Towards novel processes for the fine-chemical and pharmaceutical industries

In response to the need in the pharmaceutical industry for more complex, chiral molecules, fine-chemical companies are embracing new manufacturing technologies to produce compounds of these specifications. In particular, recent developments in biocatalysis combined with novel process engineering are providing improved methods for the production of valuable chemical intermediates.     

冲击生物技术第三次浪潮

工业生物催化是继医药、农业之后的生物技术第三次浪潮。从21世纪化学工业发展的前沿特点,介绍生物催化加工过程及生产方式,主要解决传统产业改造和新的应用领域的开拓,提出发展生物催化产业的策略和加强支持力度的建设。     

Biocatalysis for pharmaceutical intermediates: the future is now

Biocatalysis is continuing to gain momentum and is now becoming a key component in the toolbox of the process chemist, with a place alongside chemocatalysis and chromatographic separations. The pharmaceutical industry demands a speed of development that must be on a parallel with conventional chemistry and high optical purity for complex compounds with multiple chiral centres. This review describes how these demands are being addressed to make biocatalysis successful, particularly by the use of micro-scale technology for high-speed catalyst screening and process development alongside discipline integration of biology and engineering with chemistry. Developments in recombinant technology will further expand the repertoire of biocatalysis in the coming years to new chemistries and enable catalyst design to fit the process. Further development of biocatalysis for green chemistry and high productivity processes can also be expected.     

A window into biocatalysis and biotransformations

Eight papers were presented in this year's symposium "Advances in Biocatalysis" at the 232nd ACS National Meeting, accentuating the most recent development in biocatalysis. Researchers from both industry and academia are addressing several fundamental problems in biocatalysis, including the limited number of commercially available enzymes that can be provided in bulk quantities, the limited enzyme stability and activity in nonaqueous environments, and the permeability issue and cell localization problems in whole-cell systems. A trend that can be discerned from these eight talks is the infusion of new tools and technologies in addressing various challenges facing biocatalysis. Nanotechnology, bioinformatics, cellular membrane engineering and metabolic engineering (for engineering whole-cell catalysts), and protein engineering (to improve enzymes and create novel enzymes) are becoming more routinely used in research laboratories and are providing satisfactory solutions to the problems in biocatalysis. Significant progress in various aspects of biocatalysis from discovery to industrial applications was highlighted in this symposium.     

Microbial steroid transformations: current state and prospects

Studies of steroid modifications catalyzed by microbial whole cells represent a well-established research area in white biotechnology. Still, advances over the last decade in genetic and metabolic engineering, whole-cell biocatalysis in non-conventional media, and process monitoring raised research in this field to a new level. This review summarizes the data on microbial steroid conversion obtained since 2003. The key reactions of structural steroid functionalization by microorganisms are highlighted including sterol side-chain degradation, hydroxylation at various positions of the steroid core, and redox reactions. We also describe methods for enhancement of bioprocess productivity, selectivity of target reactions, and application of microbial transformations for production of valuable pharmaceutical ingredients and precursors. Challenges and prospects of whole-cell biocatalysis applications in steroid industry are discussed.     

The production of fine chemicals by biotransformations

Today, biocatalysis is a standard technology for the production of chemicals. An analysis of 134 industrial biotransformations reveals that hydrolases (44%) and redox biocatalysts (30%) are the most prominent categories. Most products are chiral (89%) and are used as fine chemicals. In the chemical industry, successful product developments involve on average a yield of 78%, a volumetric productivity of 15.5 g/(L.h) and a final product concentration of 108 g/L. By contrast, the pharmaceutical industry focuses on time-to-market. The implications of this for future research and development on biocatalysis are discussed.     

Current state and perspectives of penicillin G acylase-based biocatalyses

In the course of more than 60-year history, penicillin G acylase (PGA) gained a unique position among enzymes used by pharmaceutical industry for production of β-lactam antibiotics. Kinetically controlled enzymatic syntheses of cephalosporins of novel generations in which PGA catalyzes coupling of activated acyl donor with nucleophile belong among the latest large-scale applications. Contrary to rather specific roles of other enzymes involved in β-lactam biocatalyses, the PGA seems to have the greatest potential. On the laboratory scale, other applications with industrial potential were described, e.g., directed evolution of the enzyme to meet specific demands of industrial processes or its modification into the enzyme catalyzing reactions with novel substrates. The fact that β-lactams represent the most important group of antibiotics comprising 65 % of the world antibiotic market explains such a tremendous and continuous interest in this enzyme. Indeed, the annual consumption of PGA has recently been estimated to range from 10 to 30 million tons. The application potential of the enzyme goes beyond the β-lactam biocatalysis due to its enantioselectivity and promiscuity: the PGA can be used for the production of achiral and chiral compounds convenient for the preparation of synthons and active pharmaceutical ingrediences, respectively. These biocatalyses, however, still wait for large-scale application.     

