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1.
Biotechnol Bioeng ; 121(3): 971-979, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38088450

RESUMO

The methylotrophic yeast Pichia pastoris (Komagataella phaffii) is a highly distinguished expression platform for the excellent synthesis of various heterologous proteins in recent years. With the advantages of high-density fermentation, P. pastoris can produce gram amounts of recombinant proteins. While not every protein of interest can be expressed to such high titers, such as Baeyer-Villiger monooxygenase (BVMO) (AcPSMO) which is responsible for pyrazole sulfide asymmetric oxidation. In this work, an excellent yeast expression system was established to facilitate efficient AcPSMO expression, which exhibited 9.5-fold enhanced secretion. Subsequently, an ultrahigh throughput screening method based on fluorescence-activated cell sorting by fusing super folder green fluorescent protein (sfGFP) in the C-terminal of AcPSMO was developed, and directed evolution was performed. The protein expression level of the superior mutant AcPSMOP1 (S58T/T252P/E336N/H456D) reached 84.6 mg/L at 100 mL shaking flask, which was 4.7 times higher than the levels obtained with the wild-type. Finally, the optimized chassis cells were used for high-density fermentation on a 5-L scale, and AcPSMOP1 protein yield of 3.4 g/L was achieved, representing approximately 85% of the total protein secreted. By directly employing the pH-adjusted supernatant as a biocatalyst, 20 g/L pyrmetazole sulfide was completely transformed into the corresponding (S)-sulfoxide, with a 78.8% isolated yield. This work confers dramatic benefits for efficient secretion of other BVMOs in P. pastoris.


Assuntos
Oxigenases de Função Mista , Pichia , Saccharomycetales , Oxigenases de Função Mista/metabolismo , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/metabolismo , Sulfóxidos/metabolismo , Sulfetos/metabolismo
2.
Biochemistry ; 62(22): 3214-3221, 2023 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-37902563

RESUMO

Cytochrome P450 monooxygenases (CYP450s) play an important role in the biosynthesis of natural products by activating inert C-H bonds and inserting hydroxyl groups. However, the activities of most plant-derived CYP450s are extremely low, limiting the heterologous biosynthesis of natural products. Traditional enzyme engineering methods, either rational or screening-based, are not suitable for CYP450s because of the lack of crystal structures and high-throughput screening methods for this class of enzymes. CYP725A4 is the first hydroxylase involved in the biosynthesis pathway of Taxol. Its low activity, promiscuity, and multispecificity make it a bottleneck in Taxol biosynthesis. Here, we identified key amino acids that affect the in vivo activity of CYP725A4 by constructing the ancestral enzymes of CYP725A4. We obtained positive mutants that showed an improved yield of hydroxylated products based on the key amino acids identified, providing guidance for the modification of other CYP450s involved in the biosynthesis of natural products.


Assuntos
Aminoácidos , Produtos Biológicos , Aminoácidos/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Paclitaxel/química , Paclitaxel/metabolismo
3.
Chembiochem ; 24(20): e202300390, 2023 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-37455264

RESUMO

Nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH) constitute major hydrogen donors for oxidative/reductive bio-transformations. NAD(P)H regeneration systems coupled with formate dehydrogenases (FDHs) represent a dreamful method. However, most of the native FDHs are NAD+ -dependent and suffer from insufficient reactivity compared to other enzymatic tools, such as glucose dehydrogenase. An efficient and competitive NADP+ -utilizing FDH necessitates the availability and robustness of NADPH regeneration systems. Herein, we report the engineering of a new FDH from Candida dubliniensis (CdFDH), which showed no strict NAD+ preference by a structure-guided rational/semi-rational design. A combinatorial mutant CdFDH-M4 (D197Q/Y198R/Q199N/A372S/K371T/▵Q375/K167R/H16L/K159R) exhibited 75-fold intensification of catalytic efficiency (kcat /Km ). Moreover, CdFDH-M4 has been successfully employed in diverse asymmetric oxidative/reductive processes with cofactor total turnover numbers (TTNs) ranging from 135 to 986, making it potentially useful for NADPH-required biocatalytic transformations.


