Ionic liquids in biotechnology: applications and perspectives for biotransformations

Ionic liquids are considered as an alternative to organic solvents for catalysis. The literature in this field is reviewed with focus on advantageous use of ionic liquids in biocatalysis and biotransformations. The overview reveals that the exploration and mapping of ionic liquids with respect to biocatalysis is still sketchy. It is apparent that advantages can be gained in view of activity, stability and selectivity. Furthermore, integration of reaction and separation has a high potential in the field. The review presents quantitative data on the productivities, space–time yields, as well as stability as far as they can be extracted from the literature. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

幽门螺旋菌在双相培养基中生长特性的研究

本文观察了幽门螺旋菌在固体及液体—固体双相培养基上生长的特性,37℃72小时培养后,定量实验表明,固体培养基上菌数为1.02×10~2cfu/ml,而双相培养基上则为1.14×10~4cfu/ml;用血液双相培养基,对CP进行了生长曲线的测定,从0小时加入1.02×10~2cfu/ml开始,按每天平皿活菌计数,共5天(24、48、72、96、120、144h)分别为2.5×10~2cfu/ml,4.97×10~2cfu/ml,1.14×10~4cfu/ml,1.24×10~5cfu/ml,2.87×10~5cfu/ml,9.75×10~4cfu/ml以后逐日下降;液相培养液24—144小时pH测定,pH由7.2下降至6.4—6.9。     

Fabricating polystyrene fiber-dehydrogenase assemble as a functional biocatalyst

Immobilization of the enzymes on nano-structured materials is a promising approach to enhance enzyme stabilization, activation and reusability. This study aimed to develop polystyrene fiber-enzyme assembles to catalyze model formaldehyde to methanol dehydrogenation reaction, which is an essential step for bioconversion of CO₂ to a renewable bioenergy. We fabricated and modified electrospun polystyrene fibers, which showed high capability to immobilize dehydrogenase for the fiber-enzyme assembles. Results from evaluation of biochemical activities of the fiber-enzyme assemble showed that nitriation with the nitric/sulfuric acid ratio (v/v, 10:1) and silanization treatment delivered desirable enzyme activity and long-term storage stability, showing great promising toward future large-scale applications.     

Biofilter performance and characterization of a biocatalyst degrading alkylbenzene gases

A biofilter treating alkylbenzene vapors was characterized for its optimal running conditions and kinetic parame-ters. Kinetics of the continuous biofilter were compared to batch kinetic data obtained with biofilm samples as well as with defined microbial consortia and with pure culture isolates from the biofilter. Both bacteria and fungi were present in the bioreactor. Five strains were isolated. Two bacteria, Bacillus and Pseudomonas, were shown to be dominant, as well as a Trichosporon strain which could, however, hardly grow on alkylbenzenes in pure culture. The remaining two strains were most often overgrown by the other three organisms in liquid phase batch cultures μ _max, K_S, K_I values and biodegradation rates were calculated and compared for the difterent mixed and pure cultures. Since filter bed acidification was observed during biofiltration studies reaching a pH of about 4, experiments were also undertaken to study the influence of pH on performance of the different cultures. Biodegradation and growth were possible in all cases, over the pH range 3.5–7.0 at appreciable rates, both with mixed cultures and with pure bacterial cultures. Under certain conditions, microbial activity was even observed in the presence of alkylbenzenes down to pH 2.5 with mixed cultures, which is quite unusual and explains the ability of the present biocatalyst to remove alkylbenzenes with high efficiency in biofilters under acidic conditions.     

