Biocatalysis for industrial production of fine chemicals

Chiral intermediates constitute a significant part of the fine chemicals market, which is strongly influenced by trends in the pharmaceutical industries, where approximately 70% of pharmaceuticals are expected to be enantiomerically pure in the next century as compared to 25% today. The main technologies by which enantiomerically pure ingredients are obtained today are (dynamic) resolutions of racemic mixtures. Asymmetric syntheses are being developed, but their applications in industry are still under represented. Biotechnological methods, resolutions as well as asymmetric syntheses, are becoming increasingly important in the industrial production of fine chemicals.     

芳香族化合物微生物代谢工程研究进展

代谢工程从20世纪90年代初期发展至今已有近30年历史,对微生物菌种改良和选育工作起到了极大的推动作用.芳香族化合物是一类可以通过微生物发酵生产的化学品,广泛应用于医药、食品、饲料和材料等领域.利用代谢工程手段对莽草酸和芳香族氨基酸合成途径进行理性改造,微生物细胞可以定向地大量积累人们需要的各种芳香族化合物.笔者对近3...     

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.     

Metabolic engineering of plant secondary metabolite pathways for the production of fine chemicals

The technology of large-scale plant cell culture is feasible for the industrial production of plant-derived fine chemicals. Due to low or no productivity of the desired compounds the economy is only in a few cases favorable. Various approaches are studied to increase yields, these encompass screening and selection of high producing cell lines, media optimization, elicitation, culturing of differentiated cells (organ cultures), immobilization. In recent years metabolic engineering has opened a new promising perspectives for improved production in a plant or plant cell culture.     

Genetic engineering as a powerful tool to improve probiotic strains

Over the last decade, there has been increasing interest in the use of probiotic microorganisms. However, certain doubts have arisen around probiotics, because of the beneficial effects of these microorganisms are not clear yet, and in many occasions those beneficial effects have not been proven. Therefore, it would be of interest if these probiotic strains were able to acquire new attributes to allow them improve and increase their beneficial characteristics. Genetic engineering can be used for human applications; for instance, the resistance to antibiotics is removed and the probiotic bacteria are modified in its own DNA. This process can be achieved by: (1) the use of food-grade vectors derived from lactic acid bacteria and/or bifidobacteria cryptic plasmids, (2) the genes integration or deletion in the chromosome of the probiotic strain, by site-specific recombination using the attP/integrase system, or by homologous recombination, using either suicide vectors, (3) using the clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-associated (Cas) nuclease. Through genetic engineering, the knowledge of probiotic strains as well as its use could be improved, and the doubts about probiotics could be crumped.     

腈转化酶在精细化学品生产中的应用

腈化合物是一类重要的用于合成多种精细化学品的化合物,它们容易制备,并且可以合成多种化合物。传统化学水解方法将腈化合物转化为相应的羧酸或酰胺通常需要高温、强酸、强碱等相对苛刻的条件,腈转化酶(腈水解酶、腈水合酶和酰胺酶)由于其生物催化过程具有高效、高选择性、条件温和等特点,在精细化学品的合成中越来越受到人们的关注。许多腈转化酶已经被开发出来并用于精细化学品的生产。以下介绍了腈转化酶在医药及中间体、农药及中间体、食品与饲料添加剂等精细化学品生产中的应用。随着研究的不断深入,将会有更多的腈转化酶被开发出来并用于精细化学品的生产。     

Synthetic affinity ligands as a novel tool to improve protein stability

Cutinase from Fusarium solani pisi is the model-system for a new approach to assess and enhance protein stability based on the use of synthetic triazine-scaffolded affinity ligands as a novel protein-stabilizing tool. The active site of cutinase is excluded from the main surface regions postulated to be involved in early protein's thermal unfolding events. Hence, these regions are suitable targets for binding complementary affinity ligands with a potential stabilizing effect. A random solid-phase combinatorial library of triazine-bisubstituted molecules was screened for binding cutinase by a rapid fluorescence-based method and affinity chromatography. The best binding substituents were combined with those previously selected by screening a rationally designed library. A second-generation solid-phase biased library was designed and synthesized, following a semi-rational methodology. A dual screening of this library enabled the selection of ligands binding cutinase with higher affinity while retaining its functionality. These compounds were utilized for thermostability assessment with adsorbed cutinase at 60 degrees C and pH 8.0. When bound to different types of ligands, the enzyme showed markedly distinct activity retention profiles, with some synthetic affinity ligands displaying a stabilizing effect on cutinase and others a clearly destabilizing effect, when compared with the free enzyme.     