Assuntos
Formiato Desidrogenases , NAD , NADP/metabolismo , NAD/metabolismo , Formiato Desidrogenases/genética , Formiato Desidrogenases/metabolismo , Engenharia de Proteínas/métodos , Oxirredução
4.
Chembiochem ; 24(23): e202300582, 2023 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-37728423

RESUMO

(R)-ß-piperonyl-γ-butyrolactones are key building blocks for the synthesis of podophyllotoxin, which have demonstrated remarkable potential in cancer treatment. Baeyer-Villiger monooxygenases (BVMOs)-mediated asymmetric oxidation is a green approach to produce chiral lactones. While several BVMOs were able to oxidize the corresponding cyclobutanone, most BVMOs gave the (S) enantiomer while Cyclohexanone monooxygenase (CHMO) from Brevibacterium sp. HCU1 gave (R) enantiomer, but with a low enantioselectivity (75 % ee). In this study, we use a strategy called "focused rational iterative site-specific mutagenesis" (FRISM) at residues ranging from 6 Šfrom substrate. The mutations by using a restricted set of rationally chosen amino acids allow the formation of a small mutant library. By generating and screening less than 60 variants, we achieved a high ee of 96.8 %. Coupled with the cofactor regeneration system, 9.3 mM substrate was converted completely in a 100-mL scale reaction. Therefore, our work reveals a promising synthetic method for (R)-ß-piperonyl-γ-butyrolactone with the highest enantioselectivity, and provides a new opportunity for the chem-enzymatic synthesis of podophyllotoxin.


Assuntos
Oxigenases , Podofilotoxina , Oxigenases/metabolismo , Oxigenases de Função Mista/metabolismo , Oxirredução , Especificidade por Substrato
5.
Biotechnol Bioeng ; 120(7): 1773-1783, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37130074

RESUMO

The key precursors for nylon synthesis, that is, 6-aminocaproic acid (6-ACA) and 1,6-hexamethylenediamine (HMD), are produced from petroleum-based feedstocks. A sustainable biocatalytic alternative method from bio-based adipic acid has been demonstrated recently. However, the low efficiency and specificity of carboxylic acid reductases (CARs) used in the process hampers its further application. Herein, we describe a highly accurate protein structure prediction-based virtual screening method for the discovery of new CARs, which relies on near attack conformation frequency and the Rosetta Energy Score. Through virtual screening and functional detection, five new CARs were selected, each with a broad substrate scope and the highest activities toward various di- and ω-aminated carboxylic acids. Compared with the reported CARs, KiCAR was highly specific with regard to adipic acid without detectable activity to 6-ACA, indicating a potential for 6-ACA biosynthesis. In addition, MabCAR3 had a lower Km with regard to 6-ACA than the previously validated CAR MAB4714, resulting in twice conversion in the enzymatic cascade synthesis of HMD. The present work highlights the use of structure-based virtual screening for the rapid discovery of pertinent new biocatalysts.


Assuntos
Ácido Aminocaproico , Oxirredutases , Oxirredutases/metabolismo , Adipatos
6.
Appl Microbiol Biotechnol ; 107(18): 5727-5737, 2023 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-37477695