Comparison of cyclohexanone monooxygenase as an isolated enzyme and whole cell biocatalyst for the enantioselective oxidation of 1,3-dithiane

Both whole cells of recombinant Escherichia coli TOP10, overexpressing cyclohexanone monooxygenase (CHMO) and isolated cyclohexanone monooxygenase, were used to carry out the enantioselective oxidation of 1,3-dithiane (1) to (R)-1,3-dithiane-1-oxide (2). The two biocatalysts were evaluated under various experimental conditions (e.g., shaken flask or bioreactor; non-bound or resin-adsorbed substrate; different substrate concentrations) in terms of volumetric productivity and enantioselectivity. While productivity was similar in the two cases (up to 0.58 g L⁻¹ h⁻¹), the optical purity of the product was much higher with the isolated enzyme (up to 98% e.e.) than with the whole cell biocatalyst (up to 85% e.e.).     

Bacterial Anabaena variabilis phenylalanine ammonia lyase: A biocatalyst with broad substrate specificity

Phenylalanine ammonia lyases (PALs) catalyse the regio- and stereoselective hydroamination of cinnamic acid analogues to yield optically enriched α-amino acids. Herein, we demonstrate that a bacterial PAL from Anabaena variabilis (AvPAL) displays significantly higher activity towards a series of non-natural substrates than previously described eukaryotic PALs. Biotransformations performed on a preparative scale led to the synthesis of the 2-chloro- and 4-trifluoromethyl-phenylalanine derivatives in excellent ee, highlighting the enormous potential of bacterial PALs as biocatalysts for the synthesis of high value, non-natural amino acids.     

运动细胞自适应性过程中双相性现象的分子机制及动态数值模拟

盘基网柄菌细胞(Dictyostelium)和白细胞(leukocyte)等真核运动细胞受到外界信号刺激时,在最初的1～2 min内,胞内信号转导的首要成员PI(3,4,5)P3的浓度随时间变化呈现"双相性"(biphasic adaptation),即先后出现一大一小两个峰值,然后平息。为解释这一现象,特别是第二个峰值产生的原因,根据已有实验资料,分析了有关分子机制,建立了相应的数学模型。其中,PI(3,4,5)P3及其激活酶和抑制酶的浓度变化由一组耦合的非稳态反应-扩散方程描述,外界刺激及效应因子(如Rac和Scar/WAVE)的相互激励包含在源项中,并由蒙特-卡诺(Monte-Carlo)法处理,数值模拟结果与已有实验一致。研究发现,质膜上处于激活态的效应因子Scar/WAVE是影响PI(3,4,5)P3第二个峰值的关键,起正反馈作用。在受到胞外信号刺激后的前期,Scar/WAVE的激活态浓度受到小G蛋白Rac活性的抑制,后期反过来受到PI(3,4,5)P3的抑制,从而始终处于较低水平,这使得第二个峰值较小;当Scar/WAVE的总浓度低于0.005μmol/L后,PI(3,4,5)P3不会出现第二个峰值。由于Scar/WAVE是肌动蛋白结合蛋白,可以推测:许多经肌动蛋白合成抑制剂处理过的盘基网柄菌细胞在实验中仍然出现"双相性",应与此时的细胞骨架活性未被完全抑制有关。     

A new biocatalyst: Penicillin G acylase immobilized in sol-gel micro-particles with magnetic properties

The present work focuses on the development and basic characterization of a new magnetic biocatalyst, namely penicillin G acylase (PGA), immobilized in sol-gel matrices with magnetic properties, ultimately aimed for application in cephalexin (CEX) synthesis. A mechanically stable carrier, based on porous xerogels silica matrixes starting from tetramethoxysilane (TMOS), was prepared leading to micro-carriers with medium sized particles of 30 μm, as determined by scanning electron microscopy. An immobilization yield of 95–100% and a recovered activity of 50–65% at 37°C, as determined by penicillin G (PG) hydrolysis (pH STAT method), were observed. These results clearly exceed those reported in a previous work on PGA immobilization in sol-gel, where only 10% of activity was recovered. The values of activity were kept constant for 6 months. Immobilized PGA (682 U/g_{dry weight}) retained high specific activity throughout ten consecutive runs for PG hydrolysis, suggesting adequate biocatalyst stability. The CEX synthesis was performed at 14°C, using the free and immobilized PGA in aqueous medium. Phenylglycine methyl ester was used as acyl donor at 90 mM and 7-aminodeacetoxycephalosporanic acid was the limiting substrate at 30 mM. The CEX stoichiometric yield after 1-h reaction was close to 68% (23 mM CEX/h) and 65% (19 mM CEX/h), respectively.     

