cobalt-type NHase was immobilized in MOF ZIF-67, which is shown as a potential nanocatalyst for the large-scale industrial preparation of nicotinamide.

Recombinant expression and molecular insights into the catalytic mechanism of an NADPH-dependent conjugated polyketone reductase for the asymmetric synthesis of (R)-pantolactone.

(R)-pantolactone is a key chiral intermediate for synthesizing calcium (R)-pantothenate. The commercial synthesis of (R)-pantolactone is performed through the resolution of racemic pantolactone using lactonase-catalyzed enantioselective hydrolysis. The process needs highly toxic hydrogen cyanide and a tedious dynamic kinetic resolution. In this study, we investigated an alternative method to prepare (R)-pantolactone through asymmetric reduction of ketopantolactone (KPL). An NADPH-dependent conjugated polyketone reductase gene from Candida dubliniensis CD36 (CduCPR) was functionally overexpressed in Escherichia coli BL21 (DE3). Recombinant CduCPR belonged to the aldo-keto reductase superfamily, and showed high catalytic activity and stereoselectivity using KPL as the substrate. In a continuous feeding reaction, 200 mM ketopantolactone was reduced to (R)-pantolactone with 98% conversion and 99% enantiomeric excess (e.e.) within 2.0 h. The catalytic mechanism was further investigated. Tyr66 functions as a proton donor following hydrogen transfer from NADPH. Thr30 and His128 are critical residues to bind and orient KPL. Therefore, the recombinant CduCPR from C. dubliniensis exhibited potential application in the asymmetric synthesis of (R)-pantolactone.

Discovery of a new NADPH-dependent aldo-keto reductase from Candida orthopsilosis catalyzing the stereospecific synthesis of (R)-pantolactone by genome mining.

(R)-Pantolactone (PL) is a key chiral intermediate for the synthesis of calcium (R)-pantothenate and (R)-panthenol used as food additives. The commercial production of (R)-pantothenate is performed by the resolution of racemic pantothenate, which is synthesized through an aldol condensation and a cyanation reaction. In this study, we investigated another synthetic method of (R)-pantothenate through the stereoselective reduction of ketopantoyl lactone (KPL) by aldo-keto reductase (AKR). A series of conjugated polyketone reductases (CPRs) were discovered from GenBank database by genome mining approach. The putative CPR gene from Candida orthopsilosis Co 90-125 (CorCPR) was cloned and functionally expressed in Escherichia coli BL21 (DE3). The optimum pH and temperature of recombinant CorCPR were 6.0-7.0 and 40 ℃, respectively. The Km and vmax toward KPL were1.3 mM and 227.3 µmol/min/mg protein, respectively. The conserved sequences suggest that CorCPR belongs to AKR3C family of AKR superfamily. Furthermore, a catalytic tetrad was proposed, and the detailed mechanism was clarified by molecular docking. In a batch reaction, 50 mM KPL was reduced to (R)-PL with 99% conversion and > 99% enantiomeric excess within 5 h. The recombinant CorCPR from C. orthopsilosis shows potential application in the asymmetric synthesis of (R)-pantothenate preparation.

Evidence for the participation of an extra α-helix at ß-subunit surface in the thermal stability of Co-type nitrile hydratase.

Nitrile hydratase (NHase) has attracted considerable attention since it can efficiently catalyze the hydration of nitriles to valuable amides. However, the poor stability of NHase is one of the main drawbacks in the industrial application. In this study, we compared the structural difference between Fe-type and Co-type NHase and found that an extra α helix existed at the ß-subunit surface of Co-type NHase (defined as the ß-6th helix). Then, the effects of the ß-6th helix were investigated on the thermal stability and the catalytic kinetics of a Co-type NHase from Aurantimonas manganoxydans ATCC BAA-1229 (NHase1229). When the ß-6th helix was deleted or disrupted, the thermal stability of NHase1229 was reduced to 17.6 and 12.9% of that of wild NHase1229, respectively. Thus, the ß-6th helix is important for the thermal stability of Co-type NHase. Based on the structural characteristics of Co-type NHase, the ß-6th helix may be interacted with another helix at the α-subunit (defined as the α-2nd helix) by hydrophobic network just as a "magnetic suction buckle" on the enzyme surface to stabilize the binding of α- and ß-subunits. The ß-6th helix is located at the mouth of the substrate and product tunnel, so it plays crucial roles in catalytic process. Furthermore, the ß-6th helix in NHase1229 was swapped with a thermophilic NHase fragment from Pseudonocardia thermophila JCM3095 (NHase1229-Swap). The thermal stability of NHase1229-Swap was significantly improved, and the half-life was approximately 2.4-fold at 40 °C than that of the wild NHase1229. The knowledge is useful for improving the stability of NHases by restriction fragment swapping.