Methane as a carbon substrate for the production of microbial cells

The cultivation of aerobic, methane-utilizing, microbial cells by submerged culture techniques, in an entirely mineral salts medium, with a view to their use as an edible protein source is discussed. Particular emphasis is placed on the potentially explosive nature of gaseous mixtures containing methane and oxygen. The experiments described investigate if fully safe operation at all times, by oxygen concentration control, is possible in agitated and sparged batch fermentors. Appreciable wastage of methane is prevented by gaseous-phase recirculation. It is concluded that fully safe operation is possible, cultures being able to grow exponentially without substrate limitation by the gaseous-phase nutrients.     

Harnessing eugenol as a substrate for production of aromatic compounds with recombinant strains of Amycolatopsis sp. HR167

To harness eugenol as cheap substrate for the biotechnological production of aromatic compounds, the vanillyl alcohol oxidase gene (vaoA) from Penicillium simplicissimum CBS 170.90 was cloned in an expression vector suitable for Gram-positive bacteria and expressed in the vanillin-tolerant Gram-positive strain Amycolatopsis sp. HR167. Recombinant strains harboring hybrid plasmid pRLE6SKvaom exhibited a specific vanillyl alcohol oxidase activity of 1.1U/g protein. Moreover, this strain had gained the ability to grow on eugenol as sole carbon source. The intermediates coniferyl alcohol, coniferyl aldehyde, ferulic acid, guajacol, and vanillic acid were detected as excreted compounds during growth on eugenol, whereas vanillin could only be detected in trace amounts. Resting cells of Amycolatopsis sp. HR167 (pRLE6SKvaom) produced coniferyl alcohol from eugenol with a maximum conversion rate of about 2.3 mmol/h/l of culture, and a maximum coniferyl alcohol concentration of 4.7 g/1 was obtained after 16 h biotransformation without further optimization. Beside coniferyl alcohol, traces of coniferyl aldehyde and ferulic acid were also detected.     

Testicular morphometry as a tool to evaluate the efficiency of immunocastration in lambs

This study aimed to evaluate the efficiency of immunocastration in lambs using testicular morphometry. Thirty lambs were randomly divided into two treatments (subcutaneous administration of 1.0 mL and 0.5 mL of an anti-GnRH vaccine) and a control group (1.0 mL saline solution). The animals were vaccinated at four months of age, received a second dose 30 days later, and were slaughtered 90 days after the first vaccine dose. After slaughter, testicles were collected, and samples were removed for histological processing and evaluation of testicular morphometric parameters. Analysis of variance, Tukey’s test, and Kruskal–Wallis test were performed, with a 5% level of significance. There was a reduction in testicular weight, gonadosomatic index, seminiferous tubule diameter, germinal epithelium height, leydigosomatic index, and total tubule length. The total length per testicular gram increased in the immunocastrated group. Intrinsic spermatogenesis yield, Sertoli cell indices, and estimates of sperm and Sertoli cell production were reduced in the immunized groups (P < 0.05). The anti-GnRH vaccine in lambs at doses of 1.0 mL and 0.5 mL is sufficient to promote immunocastration, verified through severe changes in testicular morphometry from animals.     

Strategies and agronomic interventions to improve the phosphorus-use efficiency of farming systems

Phosphorus (P)-deficiency is a significant challenge for agricultural productivity on many highly P-sorbing weathered and tropical soils throughout the world. On these soils it can be necessary to apply up to five-fold more P as fertiliser than is exported in products. Given the finite nature of global P resources, it is important that such inefficiencies be addressed. For low P-sorbing soils, P-efficient farming systems will also assist attempts to reduce pollution associated with P losses to the environment. P-balance inefficiency of farms is associated with loss of P in erosion, runoff or leaching, uneven dispersal of animal excreta, and accumulation of P as sparingly-available phosphate and organic P in the soil. In many cases it is possible to minimise P losses in runoff or erosion. Uneven dispersal of P in excreta typically amounts to ~5% of P-fertiliser inputs. However, the rate of P accumulation in moderate to highly P-sorbing soils is a major contributor to inefficient P-fertiliser use. We discuss the causal edaphic, plant and microbial factors in the context of soil P management, P cycling and productivity goals of farms. Management interventions that can alter P-use efficiency are explored, including better targeted P-fertiliser use, organic amendments, removing other constraints to yield, zone management, use of plants with low critical-P requirements, and modified farming systems. Higher productivity in low-P soils, or lower P inputs in fertilised agricultural systems can be achieved by various interventions, but it is also critically important to understand the agroecology of plant P nutrition within farming systems for improvements in P-use efficiency to be realised.     