Directed evolution of a thermostable l-aminoacylase biocatalyst

Enzymes from extreme environments possess highly desirable traits of activity and stability for application under process conditions. One such example is l-aminoacylase (E.C. 3.5.1.14) from Thermococcus litoralis (TliACY), which catalyzes the enantioselective amide hydrolysis of N-protected l-amino acids, useful for resolving racemic mixtures in the preparation of chiral intermediates. Variants of this enzyme with improved activity and altered substrate preference are highly desirable. We have created a structural homology model of the enzyme and applied various two different directed evolution strategies to identify improved variants. Mutants P237S and F251Y were 2.4-fold more active towards N-benzoyl valine relative to the wild type at 65 °C. F251 mutations to basic residues resulted in 4.5-11-fold shifts in the substrate preference towards N-benzoyl phenylalanine relative to N-benzoyl valine. The substrate preference of wild type decreases with increasingly branched and sterically hindered substrates. However, the mutant S100T/M106K disrupted this simple trend by selectively improving the substrate preference for N-benzoyl valine, with a >30-fold shift in the ratio of k_cat values for N-benzoyl valine and N-benzoyl phenylalanine. Mutations that favoured N-benzoyl-phenylalanine appeared at the active site entrance, whereas those improving activity towards N-benzoyl-valine occurred in the hinge region loops linking the dimerization and zinc-binding domains in each monomer. These observations support a previously proposed substrate induced conformational transition between open and closed forms of aminoacylases.     

Systematic Approach to Solvent Selection for Biphasic Systems with a Combination of COSMO‐RS and a Dynamic Modeling Tool

Biphasic aqueous‐organic systems are important reaction systems for catalytic processes. This is especially true for biocatalysis where the range of accessible products can be significantly extended. In such systems, the aqueous phase is the reactive phase in which the biocatalyst is dissolved and the organic phase is nonreactive and acts as substrate reservoir and as in situ product extraction solvent. Here, the choice of the nonreactive phase is highly important for the overall performance of the system. In this contribution, a systematic approach to solvent selection for biphasic aqueous‐organic systems is presented with respect to partition coefficients. The model reaction is the stereoselective carbon‐carbon coupling of two 3,5‐dimethoxy‐benzaldehyde molecules to (R)‐3,3',5,5'‐tetramethoxy‐benzoin catalyzed by benzaldehyde lyase (EC 4.1.2.38) from Pseudomonas fluorescens. A systematic approach to solvent selection consisting of two steps is proposed: Firstly, the conductor‐like screening model for real solvents (COSMO‐RS) is used to facilitate a fast solvent screening. Since this is an ab initio approach it allows a pre‐screening without laborious experimental input. The proposed ranking of solvents, based on the ratio of partition coefficients at infinite dilution, is a sound basis for the successive steps. Secondly, a dynamic model is fitted to experimental data in order to obtain detailed and reliable results for mass transfer and partition coefficients. Therefore, the method makes efficient use of the experimental data and substantiates quantitative results with guided experiments.     

纳米双相磷酸钙陶瓷支架材料肌内降解性能的实验研究

目的:纳米双相磷酸钙陶瓷(Biphasic calcium phosphate nanocomposite,NanoBCP)支架是一种新型支架材料,具有三维立体多孔结构,孔隙率可达60%～80%。本研究观察了纳米双相磷酸钙陶瓷肌内降解情况。方法:将NanoBCP制备为5mm×5mm×1.5mm大小各8块的支架植入SD大鼠腿部肌袋内,相同孔径、孔隙率的羟基磷灰石(Hydroxyapatite,HA)及普通双相磷酸钙陶瓷(Biphasic calciam phosphate,BCP)作为对照,于4、12、24周取材,测定材料降解率(失重率),从大体、组织学观察以了解材料降解情况。结果:材料肌内植入后降解率测定结果:NanoBCP降解率为32%,BCP的降解率为13%,HA的降解率为3%。组织学观察发现,NanoBCP肌内植入24周后,大部分NanoBCP支架已经将解,并且将解的碎片已埋入纤维结缔组织里。结论:NanoBCP与BCP、HA相比有良好的降解性能。     