RESUMO

Cytochrome P450 monooxygenases CYP120As are the unique non-membrane P450s, which are extensively involved in retinoid biodegradation. As the O-functionalized 1,3,3-trimethylcyclohex-1-ene moiety exists in many bioactive compounds which could only be catalyzed by Class II P450s, exploration of the catalytic repertoire of CYP120As is therefore highly attractive. However, up to date, only one bacteriogenic candidate (CYP120A1) was demonstrated for the hydroxylation of C16 and C17 of retinoic acid, by utilizing the integral membrane protein cytochrome P450 reductase redox partner for the electron transfer. Herein, we provided an efficient prokaryotic functional expression system of CYP120As in E. coli by expression of the CYP120A1 coupled with several reductase partners. Fusion redox partners to the C-terminal of the heme-domain are also working on other CYP120A members. Among them, the fusion protein of CYP120A29 and FAD/FMN reductase from Bacillus megaterium P450BM3 (CYP101A2) showed the highest expression level. Based on the available translational fusion systems, the regioselectivity and the substrate scope of the CYP120As have also been explored. This work represents a good starting point for further expanding the catalytic potential of CYP120 family. KEY POINTS: • Characterization of CYP120As in E. coli is firstly achieved by constructing fusion proteins. • The feasibility of three P450 reductase domains to CYP120As was evaluated. • Hydroxylated products of retinoic acid by six CYP120As were sorted and analyzed.


Assuntos
Proteínas de Bactérias , Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Bactérias/metabolismo , Oxirredução , Transporte de Elétrons , NADPH-Ferri-Hemoproteína Redutase/genética , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Tretinoína/metabolismo
7.
Chembiochem ; 23(16): e202200228, 2022 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-35639013

RESUMO

Baeyer-Villiger monooxygenases (BVMOs) are important biocatalysts for the enzymatic synthesis of chiral sulfoxides, including chiral sulfoxide-type proton pump inhibitors for the treatment of gastrointestinal diseases. However, native BVMOs are not yet suitable for practical application due to their unsatisfactory activity and thermostability. Although protein engineering approaches can help address these issues, few feasible high-throughput methods are available for the engineering of such enzymes. Herein, a colorimetric detection method to distinguish sulfoxides from sulfides and sulfones was developed for prazole sulfide monooxygenases. Directed evolution enabled by this method has identified a prazole sulfide monooxygenase CbBVMO variant with improved activity and thermostability that catalyzes the asymmetric oxidation of lansoprazole sulfide. A 71.3 % increase in conversion and 6 °C enhancement in the melting point were achieved compared with the wild-type enzyme. This new method is feasible for high-throughput screening of prazole sulfide monooxygenase variants with improved activity, thermostability, and/or substrate specificity.


Assuntos
Ensaios de Triagem em Larga Escala , Oxigenases de Função Mista , Biocatálise , Colorimetria , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Oxirredução , Especificidade por Substrato , Sulfetos/metabolismo , Sulfóxidos/metabolismo
8.
Chembiochem ; 22(6): 996-1000, 2021 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-33146944

RESUMO

Chiral cyanohydrins are useful intermediates in the pharmaceutical and agricultural industries. In nature, hydroxynitrile lyases (HNLs) are a kind of elegant tool for enantioselective hydrocyanation of carbonyl compounds. However, currently available methods for demonstrating hydrocyanation are still stalled at precise, but low-throughput, GC or HPLC analyses. Herein, we report a chromogenic high-throughput screening (HTS) method that is feasible for the cyanohydrin synthesis reaction. This method was highly anti-interference and sensitive, and could be used to directly profile the substrate scope of HNLs either in cell-free extract or fermentation clear broth. This HTS method was also validated by generating new variants of PcHNL5 that presented higher catalytic efficiency and stronger acidic tolerance in variant libraries.


Assuntos
Aldeído Liases/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Nitrilas/metabolismo , Aldeídos/química , Aldeídos/metabolismo , Biocatálise , Evolução Molecular Direcionada , Escherichia coli/enzimologia , Nitrilas/química , Estereoisomerismo , Especificidade por Substrato
9.
Biotechnol Bioeng ; 118(2): 737-744, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33073356

