Biocatalytic synthesis of non-standard amino acids by a decarboxylative aldol reaction

Enzymes are renowned for their catalytic efficiency and selectivity, but relatively few carbon-carbon bond forming enzymes have found their way into the biocatalysis toolbox. While engineering can overcome the challenges associated with C-C bond formation for some enzyme systems, the broader synthetic potential of biocatalysis is hindered by the lack of high-quality C-C bond forming transformations. Here we show that the enzyme UstD performs a highly selective decarboxylative aldol addition with diverse aldehyde substrates to make non-standard, γ-hydroxy amino acids. We increased the activity of UstD through three rounds of classic directed evolution and an additional round of computationally-guided engineering. The enzyme that emerged, UstD2.0, is efficient in a whole-cell biocatalysis format, which circumvents the need for enzyme purification, thereby facilitating its use in traditional organic settings. This new, highly stereoselective enzyme represents a unique expansion of the biosynthetic toolbox. The products are highly desirable, functionally rich bioactive γ-hydroxy amino acids that we demonstrate can be prepared stereoselectively on gram-scale. The X-ray crystal structure of UstD2.0 at 2.25 Å reveals the active site and the molecular basis for the remarkably promiscuity of this catalyst. Taking inspiration from the versatile reactivity of enamines in organic synthesis, we hypothesize that the enamine intermediate of UstD can be engineered to react with electrophiles other than aldehydes. The advent of structural information enabled by engineering of UstD2.0 provides a foundation for probing the unique mechanism of UstD and will guide efforts to expand the reactivity of this unique enzyme.

Electrochemical Characterization of a Complex FeFe Hydrogenase, the Electron-Bifurcating Hnd From Desulfovibrio fructosovorans

Hnd, an FeFe hydrogenase from Desulfovibrio fructosovorans, is a tetrameric enzyme that can perform flavin-based electron bifurcation. It couples the oxidation of H2 to both the exergonic reduction of NAD+ and the endergonic reduction of a ferredoxin. We previously showed that Hnd retains activity even when purified aerobically unlike other electron-bifurcating hydrogenases. In this study, we describe the purification of the enzyme under O2-free atmosphere and its biochemical and electrochemical characterization. Despite its complexity due to its multimeric composition, Hnd can catalytically and directly exchange electrons with an electrode. We characterized the catalytic and inhibition properties of this electron-bifurcating hydrogenase using protein film electrochemistry of Hnd by purifying Hnd aerobically or anaerobically, then comparing the electrochemical properties of the enzyme purified under the two conditions via protein film electrochemistry. Hydrogenases are usually inactivated under oxidizing conditions in the absence of dioxygen and can then be reactivated, to some extent, under reducing conditions. We demonstrate that the kinetics of this high potential inactivation/reactivation for Hnd show original properties: it depends on the enzyme purification conditions and varies with time, suggesting the coexistence and the interconversion of two forms of the enzyme. We also show that Hnd catalytic properties (Km for H2, diffusion and reaction at the active site of CO and O2) are comparable to those of standard hydrogenases (those which cannot catalyze electron bifurcation). These results suggest that the presence of the additional subunits, needed for electron bifurcation, changes neither the catalytic behavior at the active site, nor the gas diffusion kinetics but induces unusual rates of high potential inactivation/reactivation.

Optimum growth conditions of Lactobacillus brevis LIPI13-2-LAB131 in β-galactosidase enzyme production

Indah AWN, Rohmatussolihat, Rahayu WP, Setyoningrum F, Priadi G, Afianti F. 2020. Optimum growth conditions of Lactobacillus brevis LIPI13-2-LAB131 in β-galactosidase enzyme production. Biodiversitas 21: 5403-5407. Deficiency of β-galactosidase enzyme causes lactose to become undigested in gastrointestinal system, therefore the system needs further addition of external β-galactosidase. The sources β-galactosidase enzyme varies from plants, animals, and microorganisms. In industrial applications, microorganisms have become a considered potential source of β-galactosidase. Lactobacillus brevis LIPI13-2-LAB131 had high β-galactosidase enzyme activity, which was 7.93 U/mL. The aim of this research was to optimize the growth condition of L. brevis LIPI13-2-LAB131 in order to produce maximum β-galactosidase enzyme activity. This research consisted of performing optimization processes using design expert 7.0 (DX7) program with response surface methodology (RSM) and partial purification of β-galactosidase enzyme. The results of this research showed that the optimum growth conditions of L. brevis LIPI13-2-LAB131 were in 1.48% lactose level, incubation temperature of 34.91 °C, incubation time of 48.48 hours, and 2.83% inoculum size with desirability value of 0.839. The result of enzyme purification showed that value of β-galactosidase enzyme activity increased up to 22.88 ± 0.29 U/mL with purification yield of 11.65%.

