Esterase/lipase-catalyzed selective hydrolysis of d, l-menthyl esters has become one of the promising approaches for producing l-menthol, one of the most important flavoring chemicals with extensive ...uses. However, the activity and l-enantioselectivity of the biocatalyst are not sufficient for meeting the industrial requirements. Herein, a highly active
-nitrobenzyl esterase from
168 (pnbA-BS) was cloned and then engineered to enhance its l-enantioselectivity. On the basis of the strategy tailoring the steric exclusion effect and structural flexibility of the region adjacent to the substrate, the substitution of Ala400 to Pro caused a remarkable improvement in the
value from 1.0 to 466.6. The variant A400P was purified and further confirmed with strict l-enantioselectivity in the selective hydrolysis of d, l-menthyl acetate, whereas the improved l-enantioselectivity caused decreased activity. To develop an efficient, easy-to-use, and green methodology, organic solvent was omitted and substrate constant feeding was integrated into the whole-cell catalyzed system. During the catalytic process, the selective hydrolysis of 1.0 M d, l-menthyl acetate in 14 h offered a conversion of 48.9%, e.e.
value of >99%, and space-time yield of 160.52 g (l d)
.
The development of novel nanoparticles as a new generation therapeutic drug platform is an active field of chemistry and cancer research. In recent years, fullerene nanoparticles have received ...extensive attention due to their unique physical and chemical properties. Properly modified fullerene nanoparticles have excellent biocompatibility and significant anti-tumor activity, which makes them have broad application prospects in the field of cancer therapy. Therefore, understanding the anti-tumor mechanism of fullerene nanoparticles is of great significance for the design and development of anti-tumor drugs with low toxicity and high targeting. This review has focused on various anti-tumor mechanisms of fullerene derivatives and discusses their toxicity and their distribution in organisms. Finally, the review points out some urgent problems that need solution before fullerene derivatives as a new generation of anti-tumor nano-drug platform enter clinical research.
d-pantolactone is an intermediate in the synthesis of d-pantothenic acid, which is known as vitamin B
. The commercial synthesis of d-pantolactone is carried out through the selective resolution of ...dl-pantolactone catalyzed by lactone hydrolase. In contrast to a kinetic resolution approach, the deracemization of dl-pantolactone is a simpler, greener, and more sustainable way to obtain d-pantolactone with high optical purity. Herein, an efficient three-enzyme cascade was developed for the deracemization of dl-pantolactone, using l-pantolactone dehydrogenase from
(
LPLDH), conjugated polyketone reductase from
(
CPR), and glucose dehydrogenase from
(
GDH). The
LPLDH was used to catalyze the dehydrogenated l-pantolactone into ketopantolactone; the
CPR was used to further catalyze the ketopantolactone into d-pantolactone; and glucose dehydrogenase together with glucose fulfilled the function of coenzyme regeneration. All three enzymes were co-expressed in
strain BL21(DE3), which served as the whole-cell biocatalyst. Under optimized conditions, 36 h deracemization of 1.25 M dl-pantolactone d-pantolactone led to an
value of 98.6%, corresponding to productivity of 107.7 g/(l·d).
The members of the Old Yellow Enzyme (OYE) family are capable of catalyzing the asymmetric reduction of (
/
)-citral to (
)-citronellal-a key intermediate in the synthesis of L-menthol. The ...applications of OYE-mediated biotransformation are usually hampered by its insufficient enantioselectivity and low activity. Here, the (
)-enantioselectivity of Old Yellow Enzyme from
CICC1060 (OYE2y) was enhanced through protein engineering. The single mutations of OYE2y revealed that the sites R330 and P76 could act as the enantioselectivity switch of OYE2y. Site-saturation mutagenesis was conducted to generate all possible replacements for the sites R330 and P76, yielding 17 and five variants with improved (
)-enantioselectivity in the (
/
)-citral reduction, respectively. Among them, the variants R330H and P76C partly reversed the neral derived enantioselectivity from 32.66%
(
) to 71.92%
(
) and 37.50%
(
), respectively. The docking analysis of OYE2y and its variants revealed that the substitutions R330H and P76C enabled neral to bind with a flipped orientation in the active site and thus reverse the enantioselectivity. Remarkably, the double substitutions of R330H/P76M, P76G/R330H, or P76S/R330H further improved (
)-enantioselectivity to >99%
in the reduction of (
)-citral or (
/
)-citral. The results demonstrated that it was feasible to alter the enantioselectivity of OYEs through engineering key residue distant from active sites, e.g., R330 in OYE2y.