RESUMO

AcCHMO, a cyclohexanone monooxygenase from Acinetobacter calcoaceticus, is a typical Type I Baeyer-Villiger monooxygenase (BVMO). We previously obtained the AcCHMOM6 mutant, which oxidizes omeprazole sulfide (OPS) to the chiral sulfoxide drug esomeprazole. To further improve the catalytic efficiency of the AcCHMOM6 mutant, a focused mutagenesis strategy was adopted at the intersections of the FAD-binding domain, NADPH-binding domain, and α-helical domain based on structural characteristics of AcCHMO. By using focused mutagenesis and subsequent global evolution two key residues (L55 and P497) at the intersections of the domains were identified. Mutant of L55Y improved catalytic efficiency significantly, whereas the P497S mutant alleviated substrate inhibition remarkably. AcCHMOM7 (L55Y/P497S) was obtained by combining the two mutations, which increased the specific activity from 18.5 (M6) to 108 U/g, and an increase in the Ki of the substrate OPS from 34 to 265 µM. The results indicate that catalytic performance can be elevated by modification of the sensitive sites at the intersection of the domains of AcCHMO. The results also provided some insights for the engineering of other Type I BVMOs or other multidomain proteins.


Assuntos
Acinetobacter calcoaceticus/enzimologia , Proteínas de Bactérias/química , Oxigenases de Função Mista/química , Acinetobacter calcoaceticus/genética , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Oxigenases de Função Mista/genética , Mutação de Sentido Incorreto , Domínios Proteicos
10.
Metab Eng ; 47: 184-189, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29477859

RESUMO

Biocatalytic C-H amination is one of the most challenging tasks. C-H amination reaction can hardly be driven efficiently by solely one enzyme so far. Thus, enzymatic synergy represents an alternative strategy. Herein, we report an "Artificially Bioamination Pathway" for C-H amination of cyclohexane as a model substrate. Three enzymes, a monooxygenase P450BM3 mutant, an alcohol dehydrogenase ScCR from Streptomyces coelicolor and an amine dehydrogenase EsLeuDH from Exiguobacterium sibiricum, constituted a clean cascade reaction system with easy product isolation. Two independent cofactor regeneration systems were optimized to avoid interference from the endogenous NADH oxidases in the host E. coli cells. Based on a stepwise pH adjustment and ammonium supplement strategy, and using an in vitro mixture of cell-free extracts of the three enzymes, cyclohexylamine was produced in a titer of 14.9 mM, with a product content of up to 92.5%. Furthermore, designer cells coexpressing the three required enzymes were constructed and their capability of alkane bio-amination was examined. This artificially designed bioamination paves an attractive approach for enzymatic synthesis of amines from accessible and cheap alkanes.


Assuntos
Alcanos/metabolismo , Aminas Biogênicas/biossíntese , Escherichia coli , Aminação , Bacillales/enzimologia , Bacillales/genética , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Sistema Enzimático do Citocromo P-450/biossíntese , Sistema Enzimático do Citocromo P-450/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Streptomyces coelicolor/enzimologia , Streptomyces coelicolor/genética
11.
Appl Environ Microbiol ; 84(14)2018 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-29752270

RESUMO

Two Baeyer-Villiger monooxygenases (BVMOs), designated BoBVMO and AmBVMO, were discovered from Bradyrhizobium oligotrophicum and Aeromicrobium marinum, respectively. Both monooxygenases displayed novel features for catalyzing the asymmetric sulfoxidation of bulky and pharmaceutically relevant thioethers. Evolutionary relationship and sequence analysis revealed that the two BVMOs belong to the family of typical type I BVMOs and the subtype ethionamide monooxygenase. Both BVMOs are active toward medium- and long-chain aliphatic ketones as well as various thioether substrates but are ineffective toward cyclohexanone, aromatic ketones, and other typical BVMO substrates. BoBVMO and AmBVMO showed the highest activities (0.117 and 0.025 U/mg protein, respectively) toward thioanisole among the tested substrates. Furthermore, these BVMOs exhibited distinct activity and excellent stereoselectivity toward bulky and prochiral prazole thioethers, which is a unique feature of this family of BVMOs. No native enzyme has been reported for the asymmetric sulfoxidation of bulky prazole thioethers into chiral sulfoxides. The identification of BoBVMO and AmBVMO provides an important scaffold for discovering enzymes capable of asymmetrically oxidizing bulky thioether substrates by genome mining.IMPORTANCE Baeyer-Villiger monooxygenases (BVMOs) are valuable enzyme catalysts that are an alternative to the chemical Baeyer-Villiger oxidation reaction. Although BVMOs display broad substrate ranges, no native enzymes were reported to have activity toward the asymmetric oxidation of bulky prazole-like thioether substrates. Herein, we report the discovery of two type I BVMOs from Bradyrhizobium oligotrophicum (BoBVMO) and Aeromicrobium marinum (AmBVMO) which are able to catalyze the asymmetric sulfoxidation of bulky prazole thioethers (proton pump inhibitors [PPIs], a group of drugs whose main action is a pronounced and long-lasting reduction of gastric acid production). Efficient catalysis of omeprazole oxidation by BoBVMO was developed, indicating that this enzyme is a promising biocatalyst for the synthesis of bulky and pharmaceutically relevant chiral sulfoxide drugs. These results demonstrate that the newly identified enzymes are suitable templates for the discovery of more and better thioether-converting BVMOs.