Purification and thrombolytic effects in vivo of recombination Nattokinase on carrageenan-induced tail thrombosis in a rat model

Nattokinase is a serine protease with fibrinolytic activity and has been proven clinical efficacy and safety for human use by the oral route. In this study, conducted purified recombinant nattokinase from Bacillus subtillis DB104 strain and thrombolytic effects of nattokinase have been observed in vivo. The result of the enzyme purification by membrane filtration method for enzyme recovery efficiency is 82.45%. Then the enzyme was purified to Q-Sepharose ion exchange chromatography with a purification factor of 1.82 and at a yield of 66.91%. Enzyme activity was 5,457 FU/mg protein. With the carrageenan-induced tail thrombosis in a rat model, results were significantly reduced by 67.3 to 83.6% at oral doses and injected nattokinase compared with the control group at 48 h. At 72 hours the rate increased to 82-89% in the oral dose and up to 91% in the injected dose nattokinase and comparable to 90% for heparin-positive hepatitis.

Extraction of trypsin enzyme from visceral organs of anchovy (Engraulis encrasicolus) and sardine (Sardina pilchardus) and determination of some functional characteristics

In this study, nutritional composition analysis of internal organs of anchovy (Engraulis encrasicolus) and sardine (Sardina pilchardus), trypsin activity determination of trypsin enzyme obtained from internal organs, protein solubility and color values of extracted trypsin parameters examined. Nutritional composition of internal organs: the moisture content of the anchovy internal organs 78.92±0.11 %, the ash 2.26±0.55 %, the protein 10.82±2.05 %, the fat 6.49±0.55 % were estimated. The moisture content of the sardine internal organs 81.07±1.03 %, the ash 0.71±0.04 %, the protein 5.08±2.08 %, the fat 2.01±0.17 % were estimated. Enzyme activity and spesicific activity of anchovy trypsin which are 0.064 U/mg, 0.17 U/ml were estimated, respectively. Enzyme activiy of sardine trypsin as 0.051 U/ml and spesific activity as 0.19 U/mg were estimated. In the enzyme purification step, 13.6 g of 1 kg anchovy and 18.9 g of powder trypsin were obtained from 1 kg sardine.

Microbial Bioremediation of Feather Waste for Keratinase Production: An Outstanding Solution for Leather Dehairing in Tanneries

In leather industries and tanneries, large amount of wastes has been disposed; which polluting water, soil, and atmosphere and causing serious human health problems. In particular, chemical dehairing process of leather industries produces fair amount of toxic wastes. It is, thus, urgently needed to use alternative processes free from pollution. As more than 90% of keratin is contained in feather, it is desirable to develop bioremediation process using keratinolytic microorganisms. In the present investigation, therefore, we first identified Bacillus cereus and Pseudomonas sp. to be able to produce keratinase. Then, the optimization was performed to maximize the keratinase activity with respect to cultivation temperature, pH, and incubation time. Moreover, the effects of metal ions and various substrates on keratinase activity were also investigated. The result indicates that keratinase activity became maximum at 50°C for both strains, whereas the optimal pH was 10.0 for B. cereus and 7.0 for Pseudomonas sp. The highest keratinase activity of 74.66 ± 1.52 U/mL was attained by B. cereus, whereas 57.66 ± 2.52 U/mL was attained by Pseudomonas sp. Enzymatic dehairing efficiency of leathers was also compared with chemical dehairing (Na2S and CaO), where complete dehairing was achieved by treating them with crude keratinase. Partial enzyme purification was performed by acetone precipitation. Batch cultivation of B. cereus using 1 L fermentor indicates a potential candidate for large-scale keratinase production. Thus, keratinase enzyme by degrading poultry wastes (feather) can be an alternative approach to chemical dehairing in leather industries, thus preventing environmental pollution through bioremediation.

“Fishing and Hunting”—Selective Immobilization of a Recombinant Phenylalanine Ammonia-Lyase from Fermentation Media

This article overviews the numerous immobilization methods available for various biocatalysts such as whole-cells, cell fragments, lysates or enzymes which do not require preliminary enzyme purification and introduces an advanced approach avoiding the costly and time consuming downstream processes required by immobilization of purified enzyme-based biocatalysts (such as enzyme purification by chromatographic methods and dialysis). Our approach is based on silica shell coated magnetic nanoparticles as solid carriers decorated with mixed functions having either coordinative binding ability (a metal ion complexed by a chelator anchored to the surface) or covalent bond-forming ability (an epoxide attached to the surface via a proper linker) enabling a single operation enrichment and immobilization of a recombinant phenylalanine ammonia-lyase from parsley fused to a polyhistidine affinity tag.