Hui-Lei Yu

A thermostable and organic-solvent tolerant esterase from Pseudomonas putida ECU1011: Catalytic properties and performance in kinetic resolution of α-hydroxy acids

A novel esterase, rPPE01, from Pseudomonas putida ECU1011 was heterologously expressed in Escherichia coli and identified for enzymatic resolution of hydroxy acids via O-deacetylation. α-Acetoxy carboxylates were converted with approximately 50% yield and excellent enantioselectivity (E>200) at a substrate concentration of 100 mM. The half-lives of rPPE01 were 14 days at 50°C and 30 days at 30°C, indicating the enzyme has relatively high thermostability. Another remarkable advantage of rPPE01 is that both the activity and thermostability were enhanced significantly in the presence of hydrophobic alkanes and ethers. rPPE01 retained 159% of its initial activity after incubation with 50% (v/v) n-heptane at 30°C for 60 days. The attractive organic-solvent tolerance, good thermostability and high enantioselectivity towards α-acetoxy carboxylates endow rPPE01 with the potential of practical application for the production of enantiopure hydroxy acids.

An efficient bioprocess for enzymatic production of l-menthol with high ratio of substrate to catalyst using whole cells of recombinant E. coli

A gene encoding an esterase of Bacillus subtilis ECU0554 previously isolated from soil was cloned and overexpressed in Escherichia coli BL21. The recombinant esterase (recBsE) showed the best enantioselectivity (E>100) towards DL-menthyl acetate, in contrast to DL-menthyl esters propionate and butyrate. A high ratio of substrate to catalyst (S/C-ratio, ≥50) was achieved in the kinetic resolution of DL-menthyl acetate by using whole cells of recombinant E. coli BL21. Some key parameters of the biocatalytic process, including amount of cosolvent, catalyst loading and substrate loading, were optimized. Compared with the process catalyzed by wild-type whole cells of B. subtilis ECU0554, the second-generation bioprocess using whole cells of recombinant E. coli BL21 afforded a 40-fold improvement in S/C-ratio and a 75-fold improvement in the volumetric productivity per biocatalyst loading. Moreover, the substrate loading was increased up to 200 g L(-1) (∼1 M), the biocatalyst loading was reduced to 2.5 g L(-1) and the space-time yield was improved from 54 g L(-1) d(-1) to 202 g L(-1) d(-1).

A new thermostable β-glucosidase mined from Dictyoglomus thermophilum: properties and performance in octyl glucoside synthesis at high temperatures.

A new β-glucosidase (DtGH) representing 40% identity with an apple seed glycosidase (ASG) was cloned from Dictyoglomus thermophilum. DtGH showed extremely high thermostability in aqueous solution, with half-lives of 533, 44, and 5 h measured at 70, 80 and 90 °C, respectively. Therefore it was used for direct glycosylation of n-octanol at 70 °C instead of 50 °C as usually. As a result, the glucose based conversion was increased by 27%, but the time spent to reach equilibrium was decreased from 7 d to 3 d. This enzyme also exhibited excellent stability under the reaction environment, retaining 70-80% of its initial activity after 7 d of incubation at 70 °C in either 1.7 M glucose solution or octanol-aqueous (85:15, v/v) system. It could retain part of synthetic activity even in boiling water. Owing to the strong glucose-tolerance and extremely high thermostability, DtGH should be promising for various glucosides synthesis.

Highly Efficient Synthesis of (R)-3-Quinuclidinol in a Space–Time Yield of 916 g L–1 d–1 Using a New Bacterial Reductase ArQR

A new keto reductase (ArQR), identified from Agrobacterium radiobacter ECU2556, can efficiently reduce 3-quinuclidinone in excellent enantioselectivity and high space-time yield for the synthesis of (R)-3-quinuclidinol, a chiral building block of many antimuscarinic agents. This is the first time that a high yield of (R)-3-quinuclidinol up to 916 g L(-1) d(-1) using a bioreduction approach is reported.

Stereocomplementary Bioreduction of β-Ketonitrile without Ethylated Byproduct

α-Ethylation is competing with the biocatalytic reduction of aromatic β-ketonitriles in a whole-cell system. Use of two newly mined robust and stereocomplementary carbonyl reductases in a biphasic system has completely eliminated the competing byproduct. For the first time, both enantiomers of fluoroxetine precursors were obtained at 0.5 M with >99% ee and excellent chemoselectivity, without addition of any external cofactors.