Assuntos
Actinomycetales/enzimologia , Bradyrhizobium/enzimologia , Oxigenases de Função Mista/metabolismo , Sulfetos/metabolismo , Sulfóxidos/metabolismo , Sequência de Aminoácidos , Biocatálise , Clonagem Molecular , Cicloexanonas/metabolismo , Regulação Bacteriana da Expressão Gênica , Cetonas/metabolismo , Cinética , Oxigenases de Função Mista/classificação , Oxigenases de Função Mista/isolamento & purificação , Oxirredução , Filogenia , Alinhamento de Sequência , Análise de Sequência de Proteína , Especificidade por Substrato
12.
Appl Environ Microbiol ; 83(12)2017 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-28389544

RESUMO

Streptomyces coelicolor CR1 (ScCR1) has been shown to be a promising biocatalyst for the synthesis of an atorvastatin precursor, ethyl-(S)-4-chloro-3-hydroxybutyrate [(S)-CHBE]. However, limitations of ScCR1 observed for practical application include low activity and poor stability. In this work, protein engineering was employed to improve the catalytic efficiency and stability of ScCR1. First, the crystal structure of ScCR1 complexed with NADH and cosubstrate 2-propanol was solved, and the specific activity of ScCR1 was increased from 38.8 U/mg to 168 U/mg (ScCR1I158V/P168S) by structure-guided engineering. Second, directed evolution was performed to improve the stability using ScCR1I158V/P168S as a template, affording a triple mutant, ScCR1A60T/I158V/P168S, whose thermostability (T5015, defined as the temperature at which 50% of initial enzyme activity is lost following a heat treatment for 15 min) and substrate tolerance (C5015, defined as the concentration at which 50% of initial enzyme activity is lost following incubation for 15 min) were 6.2°C and 4.7-fold higher than those of the wild-type enzyme. Interestingly, the specific activity of the triple mutant was further increased to 260 U/mg. Protein modeling and docking analysis shed light on the origin of the improved activity and stability. In the asymmetric reduction of ethyl-4-chloro-3-oxobutyrate (COBE) on a 300-ml scale, 100 g/liter COBE could be completely converted by only 2 g/liter of lyophilized ScCR1A60T/I158V/P168S within 9 h, affording an excellent enantiomeric excess (ee) of >99% and a space-time yield of 255 g liter-1 day-1 These results suggest high efficiency of the protein engineering strategy and good potential of the resulting variant for efficient synthesis of the atorvastatin precursor.IMPORTANCE Application of the carbonyl reductase ScCR1 in asymmetrically synthesizing (S)-CHBE, a key precursor for the blockbuster drug Lipitor, from COBE has been hindered by its low catalytic activity and poor thermostability and substrate tolerance. In this work, protein engineering was employed to improve the catalytic efficiency and stability of ScCR1. The catalytic efficiency, thermostability, and substrate tolerance of ScCR1 were significantly improved by structure-guided engineering and directed evolution. The engineered ScCR1 may serve as a promising biocatalyst for the biosynthesis of (S)-CHBE, and the protein engineering strategy adopted in this work would serve as a useful approach for future engineering of other reductases toward potential application in organic synthesis.