Fuzzy chorotypes as a conceptual tool to improve insight into biogeographic patterns

Chorotypes--statistically significant groups of coincident distribution areas--constitute biogeographic units that are fuzzy by nature. This quality has been referred to in the literature but has not been analyzed in depth or methodologically developed. The present work redefines chorotypes as fuzzy sets from a pragmatic perspective and basically focuses on the methodological and interpretative implications of this approach. The amphibian fauna in the Iberian Peninsula was used as an example to explore the fuzzy nature of chorotypes. The method on which this article is based is a widely used technique to define chorotypes. This method involves the fuzziness that is inherent to the identification between degree of similarity and degree of membership and includes a probabilistic analysis of the classification for the objective delimitation of chorotypes. The main innovation of this paper is a procedure to analyze chorotypes as fuzzy biogeographic units. A set of fuzzy parameters to deal with the biogeographic interpretation of fuzzy chorotypes is also described. A computer program has been developed and is freely available. History may be related to the degree of fuzziness of chorotypes. In our example, with amphibian distributions in Iberia, less fuzzy chorotypes could have a historical explanation, and the internal fuzziness of chorotypes increases with their distance to hypothetical Pleistocene refugia.     

Dogs as a tool to improve bird-strike mortality estimates at wind farms

Management of avian populations near anthropogenic infrastructures, specifically wind farms, has been hampered due to biased bird fatalities estimates. Currently, these estimations are based on field surveys performed by humans, which is a method with low efficiency and accuracy. Detection dogs have been used for decades to assist humans, and their use for wildlife surveys is of increasing interest to scientists and wildlife managers. We evaluate the accuracy rates of human and dog-handler teams in real field conditions to address if dogs could be used instead of humans for bird carcass searches. Furthermore, to verify the efficiency of detection dogs (determined by the time spent to detect each bird carcass) searching for bird carcasses, we investigate the influence of several factors that affect the performance of dogs (carcass decomposition condition, distance to the target and weather conditions). Results indicate that dogs are more accurate than humans, independently of vegetation density. Furthermore, carcass decomposition condition, distances to the carcass and weather conditions significantly affect the efficiency of working dogs. The influence of these factors on detection time was minor. Results demonstrate the usefulness of dogs in field surveys to improve bird-strike mortality estimates at wind farms and other anthropogenic structures that cause bird fatalities worldwide.     

Engineering OsBAK1 gene as a molecular tool to improve rice architecture for high yield

Generating a new variety of plant with erect-leaf is a critical strategy to improve rice grain yield, as plants with this trait can be dense-planted. The erect-leaf is a significant morphological trait partially regulated by Brassinosteroids (BRs) in rice plants. So far, only a few genes can be used for molecular breeding in rice. Here, we identified OsBAK1 as a potential gene to alter rice architecture. Based on rice genome sequences, four closely related homologs of Arabidopsis BAK1 ( AtBAK1 ) gene were amplified. Phylogenetic analysis and suppression of a weak Arabidopsis mutant bri1-5 indicated that OsBAK1 (Os08g0174700) is the closest relative of AtBAK1. Genetic, physiological, and biochemical analyses all suggest that the function of OsBAK1 is conserved with AtBAK1 . Overexpression of a truncated intracellular domain of OsBAK1 , but not the extracellular domain of OsBAK1 , resulted in a dwarfed phenotype, similar to the rice BR-insensitive mutant plants. The expression of OsBAK1 changed important agricultural traits of rice such as plant height, leaf erectness, grain morphologic features, and disease resistance responses. Our results suggested that a new rice variety with erect-leaf and normal reproduction can be generated simply by suppressing the expression level of OsBAK1 . Therefore, OsBAK1 is a potential molecular breeding tool for improving rice grain yield by modifying rice architecture.     