Resolution of racemic sulfoxides with high productivity and enantioselectivity by a Rhodococcus sp. strain as an alternative to biooxidation of prochiral sulfides for efficient production of enantiopure sulfoxides

Whole cells of Rhodococcus sp. ECU0066 were used a catalyst for resolution of racemic sulfoxides, as an alternative to asymmetric oxidation of sulfides for efficient production of enantiopure sulfoxides. Racemic sulfoxides were excellent substrates for biotransformation because of their lower biotoxicity compared to sulfides. Determination of apparent kinetic parameters indicated that phenyl methyl sulfide (PMS), but not racemic phenyl methyl sulfoxide (rac-PMSO) caused substrate inhibition. (S)-PMSO was formed at a higher concentration and good enantiomeric excess (37.8 mM and 93.7% ee(S)) in a fed-batch reaction, than by an asymmetric oxidation of PMS (10 mM and 80% eeP (S)). The bacterium also displayed fairly good activity (yields, 22.7-43.2%; within 1-8 h) and enantioselectivity (ee(S)>99.0%) towards para-substituted (methyl and chloro) phenyl methyl sulfoxides and ethyl phenyl sulfoxide, indicating it could be a promising agent for synthetic applications.

An ene reductase from Clavispora lusitaniae for asymmetric reduction of activated alkenes

A putative ene reductase gene from Clavispora lusitaniae was heterologously overexpressed in Escherichia coli, and the encoded protein (ClER) was purified and characterized for its biocatalytic properties. This NADPH-dependent flavoprotein was identified with reduction activities toward a diverse range of activated alkenes including conjugated enones, enals, maleimide derivative and α,β-unsaturated carboxylic esters. The purified ClER exhibited a relatively high activity of 7.3 U mg(prot)⁻¹ for ketoisophorone while a remarkable catalytic efficiency (k(cat)/K(m)=810 s⁻¹ mM⁻¹) was obtained for 2-methyl-cinnamaldehyde due to the high affinity. A series of prochiral activated alkenes were stereoselectively reduced by ClER furnishing the corresponding saturated products in up to 99% ee. The practical applicability of ClER was further evaluated for the production of (R)-levodione, a valuable chiral compound, from ketoisophorone. Using the crude enzyme of ClER and glucose dehydrogenase (GDH), 500 mM of ketoisophorone was efficiently converted to (R)-levodione with excellent stereoselectivity (98% ee) within 1h. All these positive features demonstrate a high synthetic potential of ClER in the asymmetric reduction of activated alkenes.

Genomic Data Mining: An Efficient Way to Find New and Better Enzymes

It is top priority nowadays for biocatalysis researchers to discover novel, potent enzymes and to redesign and engineer for tailor-made enzymes. Genomic data mining, which depends on the burgeoning computational algorithms and bioinformatics tools, accelerates the process by in silico screening and constructing focused/smart mutant libraries.

Combinatorial evolution of phosphotriesterase toward a robust malathion degrader by hierarchical iteration mutagenesis

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 (T5015) of 67.6°C. Thus, PoOPHM9 appears to be an efficient and robust candidate for malathion detoxification. Biotechnol. Bioeng. 2016;113: 2350–2357.

Substrate channel evolution of an esterase for the synthesis of cilastatin

The esterase RhEst1 from Rhodococcus sp. ECU1013 has been reported for the enantioselective hydrolysis of ethyl (S)-(+)-2,2-dimethylcyclopropane carboxylate, producing the building block of cilastatin. In this work, error-prone PCR and site-directed saturation mutagenesis were applied to RhEst1 for activity improvement, with the pH-indicator assay as a high-throughput screening method. As a result, RhEst1A147I/V148F/G254A, with mutations surrounding the substrate access channel, showed a 5-fold increase in its specific activity compared with the native enzyme, as well as a 4-fold increase in protein solubility. Combined with the determination of protein structures and computational analysis, this work shows that the amino acids around the substrate channel play a more important role in the activity evolution of RhEst1 than those in the active site.