Assuntos
Oxirredutases do Álcool/química , Oxirredutases do Álcool/genética , Atorvastatina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Engenharia de Proteínas , Streptomyces coelicolor/enzimologia , Oxirredutases do Álcool/metabolismo , Atorvastatina/química , Proteínas de Bactérias/metabolismo , Biocatálise , Estabilidade Enzimática , Hidroxibutiratos/metabolismo , Cinética , Modelos Moleculares , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Especificidade por Substrato
13.
Biotechnol Bioeng ; 113(11): 2350-7, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27216910

RESUMO

Malathion is one of the most widely used organophosphorus pesticides in the United States and developing countries. Herein, we enhanced the degradation rate of malathion starting with a phosphotriesterase PoOPHM2 while also considering thermostability. In the first step, iterative saturation mutagenesis at residues lining the binding pocket (CASTing) was employed to optimize the enzyme active site for substrate binding and activity. Hot spots for enhancing activity were then discovered through epPCR-based random mutagenesis, and these beneficial mutations were then recombined by DNA shuffling. Finally, guided by in silico energy calculations (FoldX), thermostability of the variant was improved. The mutations extend from the core region to the enzyme surface during the evolutionary pathway. After screening <9,000 mutants, the best variant PoOPHM9 showed 25-fold higher activity than wild-type PoOPHM2 , with a thermostability (T50 (15) ) of 67.6°C. Thus, PoOPHM9 appears to be an efficient and robust candidate for malathion detoxification. Biotechnol. Bioeng. 2016;113: 2350-2357. © 2016 Wiley Periodicals, Inc.


Assuntos
Evolução Molecular Direcionada/métodos , Malation/química , Simulação de Acoplamento Molecular/métodos , Mutagênese Sítio-Dirigida/métodos , Hidrolases de Triester Fosfórico/química , Hidrolases de Triester Fosfórico/genética , Sítios de Ligação , Biodegradação Ambiental , Técnicas de Química Combinatória/métodos , Ativação Enzimática/genética , Ligação Proteica , Engenharia de Proteínas/métodos , Relação Estrutura-Atividade
14.
Chembiochem ; 15(16): 2443-9, 2014 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-25236924

RESUMO

A new member of the CYP116B subfamily-P450LaMO-was discovered in Labrenzia aggregata by genomic data mining. It was successfully overexpressed in Escherichia coli, purified, and subsequently characterized spectroscopically, and its catalytic properties were assessed. Substrate profiling of the P450LaMO revealed that it was a versatile catalyst, exhibiting hydroxylation and epoxidation activities as well as O-dealkylation and asymmetric sulfoxidation activities. Diverse compounds, including alkylbenzenes, aromatic bicyclic molecules, and terpenoids, were shown to be hydroxylated by P450LaMO. Such diverse catalytic activities are uncommon for the bacterial P450s, and the P450LaMO-mediated stereoselective hydroxylation of inactivated C-H bonds-ubiquitous and relatively unreactive in organic molecules-is particularly unusual. The self-sufficient nature of P450LaMO, coupled with its broad substrate range, highlights it as an ideal template for directed evolution towards various applications.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Rhodobacteraceae/enzimologia , Sequência de Aminoácidos , Biocatálise , Sistema Enzimático do Citocromo P-450/genética , Escherichia coli/metabolismo , Hidroxilação , Dados de Sequência Molecular , Engenharia de Proteínas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Alinhamento de Sequência , Especificidade por Substrato
15.
Bioprocess Biosyst Eng ; 37(7): 1241-8, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24317427

