New opportunities for biocatalysis: making pharmaceutical processes greener

The pharmaceutical industry requires synthetic routes to be environmentally compatible as well as to fulfill the demands of process economics and product specification and to continually reduce development times. Biocatalysis has the potential to deliver 'greener' chemical syntheses, and in this review some of these opportunities are outlined and outstanding challenges presented. Future development will require research targeted towards increased commercial availability of key enzymes, as well as the improvement of enzyme stability and substrate repertoire, to fully realize the potential of biocatalysis for making pharmaceutical processes greener.     

Production of fine chemicals using biocatalysis

Presently, a large number of biotransformations are carried out on an industrial scale and are discussed in a fast increasing number of reviews. Besides this, a significant number of biotransformations have been investigated over the past year, from degrading to transforming and synthetic reactions. The development of more specific and stable biocatalysts, either isolated enzymes or whole cells, generated by the new methods of genetic engineering and improved by reaction engineering have led to new industrial biotransformations.     

Controlled-release biocatalysis for the synthesis of D-phenylglycine

An isolated and immobilised aminotransferase cloned from Pseudomonas stutzeri ST-201 into Escherichia coli was used to synthesise d-phenylglycine. The reaction was characterised by an unfavourable equilibrium constant and substrate inhibition. The use of a controlled-release system via the use of Amberlite (IRA 400)-adsorbed benzoylformate proved a useful technique to circumvent these issues. This resulted in a four-fold improvement in product concentration achievable to yield a final d-phenylglycine concentration of 10.25 mg/ml.     

Trends and innovations in industrial biocatalysis for the production of fine chemicals

Biocatalysis has become an established technology for the industrial manufacture of fine chemicals. In recent years, a multitude of chemical companies have embraced biocatalysis for the manufacture of desired stereoisomers, and new or improved methods for the synthesis of enantiomerically pure alpha- and beta-amino acids, amines, amides, peptides, nitriles, alcohols, organic acids and epoxides have emerged. Furthermore, the selectivity and mild operational conditions of biocatalysts are increasingly applied in industry to modify complex target molecules. These recent innovations in the manufacture of industrial fine chemicals using biocatalysis are discussed from an industrial perspective.     

手性医药化学品生物催化合成进展与实践

手性生物催化是利用生物催化剂对手性分子构型的识别能力进行选择性催化的新型物质加工过程,具有催化效率高、选择性强和反应条件温和等优势。近十年来,生物催化技术快速崛起,树立了多个大品种原料药过程替代的成功范例,成为手性医药化学品绿色制造不可或缺的重要工具。笔者分析了生物催化商业和学术发展的新动向,并结合笔者在手性药物生物催化合成的产业化开发实践,指出了今后的发展方向。     

Molecular modeling and biocatalysis: explanations, predictions, limitations, and opportunities

Rapid advances in structural biology have revealed the three-dimensional structures of many biocatalysts. Molecular modeling is the tool that links these structures with experimental observations. As a qualitative tool, current modeling methods are extremely useful. They can explain, on a molecular level, unusual features of reactions. They can predict how to increase the selectivity either by substrate modification or by site-directed mutagenesis. Quantitative predictions, for example the degree of enantioselectivity, are still not reliable, however. Modeling is limited also by the availability of three-dimensional structures. Most current modeling involves hydrolases, especially proteases and lipases, but structures for other types of enzymes are starting to appear.     

手性医药化学品的绿色生物合成

手性在自然界中普遍存在,与生命现象密切相关,也显著影响物质的性能。手性医药化学品的化学合成存在原子经济性、过程经济性差、环境污染和资源浪费严重等问题。生物合成技术具有过程绿色、选择性好等优势。近年来,生物合成技术在手性医药化学品合成关键酶的选择、催化机制解析、光学纯手性中间体合成途径构建、工艺开发及放大生产等方面均取得长足进步,有望解决手性中心构筑复杂、光学纯度低、污染大等手性化学品制造的瓶颈问题,推动我国医药行业的绿色可持续发展。本文主要总结了中国科学院天津工业生物技术研究所成立以来在手性医药化学品生物催化合成方面的一些研究进展。     