The recombinant carbonyl reductase from
WZ010 (ReCR) demonstrated strict (
)-stereoselectivity and catalyzed the irreversible reduction of
-Boc-3-piperidone (NBPO) to (
)-
-Boc-3-hydroxypiperidine (
...)-NBHP, a key chiral intermediate in the synthesis of ibrutinib. The NAD(H)-specific enzyme was active within broad ranges of pH and temperature and had remarkable activity in the presence of higher concentration of organic solvents. The amino acid residue at position 54 was critical for the activity and the substitution of Tyr54 to Phe significantly enhanced the catalytic efficiency of ReCR. The
/
values of ReCR Y54F for NBPO, (
/
)-2-octanol, and 2-propanol were 49.17 s
mM
, 56.56 s
mM
, and 20.69 s
mM
, respectively. In addition, the (
)-NBHP yield was as high as 95.92% when whole cells of
overexpressing ReCR variant Y54F catalyzed the asymmetric reduction of 1.5 M NBPO for 12 h in the aqueous/(
/
)-2-octanol biphasic system, demonstrating the great potential of ReCR variant Y54F for practical applications.
The gene encoding a (2R,3R)-2,3-butanediol dehydrogenase from Rhodococcus erythropolis WZ010 (ReBDH) was over-expressed in Escherichia coli and the resulting recombinant ReBDH was successfully ...purified by Ni-affinity chromatography. The purified ReBDH in the native form was found to exist as a monomer with a calculated subunit size of 37180, belonging to the family of the zinc-containing alcohol dehydrogenases. The enzyme was NAD(H)-specific and its optimal activity for acetoin reduction was observed at pH 6.5 and 55 °C. The optimal pH and temperature for 2,3-butanediol oxidation were pH 10 and 45 °C, respectively. The enzyme activity was inhibited by ethylenediaminetetraacetic acid (EDTA) or metal ions Al3+, Zn2+, Fe2+, Cu2+ and Ag+, while the addition of 10% (v/v) dimethyl sulfoxide (DMSO) in the reaction mixture increased the activity by 161.2%. Kinetic parameters of the enzyme showed lower Km values and higher catalytic efficiency for diacetyl and NADH in comparison to those for (2R,3R)-2,3-butanediol and NAD+. The activity of acetoin reduction was 7.7 times higher than that of (2R,3R)-2,3-butanediol oxidation when ReBDH was assayed at pH 7.0, suggesting that ReBDH-catalyzed reaction in vivo might favor (2R,3R)-2,3-butanediol formation rather than (2R,3R)-2,3-butanediol oxidation. The enzyme displayed absolute stereospecificity in the reduction of diacetyl to (2R,3R)-2,3-butanediol via (R)-acetoin, demonstrating its potential application on the synthesis of (R)-chiral alcohols.
α,β-Unsaturated aldehydes are widely used in the organic synthesis of fine chemicals for application in products such as flavoring agents, fragrances and pharmaceuticals. In the selective oxidation ...of α,β-unsaturated alcohols to the corresponding α,β-unsaturated aldehydes, it remains challenging to overcome poor selectivity, overoxidation and a low atom efficiency in chemical routes.