RESUMO

Recombinant Escherichia coli cells expressing Alcaligenes sp. nitrilase were simply immobilized by direct cross-linking using glutaraldehyde. About 85 % of the total nitrilase activity was recovered under the optimal cross-linking conditions. The thermal stabilities of the cross-linked cells measured at 30, 40 and 50 °C were 4.5-, 5.3-, and 5.1-fold those of the free cells, respectively. The concentration of (R)-(-)-mandelic acid reached 280 mM after merely 2 h transformation with the immobilized cells using 300 mM mandelonitrile as substrate, affording an extremely high productivity of 510.7 g L(-1) d(-1). In addition, operational stability of the immobilized cells was obviously superior to that of free cells, without significant activity loss after 15 cycles of batch reactions or 8 cycles of repeated fed-batch reactions. Therefore, the easy preparation and robust characteristics of the immobilized biocatalyst make it a very promising biocatalyst for high-performance and low-cost production of optically pure (R)-(-)-mandelic acid.


Assuntos
Alcaligenes/enzimologia , Aminoidrolases/química , Escherichia coli/metabolismo , Glutaral/química , Ácidos Mandélicos/química , Acetonitrilas/química , Catálise , Reagentes de Ligações Cruzadas/química , Relação Dose-Resposta a Droga , Enzimas/química , Enzimas Imobilizadas , Concentração de Íons de Hidrogênio , Hidrólise , Temperatura , Fatores de Tempo
16.
ChemSusChem ; 17(6): e202400204, 2024 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-38369946

RESUMO

Invited for this issue's cover is the group of Huilei Yu at the East China University of Science and Technology. The image shows a sustainable biosynthesis route to nylon monomers from bio-based substrate α, ω-dicarboxylic acids. The Research Article itself is available at 10.1002/cssc.202301477.


Assuntos
Diaminas , Ácidos Graxos , Aminoácidos , China
17.
ChemSusChem ; 17(6): e202301477, 2024 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-38117609

RESUMO

Aliphatic ω-amino fatty acids (ω-AFAs) and α,ω-diamines (α,ω-DMs) are essential monomers for the production of nylons. Development of a sustainable biosynthesis route for ω-AFAs and α,ω-DMs is crucial in addressing the challenges posed by climate change. Herein, we constructed an unprecedented thermodynamically favorable multi-enzyme cascade (TherFavMEC) for the efficient sustainable biosynthesis of ω-AFAs and α,ω-DMs from cheap α,ω-dicarboxylic acids (α,ω-DAs). This TherFavMEC was developed by incorporating bioretrosynthesis analysis tools, reaction Gibbs free energy calculations, thermodynamic equilibrium shift strategies and cofactor (NADPH&ATP) regeneration systems. The molar yield of 6-aminohexanoic acid (6-ACA) from adipic acid (AA) was 92.3 %, while the molar yield from 6-ACA to 1,6-hexanediamine (1,6-HMD) was 96.1 %, which were significantly higher than those of previously reported routes. Furthermore, the biosynthesis of ω-AFAs and α,ω-DMs from 20.0 mM α,ω-DAs (C6-C9) was also performed, giving 11.2 mM 1,6-HMD (56.0 % yield), 14.8 mM 1,7-heptanediamine (74.0 % yield), 17.4 mM 1,8-octanediamine (87.0 % yield), and 19.7 mM 1,9-nonanediamine (98.5 % yield), respectively. The titers of 1,9-nonanediamine, 1,8-octanediamine, 1,7-heptanediamine and 1,6-HMD were improved by 328-fold, 1740-fold, 87-fold and 3.8-fold compared to previous work. Therefore, this work holds great potential for the bioproduction of ω-AFAs and α,ω-DMs.


