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Artificial biorefinery of oleic acid into 1,10-decanedioic acid represents a revolutionizing route to the sustainable production of chemically difficult-to-make bifunctional chemicals. However, the carbon atom economy is extremely low (56%) due to the formation of unifunctional

Background (−)-Limonene, one of cyclic monoterpenes, is an important renewable compound used widely as a key building block for the synthesis of new biologically active molecules and fine chemicals. (−)-Perillamine, as derived from (−)-limonene, is such a useful synthon for constructing more complicated and functionally relevant chemicals. Aim We aimed to report a more sustainable and more efficient method for the regiospecific C-H amination of (−)-limonene into (−)-perillamine. Results Here we report an artificial penta-enzymatic cascade system for the transformation of the cheap and easily available (−)-limonene into (−)-perillamine for the first time. This system is composed of cytochrome P450 monooxygenase, alcohol dehydrogenase and w-transaminase for the main reactions, as well as formate dehydrogenase and NADH oxidase for cofactor recycling. After optimization of the multi-enzymatic cascade system, 10 mM (−)-limonene was smoothly converted into 5.4 mM (−)-perillamine in a one-pot-two-step biotransformation, indicating the feasibility of multi-enzymatic C7-regiospecific amination of the inert C-H bond of (−)-limonene. This method represents a concise and efficient route for the biocatalytic synthesis of derivatives from similar natural products.

Oxidoreductase-mediated biotransformation often requires consumption of a secondary sacrificial co-substrate and an additional auxiliary enzyme to drive the cofactor regeneration, which results in generation of unwanted by-product. Herein, we report a highly atom-economic self-sufficient hydride-shuttling cascade to concurrently obtain two pharmaceutically important building blocks (7,12-dioxo-lithocholic acid and L-tert-leucine) in which oxidation of cholic acid (CA) and reductive amination of trimethylpyruvic acid were integrated for redox self-recycling. In this cascade, the cofactor acts as a hydride shuttle that interconnects the two synthetically relevant reactions at the cost of only inorganic ammonium as the sacrificial agent and generates water as the greenest by-product. The preparative biotransformation using a whole-cell biocatalyst in the absence of any exogenous cofactor displayed a space–time yield of 768 g L−1 d−1 and a total turnover number (TTN) of 20 363 for NAD+ recycling. This represents the highest cofactor TTN reported to date for the bio-oxidation of CA, indicating the great potential of this cofactor and redox self-sufficient bioprocess for cost-effective and sustainable biomanufacturing of high-value-added products.

Baeyer-Villiger monooxygenases (BVMOs) are remarkable biocatalysts for the Baeyer-Villiger oxidation of ketones to generate esters or lactones. The regioselectivity of BVMOs is essential for determining the ratio of the two regioisomeric products ("normal" and "abnormal") when catalyzing asymmetric ketone substrates. Starting from a known normal-preferring BVMO sequence from Pseudomonas putida KT2440 (PpBVMO), a novel BVMO from Gordonia sihwensis (GsBVMO) with higher normal regioselectivity (up to 97/3) was identified. Furthermore, protein engineering increased the specificity constant (kcat /KM ) 8.9-fold to 484 s-1 mM-1 for 10-ketostearic acid derived from oleic acid. Consequently, by using the variant GsBVMOC308L as an efficient biocatalyst, 10-ketostearic acid was efficiently transformed into 9-(nonanoyloxy)nonanoic acid, with a space-time yield of 60.5 g L-1 d-1 . This study showed that the mutant with higher regioselectivity and catalytic efficiency could be applied to prepare medium-chain ω-hydroxy fatty acids through biotransformation of long-chain aliphatic keto acids derived from renewable plant oils.

Glucose dehydrogenase (GDH) is a general tool for driving nicotinamide (NAD(P)H) regeneration in synthetic biochemistry. An increasing number of synthetic bioreactions are carried out in media containing high amounts of organic cosolvents or hydrophobic substrates/products, which often denature native enzymes, including those for cofactor regeneration. In this work, we attempted to improve the chemical stability of Bacillus megaterium GDH (BmGDHM0 ) in the presence of large amounts of 1-phenylethanol by directed evolution. Among the resulting mutants, BmGDHM6 (Q252L/E170K/S100P/K166R/V72I/K137R) exhibited a 9.2-fold increase in tolerance against 10 % (v/v) 1-phenylethanol. Moreover, BmGDHM6 was also more stable than BmGDHM0 when exposed to hydrophobic and enzyme-inactivating compounds such as acetophenone, ethyl 2-oxo-4-phenylbutyrate, and ethyl (R)-2-hydroxy-4-phenylbutyrate. Coupled with a Candida glabrata carbonyl reductase, BmGDHM6 was successfully used for the asymmetric reduction of deactivating ethyl 2-oxo-4-phenylbutyrate with total turnover number of 1800 for the nicotinamide cofactor, thus making it attractive for commercial application. Overall, the evolution of chemically robust GDH facilitates its wider use as a general tool for NAD(P)H regeneration in biocatalysis.