Kinetic characterization of chiral biocatalysis of cycloarenes by the camphor 5-monooxygenase enzyme system

Redox enzyme mediated biocatalysis has the potential to regio- and stereo-specifically oxidize hydrocarbons producing valuable products with minimal by-product formation. In vitro reactions of the camphor (cytochrome P-450) 5-monooxygenase enzyme system with naphthalene-like substrates yield stereospecifically hydroxylated products from nonactivated hydrocarbons. Specifically, the enzyme system catalyzes the essentially stereospecific conversion of the cycloarene, tetralin (1,2,3,4-tetrahydronaphthalene) to (R)-1-tetralol ((R)-(−)-1,2,3,4-tetrahydro-1-naphthol). It is shown that this reaction obeys Michaelis–Menten kinetics and that interactions between the enzyme subunits are not affected by the identity of the substrate. This subunit independence extends to the efficiency of NADH usage by the enzyme system—subunit ratios do not effect efficiency, but substrate identity does. Tetralin is converted at an efficiency of 13±3%, whereas (R)-1-tetralol is converted at 7.8±0.7%. A model of this system based on Michaelis–Menten parameters for one subunit (Pdx: K_M=10.2±2 μM) and both substrates (tetralin: K_M=66±26 μM, ν_max=0.11±0.04 s⁻¹, and (R)-1-tetralol: K_M=2800±1300 μM, ν_max=0.83±0.22 s⁻¹) is presented and used to predict the consumption and production of all substrates, products and cofactors.     

Biocatalytic synthesis of intermediates for the synthesis of chiral drug substances

There has been an increasing awareness of the enormous potential of microorganisms and enzymes for the transformation of synthetic chemicals with high chemo-, regio- and enantioselectivity. Chiral intermediates and fine chemicals are in high demand, both from the pharmaceutical and agrochemical industries, for the preparation of bulk drug substances and agricultural products. Biocatalytic processes have been described for the synthesis of chiral intermediates for beta3- and beta2-receptor agonists, antihypertensive drugs, antiviral agents, melatonin receptor agonists, anticholesterol and anticancer drugs, and drugs to treat Alzheimer's disease.     

Trigonopsis variabilis D-amino acid oxidase: control of protein quality and opportunities for biocatalysis through production in Escherichia coli

Trigonopsis variabilis D-amino acid oxidase accounts for 35% of Escherichia coli protein when added D-methionine suppresses the toxic activity of the recombinant product. Permeabilized E. coli cells are reusable and stabilized enzyme preparations. The purified oxidase lacks the microheterogeneity of the natural enzyme. Oriented immobilization of a chimeric oxidase maintains 80% of the original activity in microparticle-bound enzymes.     

Efficient synthesis of simvastatin by use of whole-cell biocatalysis

Simvastatin is a semisynthetic derivative of the fungal polyketide lovastatin and is an important drug for lowering cholesterol levels in adults. We have developed a one-step, whole-cell biocatalytic process for the synthesis of simvastatin from monacolin J. By using an Escherichia coli strain overexpressing the previously discovered acyltransferase LovD (X. Xie, K. Watanabe, W. A. Wojcicki, C. C. Wang, and Y. Tang, Chem. Biol. 13:1161-1169, 2006), we were able to achieve >99% conversion of monacolin J to simvastatin without the use of any chemical protection steps. The key finding was a membrane-permeable substrate, alpha-dimethylbutyryl-S-methyl-mercaptopropionate, that was efficiently utilized by LovD as the acyl donor. The process was scaled up for gram-scale synthesis of simvastatin. We also demonstrated that simvastatin synthesized via this method can be readily purified from the fermentation broth with >90% recovery and >98% purity as determined by high-performance liquid chromatography. Bioconversion using high-cell-density, fed-batch fermentation was also examined. The whole-cell biocatalysis can therefore be an attractive alternative to currently used multistep semisynthetic transformations.     