Assuntos
Aminoácidos , Diaminas , Ácidos Dicarboxílicos , Ácidos Graxos
18.
Appl Microbiol Biotechnol ; 97(14): 6293-300, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23143530

RESUMO

A new strain, Enterobacter sp. ECU1107, was identified among over 200 soil isolates using a two-step screening strategy for the enantioselective synthesis of (2S,3R)-3-phenylglycidate methyl ester (PGM), a key intermediate for production of a potent anticancer drug Taxol®. An organic-aqueous biphasic system was employed to reduce spontaneous hydrolysis of the substrate PGM and isooctane was found to be the most suitable organic solvent. The temperature and pH optima of the whole cell-mediated bioreaction were 40 °C and 6.0, respectively. Under these reaction conditions, the enantiomeric excess (ee(s)) of (2S,3R)-PGM recovered was greater than 99 % at approximately 50 % conversion. The total substrate loading in batch reaction could reach 600 mM. By using whole cells of Enterobacter sp. ECU1107, (2S,3R)-PGM was successfully prepared in decagram scale in a 1.0-l mechanically stirred reactor, affording the chiral epoxy ester in >99 % ee s and 43.5 % molar yield based on the initial load of racemic substrate.


Assuntos
Antineoplásicos/metabolismo , Proteínas de Bactérias/metabolismo , Enterobacter/enzimologia , Esterases/metabolismo , Paclitaxel/biossíntese , Propionatos/metabolismo , Antineoplásicos/química , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biocatálise , Enterobacter/classificação , Enterobacter/genética , Enterobacter/isolamento & purificação , Estabilidade Enzimática , Esterases/química , Esterases/genética , Cinética , Dados de Sequência Molecular , Estrutura Molecular , Paclitaxel/química , Filogenia , Microbiologia do Solo , Estereoisomerismo
19.
Appl Microbiol Biotechnol ; 97(24): 10329-37, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24092008

RESUMO

Enantiopure sulfoxides can be prepared via the asymmetric oxidation of sulfides using sulfide monooxygenases. The n-octane-water biphasic system was chosen for the bio-oxidation of a water-insoluble phenyl methyl sulfide (PMS) by Rhodococcus sp. CCZU10-1. In this n-octane-water system, the optimum reaction conditions were obtained. (S)-phenyl methyl sulfoxide ((S)-PMSO) with >99.9 % enantiomeric excess formed at 55.3 mM in the n-octane-water biphasic system. Using fed-batch method, a total of 118 mM (S)-PMSO accumulated in 1-L reaction mixture after the 7th feed, and no (R)-PMSO and sulfone were detected. Moreover, Rhodococcus sp. CCZU10-1 displayed fairly good activity and enantioselectivity toward other sulfides. In conclusion, Rhodococcus sp. CCZU10-1 is a promising biocatalyst for synthesizing highly optically active sulfoxides.


Assuntos
Octanos , Rhodococcus/metabolismo , Solventes , Sulfetos/metabolismo , Sulfóxidos/metabolismo , Água , Biotransformação , Oxirredução , Estereoisomerismo
20.
Chem Commun (Camb) ; 59(98): 14571-14574, 2023 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-37987314

RESUMO

Baeyer-Villiger monooxygenases (BVMOs) are able to catalyse the asymmetric oxidation of sulfides. This property has made them attractive catalysts for the synthesis of chiral sulfoxide drugs. Here, we have designed and synthesised an exhaustive combinatorial mutant library of the previously identified lansoprazole sulfide monooxygenase CbBVMOV1. From this synthetic combinatorial mutant library, the best mutant, CbBVMOV3, was selected with a specific activity of approximately 1 U mg-1 for lansoprazole sulfoxides. We then optimised the reaction conditions of a two-phase system, achieving the enzymatic asymmetric synthesis of (R)-lansoprazole in a space-time yield of 213 g L-1 d-1 and an enantiomeric excess of >99% (R) with no detectable by-products. In addition, CbBVMOV3 showed higher activity towards other prazole sulfides. These results indicate the potential application of CbBVMO in the chiral sulfoxide drug industry.


Assuntos
Oxigenases de Função Mista , Sulfóxidos , Oxigenases de Função Mista/metabolismo , Oxirredução , Sulfetos , Lansoprazol
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