Microbial synthesis of plant-based (-)-menthol is of great interest because of its high demand (≈30 kiloton per year) as well as unique odor and cooling characteristics. However, this remains a great challenge due to the yet unfilled gap between (-)-limonene and (+)-cis-isopulegone. Herein, the first artificial and effective system was developed for (+)-cis-isopulegone biosynthesis from (-)-limonene by recruiting two bacterial enzymes to replace their inefficient counterparts from Mentha piperita, limonene-3-hydroxylase, and isopiperitenol dehydrogenase. A cofactor self-regenerative recombinant Escherichia coli strain was constructed by introducing a formate dehydrogenase for nicotinamide adenine dinucleotide phosphate (NADPH) regeneration and an engineered microbial isopiperitenol dehydrogenase. The production of (+)-cis-isopulegone (up to 281.2 mg L-1 ) was improved by 36 times compared with that of the initial strain. This work lays a reliable foundation for the microbial synthesis of (-)-menthol.

Dehydrogenases are widely employed as biocatalysts for the production of optically pure chemicals under mild conditions. Most dehydrogenases are nicotinamide cofactor (NADPH or NADH)-dependent oxid...

The asymmetric reductive amination of ketones with ammonia using engineered amine dehydrogenases (AmDHs) is a particularly attractive and environmentally friendly method for the synthesis of chiral amines. However, one major challenge for these engineered AmDHs is their limited range of accepted substrates. Herein, several engineered AmDHs were developed through the evolution of naturally occurring leucine dehydrogenases, which displayed good amination activity toward aliphatic ketones but restricted catalytic scope for short-chain substrates. Computational analysis helped identify two residues, located at the distal end of the substrate-binding cavity, that generate steric hindrance and prevent the binding of bulky aliphatic ketones. By fine-tuning these two key hotspots, the resulting AmDH mutants are able to accept previously inaccessible bulky substrates. More importantly, the mutations were also proved applicable for expanding the substrate scope of other homologous AmDHs with sequence identities as ...

A novel carbonyl reductase from Serratia marcescens , Sm CR, was successfully cloned and overexpressed in Escherichia coli . Sm CR could catalyze the asymmetric reduction of long-chain keto acids/esters containing remote carbonyl groups, such as 4-oxo- and 5-oxodecanoic acids, yielding chiral γ- and δ-decalactones with high enantiopurity (up to 99% ee ). This is the first report of enzymatic synthesis of ( R )-γ- and ( R )-δ-decalactones starting from γ-, δ-keto acids using free enzymes.

The biodegradation of pesticides by organophosphorus hydrolases (OPHs) requires an efficient enzyme production technology in industry. Herein, a Pichia pastoris strain was constructed for the extracellular expression of PoOPHM9, an engineered malathion-degrading enzyme. After optimization, the maximum titer and yield of fermentation reached 50.8 kU/L and 4.1 gprotein/L after 3 days, with the highest space-time yield (STY) reported so far, 640 U L–1 h–1. PoOPHM9 displayed its high activity and stability in the presence of 0.1% (w/w) plant-derived detergent. Only 0.04 mg/mL enzyme could completely remove 0.15 mM malathion in aqueous solution within 20 min. Furthermore, 12 μmol malathion on apples and cucumbers surfaces was completely removed by 0.05 mg/mL PoOPHM9 in tap water after 35 min washing. The efficient production of the highly active PoOPHM9 has cleared a major barrier to biodegradation of pesticide residues in food industry.