Isolation of an Organic-Solvent-Tolerant Cholesterol-Transforming <Emphasis Type="Italic">Bacillus</Emphasis> species,BC1 , from Coastal Sediment

Steroid transformation is of great importance in the pharmaceutical industry. The major limiting factor in this process is the extremely poor solubility of steroids in aqueous media, which lowers their transformation rate and increases costs. This problem can be overcome by using organic-solvent-tolerant bacteria (OSTB), which can carry out the desired bioconversions in an organic-solvent-saturated system. OSTB are a relatively novel group of extremophilic microbes that have developed various adaptations to withstand solvent toxicity. The aim of this study was to isolate marine bacteria producing organic-solvent-stable cholesterol-transforming enzymes. A Bacillus species, BC1, isolated from Arabian Sea sediment was found to degrade cholesterol and exhibit excellent solvent tolerance particularly to chloroform. OSTB have tremendous potential in industrial processes involving nonaqueous biocatalysis and transformation in the presence of an organic phase.     

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.     

Prerequisites for the pharmaceutical industry to develop and commercialise helminths and helminth-derived product therapy

During the past 10 years, immunologists, epidemiologists and parasitologists have made many new exciting discoveries in the field of helminth-mediated immune regulation. In addition, many animal experiments have shown that certain helminths or products derived from helminths can protect mice from developing allergic or autoimmune disease. Some clinical trials utilising Trichuris suis or Necator americanus for the treatment of allergic disorders and inflammatory bowel disease have been conducted. The outcomes of these trials suggest that they may be used to treat these disorders. However, to date no helminth therapy is routinely being applied to patients and no helminth-derived product therapy has been developed. In order to bring new drugs to the market and shoulder the enormous costs involved in developing such therapies, pharmaceutical companies need to be involved. However, currently the resources from the pharmaceutical industry devoted to this concept are relatively small and there are good reasons why the industry may have been reluctant to invest in developing these types of therapies. In this review article, the hurdles that must be overcome before the pharmaceutical industry might invest in these novel therapies are outlined.     

Lipase-catalyzed regioselective preparation of fatty acid esters of hydrocortisone

A series of fatty acid derivatives of hydrocortisone has been prepared by an enzymatic methodology. Nine 21-monoacyl products and one 3,11,17-triacetyl derivative, nine of them novel compounds, were obtained in a highly regioselective way through lipase-catalyzed esterification, transesterification and alcoholysis reactions. The influence of various reaction parameters such as acylating agent: substrate ratio, enzyme: substrate ratio, solvent, temperature and nature of acylating agent and alcohol was evaluated. Among the tested lipases, Candida antarctica lipase appeared to be the most appropriate and showed a high efficient behavior especially in a one-pot transesterification. The advantages presented by this methodology, such as mild reaction conditions and low environmental impact, make the biocatalysis a convenient way to prepare acyl derivatives of hydrocortisone. These lipophilic compounds are potential products in the pharmaceutical industry.     

Industrial biocatalysis

The number of industrial processes for the synthesis of fine and commodity chemicals, pharmaceutical and agrochemical intermediates and drug substances utilizing biological catalysts continues to grow. The combination of new molecular biology techniques, such as directed evolution and pathway engineering, with new and efficient high-throughput screening methods is poised to bolster this field and further advance the contribution of biocatalysis to the chemical and the pharmaceutical industries.     

New opportunities for biocatalysis: driving the synthesis of chiral chemicals

Various biocatalytic methods have been developed for the synthesis of chiral chemicals, which have made their synthesis more environmentally friendly and product-specific. New opportunities for biocatalysis, including new scientific developments in genomics and protein engineering technologies, novel process developments and the increased availability of useful enzymes, offer many possibilities for the manufacture of new chiral compounds and deliver greener and economically competitive processes. In this review, new opportunities for biocatalysis in the preparation of chiral molecules are outlined and highlighted.     

中国酶工程的兴旺与崛起

酶工程是生物工程的重要组成部分,工业生物催化技术被认为是继医药、农业之后的第三个浪潮。在25年中,中国在酶工程领域研究中取得很大进展,本综述集中介绍在中国酶工程会议上,酶的基因工程、酶的蛋白质工程、生物合成、微生物转化和生物传感器方面的成果和我国酶制剂工业的进展。     

Metagenomic technology and genome mining: emerging areas for exploring novel nitrilases

Nitrilase is one of the most important members in the nitrilase superfamily and it is widely used for bioproduction of commodity chemicals and pharmaceutical intermediates as well as bioremediation of nitrile-contaminated wastes. However, its application was hindered by several limitations. Searching for new nitrilases and improving their application performances are the driving force for researchers. Genetic data resources in various databases are quite rich in post-genomic era. Besides, more than 99 % of microbes in the environment are unculturable. Metagenomic technology and genome mining are thus becoming burgeoning areas and provide unprecedented opportunities for searching more useful novel nitrilases due to the abundance of already existing but unexplored gene resources, namely uncharacterized genome information in the database and unculturable microbes in the natural environment. These techniques seem to be innovative and highly efficient. This study reviews the current status and future directions of metagenomics and genome mining in nitrilase exploration. Moreover, it discussed their utilization in coping with the challenges for nitrilase application. In the next several years, with the rapid development of nitrile biocatalysis, these two techniques would be bound to attract increasing attentions and even become a dominant trend for finding more novel nitrilases. Also, this review would provide guidance for exploitation of other commercially important enzymes.     

