The accumulation of polyethylene
terephthalate (PET) in the environment
has brought an enormous threat to the global ecosystem. Although the
recently reported PET hydrolase (PETase) displays an efficient decomposition
to PET, the low activity and thermostability limit its practical applications.
Herein, we introduce a biomodification strategy by fusing a zwitterionic
polypeptide (5–30 kDa) consisting of alternating-charged glutamic
acid (E) and lysine (K) residues to the C-terminus of PETase and find
that increasing the fusion peptide length leads to the improved catalytic
performance. The product release in the degradation of highly crystallized
PET films (45.2% in crystallinity) by PETase-EK30 is promoted by over
11 times as compared with PETase. The molecular mechanism of the enhanced
catalytic performance is investigated via structural analysis, substrate
binding, and molecular simulations. Characterizations of the secondary
and tertiary structures verify a strengthened structural stability
of the EKylated PETases. Synchronous fluorescence spectra indicate
a more open substrate-binding pocket after EKylation. MD simulations
of enzyme–substrate complexes support that the EKylation induces
the exposure of hydrophobic amino acids (W185, I208, and W159) in
the substrate-binding pocket and the rotation of the benzene ring
of Y87, which promote the substrate binding kinetics. This leads to
the enhanced substrate affinity, exactly represented by the increased
association constant and the decreased binding free energy. Besides,
a shortened catalytic distance is observed from the MD simulations,
which might also contribute to the enhanced catalytic activity toward
PET degradation. The molecular insights into the enhanced enzyme performance
would benefit in extending the application of the EKylation strategy
in various enzymes.
An effective synthetic strategy for the amino-containing organic derivative of hexavanadate, TBA 2 ijV 6 O 13 {(OCH 2 ) 3 CNH 2 } 2 ], is presented. By using this method, the yield is increased extremely and crystals of the intermediate and cation-exchanged products are both obtained and structurally characterized via single-crystal X-ray diffraction, IR and 1 H-NMR.
Mutations in the human P gene result in oculocutaneous albinism type 2, the most common form of albinism. Mouse melan-p1 melanocytes, cultured from mice null at the homologous pink-eyed dilution (p) locus, exhibit defective melanin production. A variety of compounds including tyrosine, NH4Cl, bafilomycin A1, concanamycin, monensin, and nigericin are capable of restoring melanin synthesis in these cells. In the current study, we investigated the subcellular effects of bafilomycin A1 and monensin treatment of melan-p1 cells. Both agents play two roles in the processing of tyrosinase (Tyr) in melan-p1 cells. First, combined glycosidase digestion and immunoblotting analysis showed that these agents reduce levels of Tyr retained in the endoplasmic reticulum (ER) and facilitate the release of Tyr from the ER to the Golgi. Secondly, treatment with these compounds resulted in the stabilization of Tyr. Surprisingly, induction of melanin synthesis corresponds more closely with diminution of ER-retained Tyr, rather than the absolute amount of Tyr. Our results suggest that bafilomycin A1 and monensin induce melanin synthesis in melan-p1 cells mainly by facilitating Tyr processing from the ER to the Golgi by increasing the pH in either the ER or the ER-Golgi intermediate compartment.
A highly selective para C-H amination of unprotected phenols with iminoquinone acetals was realized, giving the functional phenols in good to excellent yields. Overall, this transformation is operationally simple, proceeds with readily available phenols, and has wide substrate scope and low catalyst loading. The biarylamine product is stochastically formed via [5,5]-sigmatropic rearrangement of a mixed acetal species which is generated in situ under the reaction conditions.
A psychrophilic extracellular protease was isolated from the marine bacterium Planococcus sp. M7 found in the deep-sea mud of the Southern Indian Ocean. The mature protease is about 43 kDa and contains 389 amino acids. Sequence alignment revealed that the protease whose catalytic triad was comprised of Ser224, Lys249, and Gln253 contains a catalytic module belonging to the serralysin-type protease family 41, and displays 46.55% identity with the experimentally verified serine protease from Bacillus subtilis str. 168. The enzyme displayed an alkaline mesophilic preference with an optimum pH of 10.0 and an optimum temperature of 35 °C. The enzyme retained its activity from 5 to 35 °C and was resistant to repeated freezing and thawing, but was completely inactivated at 55 °C. Calcium ions had a protective effect against thermal denaturation. More than 60% of the maximum activity was retained at pH values in the range of 5.0-11.0. Almost no activity loss was detected after 1 h of incubation at pH 8.0-10.0 and 20 °C, or with 1.0% SDS. Most important, this protease also showed good stability and compatibility with the standard enzyme-free detergent, which indicates its special interest for applications in detergent industry.
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