Ketoreductases are tools for the synthesis of chiral alcohols in industry. However, the low activity of natural enzymes often restricts their use in industrial applications. On the basis of computational analysis and previous reports, two residues (F92 and F94) probably affecting the activity of ketoreductase CgKR1 were identified. By tuning these two residues, the CgKR1-F92C/F94W variant was obtained that exhibited higher activity toward all 28 structurally diverse substrates examined than the wild-type enzyme. Among them, 13 substrates have a specific activity over 50 U mg–1 (54–775 U mg–1). Using CgKR1-F92C/F94W as a catalyst, five substrates at high loading (>100 g–1 L–1) were reduced completely in gram-scale preparative reactions. This approach provides accesses to pharmaceutically relevant chiral alcohols with high enantioselectivity (up to 99.0% ee) and high space-time yield (up to 583 g–1 L–1 day–1). Molecular dynamics simulations highlighted the crucial role of residues 92 and 94 in activity impr...

Ursodeoxycholic acid (UDCA) is the main active ingredient of natural bear bile powder with multiple pharmacological functions. 7β-Hydroxysteroid dehydrogenase (HSDH) is a key biocatalyst for the synthesis of UDCA. However, all the 7β-HSDHs reported commonly suffer from poor activity and thermostability, resulting in limited productivity of UDCA. In this study, a multiobjective directed evolution (MODE) strategy was proposed and applied to improve the activity, thermostability, and pH optimum of a 7β-HSDH. The best variant (V3–1) showed a specific activity 5.5-fold higher than and a half-life 3-fold longer than those of the wild type. In addition, the pH optimum of the variant was shifted to a weakly alkaline value. In the cascade reaction, the productivity of UDCA with V3–1 increased to 942 g L–1 day–1, in contrast to 141 g L–1 day–1 with the wild type. Therefore, this study provides a useful strategy for improving the catalytic efficiency of a key enzyme that significantly facilitated the bioproduction o...

Baeyer-Villiger monooxygenases (BVMOs) are an emerging class of promising biocatalysts for the oxidation of ketones to prepare corresponding esters or lactones. Although many BVMOs have been reported, the development of highly efficient enzymes for use in industrial applications is desirable. In this work, we identified a BVMO from Rhodococcus pyridinivorans (BVMO(Rp)) with a high affinity toward aliphatic methyl ketones (K(m) < 3.0 μM). The enzyme was highly soluble and relatively stable, with a half-life of 23 h at 30°C and pH 7.5. The most effective substrate discovered so far is 2-hexanone (k(cat) = 2.1 s(−1); K(m) = 1.5 μM). Furthermore, BVMO(Rp) exhibited excellent regioselectivity toward most aliphatic ketones, preferentially forming typical (i.e., normal) products. Using the newly identified BVMO(Rp) as the catalyst, a high concentration (26.0 g/liter; 200 mM) of methyl levulinate was completely converted to methyl 3-acetoxypropionate after 4 h, with a space-time yield of 5.4 g liter(−1) h(−1). Thus, BVMO(Rp) is a promising biocatalyst for the synthesis of 3-hydroxypropionate from readily available biobased levulinate to replace the conventional fermentation. IMPORTANCE BVMOs are emerging as a green alternative to traditional oxidants in the BV oxidation of ketones. Although many BVMOs are discovered and used in organic synthesis, few are really applied in industry, especially in the case of aliphatic ketones. Herein, a highly soluble and relatively stable monooxygenase from Rhodococcus pyridinivorans (BVMO(Rp)) was identified with high activity and excellent regioselectivity toward most aliphatic ketones. BVMO(Rp) possesses unusually high substrate loading during the catalysis of the oxidation of biobased methyl levulinate to 3-hydroxypropionic acid derivatives. This study indicates that the synthesis of 3-hydroxypropionate from readily available biobased levulinate by BVMO(Rp)-catalyzed oxidation holds great promise to replace traditional fermentation.