Two-dimensional electrophoresis as a tool for control of quality and consistency in production systems using animal cells

A nonrecombinant human melanoma cell line and recombinant chinese hamster ovary (CHO) cells were used as examples for long-termin vitro cultivation in protein-free media. The method used to monitor the consistency of protein release by these mammalian cells was two-dimensional electrophoresis with immobilized pH gradient. Secreted proteins from a melanoma cell line cultivated in a continuous fermentation system over a period of 22 months were monitored. Two-dimensional patterns of secreted proteins were compared and the stability of their composition was determined over a period of nearly 14 months, with significant pattern variation being observed after 14 months. The protein pattern from this extendedin vitro culture was compared to those of the very same melanoma cell line recultivated after being frozen in liquid nitrogen for more than 2 years. Due to the high resolution of complex polypeptide mixtures and the possibility to detect even minor differences in the composition of protein patterns, we propose the two-dimensional electrophoresis as a tool for quality assessment in animal cell culture technology.     

Integrated operation of continuous chromatography and biotransformations for the generic high yield production of fine chemicals

The rapid progress in biocatalysis in the identification and development of enzymes over the last decade has enormously enlarged the chemical reaction space that can be addressed not only in research applications, but also on industrial scale. This enables us to consider even those groups of reactions that are very promising from a synthetic point of view, but suffer from drawbacks on process level, such as an unfavourable position of the reaction equilibrium. Prominent examples stem from the aldolase-catalyzed enantioselective carbon-carbon bond forming reactions, reactions catalyzed by isomerising enzymes, and reactions that are kinetically controlled. On the other hand, continuous chromatography concepts such as the simulating moving bed technology have matured and are increasingly realized on industrial scale for the efficient separation of difficult compound mixtures - including enantiomers - with unprecedented efficiency. We propose that coupling of enzyme reactor and continuous chromatography is a very suitable and potentially generic process concept to address the thermodynamic limitations of a host of promising biotransformations. This way, it should be possible to establish novel in situ product recovery processes of unprecedented efficiency and selectivity that represent a feasible way to recruit novel biocatalysts to the industrial portfolio.     

Solubilization and conversion of hepatic adenylate cyclase to a form requiring MnATP as substrate

A general feature of membrane-bound adenylate cyclase systems is the “lability” of the basal enzyme to dispersion by detergents. A stable form of the detergentsolubilized enzyme is obtained only if the membrane-bound enzyme is first pretreated with fluoride or Gpp(NH)p. However, we have found with the basal hepatic enzyme that the lability is evident primarily when MgATP is used as substrate; substitution of MnATP for MgATP reveals that substantial basal activity survives detergent treatment. This effect is independent of the detergent; it is seen with either Lubrol PX or with deoxycholate. In addition to the altered substrate requirement, the membrane-bound and solubilized forms of the basal enzyme exhibit other differences. In contrast to the membrane-bound form, the solubilized enzyme shows (1) weak stimulation by Gpp(NH)p; (2) little inhibition by adenosine, (3) strong inhibition by P_i or PP_i, and (4) and apparent loss of the Me²⁺-reactive regulatory site. Such dissimilarities between membranebound and solubilized cyclase are not seen if the membranes are pretreated with Gpp(NH)p prior to exposure to detergents. The characteristics of the solubilized basal hepatic enzyme are similar to those of the naturally occurring soluble adenylate cyclase found in mature rat testes. It would appear that separation of adenylate cyclase from components that confer regulation by divalent cation and guanine nucleotides produces a form of the enzyme that will turnover only MnATP; this may represent the free catalytic moiety. Such preparations could be useful in reconstructing some of the regulatory functions of adenylate cyclase seen in its membrane-bound form.     

Dates as a potential substrate for single cell protein production

The use of date juice as a substrate for single cell protein production was investigated. Four strains of Saccharomyces cerevisiae and two strains of Candida utilis were examined as possible production cultures. The criteria used for screening the organisms were total cell count, total protein and decrease in soluble solids. S. cerevisiae ATCC 4111 gave the highest protein and cell production. The optimum substrate concentration was 4 - 5% soluble solids. At this concentration, 55% of the sugars was utilized. Cell mass after 12 h fermentation was 4.86 g l⁻¹. The harvested and freeze-dried cells contained 8.6% nitrogen. The best combination of nutrient supplementation was found to be 0.25% (NH₄)₂HPO₄ and 0.1% FeNH₄(SO₄)₂; addition of MgSO₄ and (NH₄)₂SO₄ did not increase cell production.