An E. coli strain coexpressing the NADP
-specific alcohol dehydrogenase YsADH and the oxygen-dependent NADPH oxidase TkNOX was constructed; these components enabled the NADP
regeneration and catalyzed the oxidation of 100 mM 3-methyl-2-buten-1-ol to 3-methyl-2-butenal with a yield of 21.3%. The oxygen supply was strengthened by introducing the hemoglobin protein VsHGB into recombinant E. coli cells and replacing the atmosphere of the reactor with pure oxygen, which increased the yield to 51.3%. To further improve catalytic performance, the E. coli cells expressing the multifunctional fusion enzyme YsADH-(GSG)-TkNOX-(GSG)-VsHGB were generated, which completely converted 250 mM 3-methyl-2-buten-1-ol to 3-methyl-2-butenal after 8 h of whole-cell oxidation. The reaction conditions for the cascade biocatalysis were optimized, in which supplementation with 0.2 mM FAD and 0.4 mM NADP
was essential for maintaining high catalytic activity. Finally, the established whole-cell system could serve as a platform for the synthesis of valuable α,β-unsaturated aldehydes through the selective oxidation of various α,β-unsaturated alcohols.
The construction of a strain expressing the fusion enzyme YsADH-(GSG)-TkNOX-(GSG)-VsHGB achieved efficient NADP
regeneration and the selective oxidation of various α,β-unsaturated alcohols to the corresponding α,β-unsaturated aldehydes. Among the available redox enzymes, the fusion enzyme YsADH-(GSG)-TkNOX-(GSG)-VsHGB has become the most recent successful example to improve catalytic performance in comparison with its separate components.
The importance of yeast old yellow enzymes is increasingly recognized for direct asymmetric reduction of (E/Z)-citral to (R)-citronellal. As one of the most performing old yellow enzymes, the enzyme ...OYE3 from Saccharomyces cerevisiae S288C exhibited complementary enantioselectivity for the reduction of (E)-citral and (Z)-citral, resulting in lower e.e. value of (R)-citronellal in the reduction of (E/Z)-citral. To develop a novel approach for the direct synthesis of enantio-pure (R)-citronellal from the reduction of (E/Z)-citral, the enzyme OYE3 was firstly modified by semi-rational design to improve its (R)-enantioselectivity. The OYE3 variants W116A and S296F showed strict (R)-enantioselectivity in the reduction of (E)-citral, and significantly reversed the (S)-enantioselectivity in the reduction of (Z)-citral. Next, the double substitution of OYE3 led to the unique variant S296F/W116G, which exhibited strict (R)-enantioselectivity in the reduction of (E)-citral and (E/Z)-citral, but was not active on (Z)-citral. Relying on its capability discriminating (E)-citral and (Z)-citral, a new cascade reaction catalyzed by the OYE3 variant S296F/W116G and glucose dehydrogenase was developed, providing the enantio-pure (R)-citronellal and the retained (Z)-citral after complete reduction of (E)-citral.
Homocysteine methyltransferase (HMT) converts homocysteine to methionine using
-methylmethionine (SMM) or
-adenosylmethionine (SAM) as methyl donors in organisms, playing an important role in ...supplying methionine for the growth and the development of plants. To better understand the functions of the
genes in plants, we conducted a wide evolution and expression analysis of these genes. Reconstruction of the phylogenetic relationship showed that the
gene family was divided into Class 1 and Class 2. In Class 1,
s were only found in seed plants, while Class 2 presented in all land plants, which hinted that the
genes might have diverged in seed plants. The analysis of gene structures and selection pressures showed that they were relatively conserved during evolution. However, type I functional divergence had been detected in the
s. Furthermore, the expression profiles of
s showed their distinct expression patterns in different tissues, in which some
s were widely expressed in various organs, whereas the others were highly expressed in some specific organs, such as seeds or leaves. Therefore, according to our results in the evolution, functional divergence, and expression, the
genes might have diverged during evolution. Further analysis in the expression patterns of
s with their methyl donors suggested that the diverged
s might be related to supply methionine for the development of plant seeds.
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