A methyl parathion hydrolase (MPH) gene, bjmpd, was cloned from a newly isolated MP-degrading bacterial strain, Burkholderia jiangsuensis MP-1T and heterologously expressed in Escherichia coli BL21 (DE3). Although the amino acid sequence of the bjmpd-encoded enzyme, named BjMPH, differed from that of MPH from Pseudomonas sp. WBC-3 (PsMPH) in only three residues, Ser132, Val247 and Ala267, a significantly higher specific activity towards MP was exhibited by BjMPH than PsMPH. Among them, Ala267 was identified as a key site affecting the catalytic efficiency, and it was rather conservative (Ala or Ser) in homologous proteins, suggesting that a simple substitution of the residue in conservative site with another conservative residue based on the consensus sequence approach might possibly enhance the catalytic efficiency of the MP-degrading enzyme. Inspired by such an observation, we identified a new mutant, BjMPHT64N, exhibiting 3.78-fold higher catalytic efficiency (kcat/KM) towards MP than its wild-type, reaching 4.20×106M-1s-1. The mutant BjMPHT64N also displayed enhanced reactivities (kcat/KM) towards other organophosphorus pesticides. Homology-modelling analysis indicates that enhanced polar contacts of the 64th residue in this mutant may contribute to stabilizing the structure of the enzyme and promote the interactions between enzyme and substrate. This study generated an efficient MP-degrading enzyme, and provides useful information for enhancing the catalytic efficiency of MPHs via conservative residue substitution based on the consensus approach.

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

Baeyer-Villiger monooxygenases (BVMOs) are versatile biocatalysts in organic synthesis that can generate esters or lactones by inserting a single oxygen atom adjacent to a carbonyl moiety. The regioselectivity of BVMOs is essential in determining the ratio of two regioisomers for converting asymmetric ketones. Herein, we report a novel BVMO from Pseudomonas aeruginosa (PaBVMO); this has been exploited for the direct synthesis of medium-chain α,ω-dicarboxylic acids through a Baeyer-Villiger oxidation-hydrolysis cascade. PaBVMO displayed the highest abnormal regioselectivity toward a variety of long-chain aliphatic keto acids (C16 -C20 ) to date, affording dicarboxylic monoesters with a ratio of up to 95 %. Upon chemical hydrolysis, α,ω-dicarboxylic acids and fatty alcohols are readily obtained without further treatment; this significantly reduces the synthetic steps of α,ω-dicarboxylic acids from renewable oils and fats.

A novel (R)-imine reductase (PlRIR) from Paenibacillus lactis was heterologously overexpressed in Escherichia coli, purified and characterized. The purified PlRIR exhibited relatively high catalytic efficiency (kcat/Km=1.58 s−1 mm−1) towards 2,3,3-trimethylindolenine. A panel of 3H-indoles and 3H-indole iodides were reduced by PlRIR to yield the corresponding products with good-to-excellent enantioselectivity (66–98 % ee). In addition, PlRIR also possesses good activities toward other types of imines such as pyrroline, tetrahydropyridine, and dihydroisoquinoline, indicating a reasonably broad substrate acceptance. In a 100 mg scale preparative reaction, 100 mm 2,3,3-trimethylindolenine was converted efficiently to afford (R)-2,3,3-trimethylindoline with 96 % ee and 81 % yield.

Structure-based directed evolution has been successfully applied to BSEV4, a variant of Bacillus subtilis esterase, for enantioselective hydrolysis of DL-menthyl esters with elevated thermostability. BSEV4 displayed only 90% enantiomeric excess (ee) in preparing L-menthol from DL-menthyl benzoate, whereas the quadruple mutant BSEV7, produced by iterative saturation mutagenesis, showed >99% ee. Partial deconvolution of BSEV7 by generating the four respective single mutants indicated that each of them only improved the enantioselectivity slightly, implying pronounced synergy when the mutations act together. BSEV9 and BSEV11, produced by site-directed mutagenesis and random mutagenesis, respectively, showed 2.5- and 20-fold higher activities than BSEV7 while retaining high E-values. With merely 0.1 g L−1 of BSEV11 loading (cell-free extract), 130 g L−1 of DL-menthyl benzoate was asymmetrically hydrolyzed within 6 h, resulting in an enantiopurity of >99% ee and a space-time yield of 138 g L−1 d−1. This suggests that it is feasible to establish an efficient bioprocess by simultaneously addressing multiple issues of biocatalyst performance using the iterative multi-target evolution approach.

