Biotransformation of nitriles to amides using soluble and immobilized nitrile hydratase from Rhodococcus erythropolis A4

A semi-purified nitrile hydratase from Rhodococcus erythropolis A4 was applied to biotransformations of 3-oxonitriles 1a–4a, 3-hydroxy-2-methylenenitriles 5a–7a, 4-hydroxy-2-methylenenitriles 8a–9a, 3-hydroxynitriles 10a–12a and 3-acyloxynitrile 13a into amides 1b–13b. Cross-linked enzyme aggregates (CLEAs) with nitrile hydratase and amidase activities (88% and 77% of the initial activities, respectively) were prepared from cell-free extract of this microorganism and used for nitrile hydration in presence of ammonium sulfate, which selectively inhibited amidase activity. The genes nha1 and nha2 coding for α and β subunits of nitrile hydratase were cloned and sequenced

Microbial P450 enzymes in biotechnology

Oxidations are key reactions in chemical syntheses. Biooxidations using fermentation processes have already conquered some niches in industrial oxidation processes, since they allow the introduction of oxygen even into non-activated carbon atoms in a sterically and optically selective manner which is difficult or impossible to achieve by synthetic organic chemistry. Biooxidation using isolated enzymes is limited to oxidases and dehydrogenases. Surprisingly, cytochrome P450 monooxygenases (CYPs) have scarcely been studied for use in biooxidations, although they are one of the largest known superfamilies of enzyme proteins. Their gene sequences have been identified in various organisms such as humans, bacteria, algae, fungi and plants. The reactions catalyzed by P450s are quite diverse and range from biosynthetic pathways (e.g. those of animal hormones and secondary plant metabolites) to the activation or biodegradation of hydrophobic xenobiotic compounds (e. g. those of various drugs in the liver of higher animals). From a practical point of view, the great potential of P450s is limited by their functional complexity, low activity, and limited stability. In addition, P450-catalyzed reactions require a constant supply of NAD(P)H which makes continuous cell-free processes very expensive. Quite recently, several groups have started to investigate cost-efficient ways which could allow the continuous supply of electrons to the heme iron. These include, for example, the use of electron mediators, direct electron supply from electrodes and enzymatic approaches. In addition, methods of protein design and directed evolution have been applied in an attempt to enhance the activity of the enzymes and improve their selectivity. The promising application of bacterial P450s as catalyzing agents in biocatalytic reactions and recent progress made in this field are covered in this review

Effect of various solvents, cations, inhibitors, reducing agents, metal chelators, and denaturing agents on the activity of PepX and PepN from <i>Lb. helveticus</i> ATCC 12046 (at 37°C in 50 mM Na₂HPO₄/KH₂PO₄ buffer, pH 6.5).

1<p>The value of 100% was determined in the presence of the corresponding solvent without the additional substance. The substance was dissolved in: ²H₂O_dd; ³DMSO; ⁴Acetone; ⁵EtOH. Triplicate measurements; the standard deviation was <5%; n.d., not determined.</p

Characterization of the Recombinant Exopeptidases PepX and PepN from <i>Lactobacillus helveticus</i> ATCC 12046 Important for Food Protein Hydrolysis

<div><p>The proline-specific X-prolyl dipeptidyl aminopeptidase (PepX; EC 3.4.14.11) and the general aminopeptidase N (PepN; EC 3.4.11.2) from <i>Lactobacillus helveticus</i> ATCC 12046 were produced recombinantly in <i>E. coli</i> BL21(DE3) via bioreactor cultivation. The maximum enzymatic activity obtained for PepX was 800 µkat_{H-Ala-Pro-<i>p</i>NA} L⁻¹, which is approx. 195-fold higher than values published previously. To the best of our knowledge, PepN was expressed in <i>E. coli</i> at high levels for the first time. The PepN activity reached 1,000 µkat_{H-Ala-<i>p</i>NA} L⁻¹. After an automated chromatographic purification, both peptidases were biochemically and kinetically characterized in detail. Substrate inhibition of PepN and product inhibition of both PepX and PepN were discovered for the first time. An apo-enzyme of the Zn²⁺-dependent PepN was generated, which could be reactivated by several metal ions in the order of Co²⁺>Zn²⁺>Mn²⁺>Ca²⁺>Mg²⁺. PepX and PepN exhibited a clear synergistic effect in casein hydrolysis studies. Here, the relative degree of hydrolysis (rDH) was increased by approx. 132%. Due to the remarkable temperature stability at 50°C and the complementary substrate specificities of both peptidases, a future application in food protein hydrolysis might be possible.</p></div

Kinetic parameters of PepN¹ using chromogenic <i>p</i>-nitroanilide substrates.

1<p>Protein concentration of the enzyme solution: 2.16 mg mL⁻¹.</p>2<p>Substrate for the standard assay.</p>3<p>The maximum substrate solubility was 3.73 mM under the assay conditions. Triplicate measurements; the standard deviation was <5%.</p

Determination of the kinetic parameters of PepN for the substrate H-Ala-<i>p</i>NA according to Michaelis-Menten analysis (A) and Lineweaver-Burk linearization (B) (each point represents the average of triplicate measurements; the standard deviation was <5%).

<p>Determination of the kinetic parameters of PepN for the substrate H-Ala-<i>p</i>NA according to Michaelis-Menten analysis (A) and Lineweaver-Burk linearization (B) (each point represents the average of triplicate measurements; the standard deviation was <5%).</p

Characterization of the purified PepX (A; C; E; G) and PepN (B; D; F; H) from <i>Lb.</i><i>helveticus</i> ATCC 12046.

<p>A and B: pH optimum; C and D: temperature optimum; E and F: thermal stability; G and H: influence of NaCl and KCl (each point represents the average of triplicate measurements; the standard deviation was <5%).</p

Increase of the relative degree of hydrolysis of a prehydrolyzed casein solution by PepX and/or PepN (each point represents the average of triplicate measurements; the standard deviation was <5%).

<p>Increase of the relative degree of hydrolysis of a prehydrolyzed casein solution by PepX and/or PepN (each point represents the average of triplicate measurements; the standard deviation was <5%).</p