Novel mannitol based non-ionic surfactants from biocatalysis: Part two: improved synthesis

The 1-O-lauroyl- -mannitol, a non-ionic surfactant, was synthesised via a chemo-enzymatic pathway starting from the 1,2:4,5-di-O-isopropylidene- -mannitol and vinyl laurate as acylation agent. The high hydrophobicity of the substrates allowed the enzymatic reaction to occur both in n-hexane and in solvent free conditions. The immobilised Candida antarctica lipase B was used as the catalyst of the enzymatic step. This enzyme acts differently depending on the position of the hydroxyls with respect to the isopropylidene groups. The acid selective hydrolysis of the isopropylidene groups gave the non-ionic surfactant without the presence of isomers.     

Enzymatic synthesis of chiral intermediates for pharmaceuticals

There has been an increasing awareness of the enormous potential of microorganisms and enzymes for the transformation of synthetic chemicals with high chemo-, regio- and enatioselective manner. Chiral intermediates are in high demand by pharmaceutical industries for the preparation bulk drug substances. In this review article, microbial/enzymatic processes for the synthesis of chiral intermediates for antihypertensive drugs, melatonin receptor agonists, and beta3-receptor receptor agonists are described.     

Biodiesel biorefinery: opportunities and challenges for microbial production of fuels and chemicals from glycerol waste

ABSTRACT: The considerable increase in biodiesel production worldwide in the last 5 years resulted in astoichiometric increased coproduction of crude glycerol. As an excess of crude glycerol hasbeen produced, its value on market was reduced and it is becoming a "waste-stream" insteadof a valuable "coproduct". The development of biorefineries, i.e. production of chemicals andpower integrated with conversion processes of biomass into biofuels, has been singled out asa way to achieve economically viable production chains, valorize residues and coproducts,and reduce industrial waste disposal. In this sense, several alternatives aimed at the use ofcrude glycerol to produce fuels and chemicals by microbial fermentation have beenevaluated. This review summarizes different strategies employed to produce biofuels andchemicals (1,3-propanediol, 2,3-butanediol, ethanol, n-butanol, organic acids, polyols andothers) by microbial fermentation of glycerol. Initially, the industrial use of each chemical isbriefly presented; then we systematically summarize and discuss the different strategies toproduce each chemical, including selection and genetic engineering of producers, andoptimization of process conditions to improve yield and productivity. Finally, the impact ofthe developments obtained until now are placed in perspective and opportunities andchallenges for using crude glycerol to the development of biodiesel-based biorefineries areconsidered. In conclusion, the microbial fermentation of glycerol represents a remarkablealternative to add value to the biodiesel production chain helping the development ofbiorefineries, which will allow this biofuel to be more competitive.     

Enantioselective synthesis of chiral sulfinates using chiral diamines

The reaction of p-toluenesulfinyl chloride with alcohols in the presence of chiral diamines was examined. Chiral sulfinates were obtained in good yields with enantioselectivity up to 76% ee.     

Enzymatic biocatalysis in chemical synthesis of pharmaceuticals (Review)

In this review we summarize the available research on enzymatic biocatalysis in the chemical synthesis of drugs. We focus on oxydoreductsases, particularly ketoreductases, that are widely used in biotechnological processes: alpha- and omega-transaminases, lipases, nitrile hydrolases, and aldolases. The potential for the extended use of novel enzymes produced via bioengineering is discussed.     

Acetohydroxyacid synthase: a new enzyme for chiral synthesis of R-phenylacetylcarbinol

We have found that acetohydroxyacid synthase (AHAS) is an efficient catalyst for the enantiospecific (> or =98% enantiomeric excess) synthesis of (R)-phenylacetylcarbinol (R-PAC) from pyruvate and benzaldehyde, despite the fact that its normal physiological role is synthesis of (S)-acetohydroxyacids from pyruvate and a second ketoacid. (R)-phenylacetylcarbinol is the precursor of important drugs having alpha and beta adrenergic properties, such as L-ephedrine, pseudoephedrine, and norephedrin. It is currently produced by whole-cell fermentations, but the use of the isolated enzyme pyruvate decarboxylase (PDC) for this purpose is the subject of active research and development efforts. Some of the AHAS isozymes of Escherichia coli have important advantages compared to PDC, including negligible acetaldehyde formation and high conversion of substrates (both pyruvate and benzaldehyde) to PAC. Acetohydroxyacid synthase isozyme I is particularly efficient. The reaction is not limited to condensation of pyruvate with benzaldehyde and other aromatic aldehydes may be used.