7β-Hydroxysteroid dehydrogenase (7β-HSDH) is a key enzyme for the efficient biosynthesis of ursodeoxycholic acid (UDCA), an effective pharmaceutical for primary biliary cirrhosis and human cholesterol gallstones. In this work, a new 7β-HSDH from Ruminococcus torques ATCC 35915 , designated as 7β-HSDH Rt , was identified and heterologously overexpressed in Escherichia coli for the enzymatic synthesis of ursodeoxycholic acid from chenodeoxycholic acid (CDCA). 7β-HSDH Rt was firstly employed in one-pot mode together with 7α-HSDH Ca , another NADPH-dependent 7α-HSDH from Clostridium absonum , to convert CDCA into UDCA without additional coenzyme regeneration. However, the final yield was limited to merely 73%, probably due to chemical equilibrium. Therefore, to enhance the UDCA yield, we alternatively adopted a two-step reaction strategy where the enzymes involved in the first reaction were simply heat-inactivated between the 1st-step reaction (dehydrogenation) and the 2nd-step reaction (hydrogenation), in order to prevent the undesired bioreduction of 7-oxo-LCA into CDCA in the 2nd step. Consequently, the analytic yield of UDCA was significantly improved up to above 98% at a substrate load of 10 mM ( ca. 4 g L −1 ), without any detectable intermediate (7-oxo-LCA) as observed in the case of one-pot reaction.

An eco-friendly and convenient preparation method for notoginsenoside ST-4 has been established by completely transforming vina-ginsenoside R7 using a recombinant glycosidase hydrolyzing enzyme (HaGH03) from Herpetosiphon aurantiacus. This enzyme specifically hydrolyzed the glucose at the C-20 position but not the external xylose or two inner glucoses at position C-3. Protein sequence BLAST revealed that HaGH03, composed of 749 amino acids and presumptively listed as a member of the family 3 glycoside hydrolases, has highest identity (48 %) identity with a thermostable β-glucosidase B, which was not known of any functions for ginsenoside transformation. The steady state kinetic parameters for purified HaGH03 measured against p-nitrophenyl β-D-glucopyranoside and vina-ginsenoside R7 were K M = 5.67 ± 0.24 μM and 0.59 ± 0.23 mM, and k cat = 69.2 ± 0.31/s and 2.15 ± 0.46/min, respectively. HaGH03 converted 2.5 mg/mL of vina-ginsenoside R7 to ST-4 with a molar yield of 100 % and a space-time yield of 104 mg/L/h in optimized conditions. These results underscore that HaGH03 has much potential for the effective preparation of target ginsenosides possessing valuable pharmacological activities. This is the first report identifying an enzyme that has the ability to transform vina-ginsenoside R7 and provides an approach to preparing rare notoginsenoside ST-4.

Ursodeoxycholic acid (UDCA) is an effective drug for the treatment of hepatitis. In this study, 7α-hydroxysteroid dehydrogenase (7α-HSDH) and lactate dehydrogenase (LDH), as well as 7β-HSDH and glucose dehydrogenase (GDH), were co-immobilized onto an epoxy-functionalized resin (ES-103) for catalyzing the synthesis of UDCA from chenodeoxycholic acid (CDCA). Through optimizing the immobilization pH, time and the loading ratio of enzymes to resin, the specific activities of immobilized LDH-7αHSDH@ES-103 and 7βHSDH-GDH@ES-103 were 43.2 U g-1 and 25.8 U g-1, respectively, which are 12- and 516-fold higher than that under the initial immobilization conditions. Continuous production of UDCA from CDCA was subsequently achieved by using immobilized LDH-7αHSDH@ES-103 and 7βHSDH-GDH@ES-103 in two serial packed bed reactors. The yield of UDCA reached nearly 100% and lasted for at least 12 h in the packed bed reactors, which is superior to the batch-wise reaction. This efficient continuous approach developed in this study may provide a feasible route for large-scale biotransformation of CDCA into UDCA.

The lipase isolated from Serratia marcescens (LipA) is a useful biocatalyst for kinetic resolution of a pharmaceutically relevant epoxyester, (±)-3-(4'-methoxyphenyl) glycidic acid methyl ester [(±)-MPGM], to afford optically pure (-)-MPGM, a key intermediate for the synthesis of diltiazem hydrochloride. Two mutants, LipAL315S and LipAS271F, were identified from the combinatorial saturation mutation library of 14 amino acid residues lining the substrate-binding pocket. LipAL315S, LipAS271F, and their combination LipAL315S/S271F showed 2.6-, 2.2-, and 4.6-fold improvements in their specific activities towards para-nitrophenyl butyrate (pNPB), respectively. Among these positive mutants, LipAS271F displayed a 3.5-fold higher specific activity towards the pharmaco substrate (±)-MPGM. Kinetic study showed that the improvement in catalytic efficiency of LipAS271F against (±)-MPGM was mainly resulted from the enhanced affinity between substrate and enzyme, as indicated by the decrease of K m. Furthermore, to address the insoluble expression issue in Escherichia coli, the homologous expression of LipA gene in S. marcescens was achieved by introducing it into an expression vector pUC18, resulting in ca. 20-fold higher lipase production. The significantly improved volumeric production and specific activity of S. marcescens lipase make it very attractive as a new-generation biocatalyst for more efficient and economical manufacturing of (-)-MPGM.