Stabilization of multimeric sucrose synthase from Acidithiobacillus caldus via immobilization and post-immobilization techniques for synthesis of UDP-glucose

Sucrose synthases (SuSys) have been attracting great interest in recent years in industrial biocatalysis. They can be used for the cost-effective production of uridine 5′-diphosphate glucose (UDP-glucose) or its in situ recycling if coupled to glycosyltransferases on the production of glycosides in the food, pharmaceutical, nutraceutical, and cosmetic industry. In this study, the homotetrameric SuSy from Acidithiobacillus caldus (SuSyAc) was immobilized-stabilized on agarose beads activated with either (i) glyoxyl groups, (ii) cyanogen bromide groups, or (iii) heterogeneously activated with both glyoxyl and positively charged amino groups. The multipoint covalent immobilization of SuSyAc on glyoxyl agarose at pH 10.0 under optimized conditions provided a significant stabilization factor at reaction conditions (pH 5.0 and 45 °C). However, this strategy did not stabilize the enzyme quaternary structure. Thus, a post-immobilization technique using functionalized polymers, such as polyethyleneimine (PEI) and dextran-aldehyde (dexCHO), was applied to cross-link all enzyme subunits. The coating of the optimal SuSyAc immobilized glyoxyl agarose with a bilayer of 25 kDa PEI and 25 kDa dexCHO completely stabilized the quaternary structure of the enzyme. Accordingly, the combination of immobilization and post-immobilization techniques led to a biocatalyst 340-fold more stable than the non-cross-linked biocatalyst, preserving 60% of its initial activity. This biocatalyst produced 256 mM of UDP-glucose in a single batch, accumulating 1 M after five reaction cycles. Therefore, this immobilized enzyme can be of great interest as a biocatalyst to synthesize UDP-glucose.

     

生物信息学在工业生物催化研究中的应用

随着石油等不可再生资源的日益减少以及环境污染问题的日益严重,应用工业生物催化技术改造或取代传统化工工艺已经成为新世纪化学工业可持续发展的研究热点。工业生物催化技术的研究对象是生物催化剂及其催化过程。近来,利用生物信息学技术进行工业生物催化研究已经越来越受到人们的重视。随着工业生物催化的发展,生物信息学将直接指导并加快新型高效生物催化剂的发现及功能改造进程。     

工业生物催化前线动态及名家观点

随着现代生物技术的进步,尤其是酶的快速筛选和活力优化技术的发展,使酶的获取更加容易、酶的操作更加简单,进而促使生物催化成为手性合成的便利工具。综述了一些著名的国际化工或制药公司最近在生物催化技术研发和应用方面的动态信息以及相关技术的一些评论,以便我国从事工业生物催化工作的相关人士能从中获得有益启示。     

White biotechnology: ready to partner and invest in

It needs three factors to build an industry: market demand, product vision and capital. White biotechnology already produces high volume products such as feed additive amino acids and specialty products like enzymes for enantioselective biocatalysis. It serves large and diverse markets in the nutrition, wellness, pharmaceutical, agricultural and chemical industry. The total volume adds up to $ 50 billion worldwide. In spite of its proven track record, white biotechnology so far did not attract as much capital as red and even green biotechnology. However, the latest finance indicators confirm the continuously growing attractiveness of investment opportunities in white biotechnology. This article discusses white biotechnology's position and potential in the finance market and success factors.     

Well-defined protein–polymer conjugates—synthesis and potential applications

During the last decades, numerous studies have focused on combining the unique catalytic/functional properties and structural characteristics of proteins and enzymes with those of synthetic molecules and macromolecules. The aim of such multidisciplinary studies is to improve the properties of the natural component, combine them with those of the synthetic, and create novel biomaterials in the nanometer scale. The specific coupling of polymers onto the protein structures has proved to be one of the most straightforward and applicable approaches in that sense. In this article, we focus on the synthetic pathways that have or can be utilized to specifically couple proteins to polymers. The different categories of well-defined protein–polymer conjugates and the effect of the polymer on the protein function are discussed. Studies have shown that the specific conjugation of a synthetic polymer to a protein conveys its physico-chemical properties and, therefore, modifies the biodistribution and solubility of the protein, making it in certain cases soluble and active in organic solvents. An overview of the applications derived from such bioconjugates in the pharmaceutical industry, biocatalysis, and supramolecular nanobiotechnology is presented at the final part of the article.