Network failures are common on the Internet, and with mission-critical services widely applied, there grows demand for the Internet to maintain the performance in possibilities of failures. However, the border gateway protocol (BGP) can not react quickly to be recovered from them, which leads to unreliable packet delivery degrading the end-to-end performance. Although much solutions were proposed to address the problem, there exist limitations. The authors designed a software defined autonomous system (AS)-level fast rerouting (SD-FRR) to efficiently recover from interdomain link failures in the administrative domain. The approach leverages the principle of software defined networking (SDN) to achieve the centralized control of the entire network. By considering routing policies and BGP decision rules, an algorithm that can automatically find a policy-compliant protection path in case of link failure was proposed. The OpenFlow forwarding rules are installed on routers to ensure data forwarding. Furthermore, to deactivate the protection path, how to remove flow entries based on prefixes was proposed. Experiments show that the proposal provides effective failure recovery and does not introduce significant control overhead to the network.

Bacillus subtilis esterase (BSE) exhibits high activity, extraordinary substrate/product tolerance and excellent enantioselectivity in the production of l-menthol through enantioselective hydrolysis of dl-menthyl acetate. However, rapid inactivation of wild-type BSE at elevated temperatures often hampers its applications. In this work, directed evolution was used to create thermostable mutants of BSE. After screening and recombination of beneficial mutations, BSEV4 was chose for the best mutant. The BSEV4 had half-lives of 462, 248 and 0.34 h at 30, 40 and 50 °C, respectively, which were 5.6, 4.1 and 2.0 folds longer than those of BSEWT. Moreover, BSEV4 showed an increase of 4.5 °C in T 50 15 and a higher temperature optimum compared with the wild-type enzyme. In the kinetic resolution of dl-menthyl acetate at 1.0 M substrate loading, BSEV4 displayed improvements in operational stability than BSEWT, leading to a 1.5-fold higher total turnover number at 45 °C. The model structure of BSEV4 with four mutations, built with a highly homologous p-nitrobenzyl esterase (PDB ID: 1QE3 ) as the template, revealed that the newly formed hydrogen bonds and ionic bonds were beneficial for enhancing the thermostability of BSE.

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.

A new thermostable β-1,4-mannanase (DtManB) cloned from Dictyoglomus thermophilum CGMCC 7283 showed the maximum activity towards hydroxypropyl guar gum at 80 °C, with a half-life of 46 h. DtManB exhibited good compatibility with various additives of fracturing fluid, retaining more than 50 % activity in all the cases tested. More importantly, premature degradation could be alleviated significantly when using DtManB as breaker, because at 27 and 50 °C it displayed merely 3.7 and 18.5 % activities compared to those at 80 °C. In a static test, 0.48 mg DtManB could break 200 mL borax cross-linked fracturing fluid dramatically at 80 °C, and merely 18 mPa s of the viscosity was detected even after the broken fluid was cooled down and only 161.4 mg L(-1) of the residue was left after the enzymatic reaction. All these positive features demonstrate the great potential of this mannanase as a new enzyme breaker for application in enhanced recovery of petroleum oil.

In this paper, a new pH/temperature-sensitive beads with semi-interpenetrating polymeric network based on sodium alginate(SA) and poly(aspartic acid) derivatives(M-E-PSI) were prepared using as drug delivery carrier. With indomethacin as a drug model，we investigated the release behaviors of indomethacin in different pH value, temperature and ratio of SA/ M-E-PSI. It turned out that the release amount of indomethacin in pH=2.1 phosphate buffer solution(PBS) was evidently higher than that in pH=7.4 PBS. And also, the release amount of indomethacin was also increased with increasing temperature and poly(aspartic acid) derivatives content in the beads.

In this study, a pH/temperature sensitive polymeric hydrogel from poly (lysine methyl ester-diacetoxy tartaric acid) and sodium alginate (PLMD-SA) was prepared via the condensation polymerization. Using indomethacin as the model drug, the controlled drug-release from the resulting pH/temperature sensitive PLMD-SA was evaluated as functions of pH, PLMD content and temperature by UV spectrophotometer. It was found that the release amount of indomethacin in pH=7.4 phosphate buffer solution (PBS) was evidently higher than in pH=2.3 PBS.

In this paper, the ammonium dichromate and copper nitrate were used as raw materials to prepare the copper chromite, and nanocopper chromite as a catalyst was used to synthesize the monomer dioxanone in diethylene glycol dehydrogenation. Under the strict conditions of no water and no oxygen, the stannous octoate was selected to catalyze ring-opening polymerization of the dioxanone to prepare the polydioxanone. And dioxanone and its polymers were characterized with IR spectroscopy,1H NMR spectroscopy, thermogravimetric analysis (TG) and differential scanning calorimetry (DSC ) measurements.

In order to describe the functional characteristics of new esterases by evaluating their capacity of releasing alcohols from the corresponding esters, and to build “visiting cards” which might guide more widespread application in other reactions, an array of 20 acetates of structurally diverse alcohols were designed and synthesized for rapidly fingerprinting the activities of newly discovered lipases or esterases. At first, two well-known commercial lipases/esterases, Candida rugosa lipase (CRL) and pig liver esterase (PLE), were employed as models to verify the feasibility of this method. Subsequently, four home-made new enzymes were tested to compare their substrate fingerprints. Three parameters were adopted for quantitative characterization of the fingerprints of enzymes. Among them, the Shannon–Wiener index was adopted for the first time to quantitatively describe the breadth of substrate spectrum, making it easy and comprehensive to evaluate the breadth of substrate spectrum of homologous enzymes.

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 screening9,000 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.

In order to search for oxidoreductases suitable for the preparation of methyl (R)-o-chloromandelate [(R)-CMM], the key intermediate for clopidogrel, the homologous proteins of Gre2p were expressed in Escherichia coli, among which CgKR1 showed the most satisfactory activity and stereoselectivity towards methyl o-chlorobenzoylformate (CBFM). Using the crude enzyme of CgKR1 and glucose dehydrogenase (GDH), as much as 300 g⋅L−1 of CBFM was almost stoichiometrically converted to (R)-CMM with excellent enantiomeric excess (98.7% ee). More importantly, the reaction could be performed without external addition of an expensive cofactor. The substrate profile indicates that keto esters serve as the most suitable substrate, which was confirmed by gram-scale preparations. Homology modeling and docking analysis revealed the molecular basis for the high stereoselectivity of CgKR1. These demonstrate not only the feasibility of in silico mining of novel enzymes based on sequence homology but also the applicability of this new reductase for the practical production of optically active (R)-CMM.

In the last 50 years the number of known natural organo-halogen compounds has grown from a dozen to more than 4000 today. The most studied group of halogenase biocatalysts are haloperoxidases, which are able to accelerate a wide range of halogenation reactions following the mode of action represented in Scheme 11.1. More recently new halogenases have been identified such as 2-oxoglutarate irondependent halogenase, nucleophilic halogenases that can introduce fluorine and FADH2 dependant halogenases. The FADH2 cofactor enzymes can halogenate substrates with excellent regioselectivity. A good example of this reaction is the synthesis of isolated 7chlorotriptophan by Walsh et al. a target synthesis that would involve many steps using traditional organic synthesis methods (Scheme 11.2). Hence this group of enzymes offers the chemical industry significant improvements in synthetic methodologies as low regio-selectivities are usually obtained in traditional halogenation reagents and these reagents are limited to reaction at specific activated sites. Halogenases are now known that regioselectively halogenate all four positions of the indole benzene ring which is not possible with chemical reagents. By replacing chloride salts by bromide salts in the reaction media bromination is possible in many cases. Dehalogenases have been known to efficiently dehalogenate activated halogen compounds and are applied on the large scale for the synthesis of (S)-chloropropionic acid as

A new reductase, CgKR2, with the ability to reduce ethyl 2-oxo-4-phenylbutyrate (OPBE) to ethyl (R)-2-hydroxy-4-phenylbutyrate ((R)-HPBE), an important chiral precursor for angiotensin-converting enzyme (ACE) inhibitors, was discovered. For the first time, (R)-HPBE with >99% ee was produced via bioreduction of OPBE at 1 M without external addition of cofactors. The space-time yield (700 g·L(-1)·d(-1)) was 27 times higher than the highest record.