Biphasic effect of red light on the growth of coleoptiles in etiolated barley seedlings

InHordeum vulgare cultivar “Kirin-choku No. 1”, the final length of intact coleoptiles of totally etiolated seedlings was approximately twice as long as that of those grown under continuous red light. The fluence response curve of the latter was biphasic; the low-energy effect was saturated by red light of ca. 50 J m⁻² which gave rise to about 40% of the maximum inhibition by continuous irradiation with red light of 1.2 W m⁻², whereas the high-energy effect was induced by irradiation for 1 hr or longer. Coleoptiles of 3-day-old seedlings were most sensitive to light causing the low-energy effect, which was repeatedly red/far-red reversible. The growth inhibition was correlated to the photometrically measured percentage of Pfr so that the maximum effect was induced by red light of 50 J m⁻² which transformed 70% of phytochrome to Pfr in the coleoptile tip. Wavelength dependence of the high-energy effect showed that monochromatic light of 400, 600 and 650 nm greatly inhibited the coleoptile growth, whereas light of 700 and 750 nm promoted it instead. The effect was also induced by intermittent irradiation with red light, and the more frequently the intermittent treatment was given, the more the growth was inhibited.     

MSCs复合双相磷酸钙陶瓷促进腰椎骨折患者脊柱融合

目的:探索自体骨髓间充质干细胞(Mesenchymal stem cells,MSCs)复合双相磷酸钙陶瓷(Biphasic calcium phosphate ceramics,BCP)在腰椎骨折患者脊柱融合中的应用。方法:收集我院住院腰椎骨折患者50例,随机分为两组:MSCs复合BCP组和自体髂骨组。MSCs复合BCP组中,体外分离培养患者自体MSCs,复合BCP材料后共聚焦显微镜观察细胞和材料复合生长状况;然后回植于胸腰椎体骨折处,采用后路腰椎椎体间融合术(PLIF),钛合金椎弓根钉棒系统作脊柱内固定。自体髂骨组中,用患者自体髂骨代替BCP骨移植物。术前、术后1、3、6、12个月行X线和CT检查,同时采用Lenke字母分级评价脊柱融合情况,LBOS评分评价临床疗效,Cobb角评价患者伤椎复位效果。结果:两组患者术后6个月Lenke字母分级法表明两组患者均为A、B级,无C、D级;LBOS评分表明MSCs复合BCP组的优良率为68%(17/25),自体髂骨组优良率为76%(19/25);Cobb角结果表明两组伤椎复位效果良好。所有数据两组差异无明显统计学意义(P0.05)。结论:MSCs复合BCP材料是一种优良的促进脊柱融合方法,可替代自体髂骨移植。     

Biphasic Liberation of Arachidonic and Stearic Acids During Cerebral Ischemia

The mode of free fatty acid (FFA) liberation from the mouse brain during ischemia was investigated at various times after decapitation and under nizofenone treatment. Normal nonischemic brain FFAs consist mainly of palmitic acid (16:0), stearic acid (18:0), and oleic acid (18:1) with smaller amounts of arachidonic acid (20:4), docosahexaenoic acid (22:6), and others. Postdecapitative ischemia induced a rapid, biphasic release of 20:4 after a short lag of less than 30 s. The first phase showed a rapid 6.4-fold increase within 1 min of decapitation, followed by the second phase involving a slow release at less than one-fifth the rate of the first phase and lasting for at least 10 min. A similar, but not so marked, biphasic liberation was observed with 18:0. However, all of the other fatty acids (16:0, 18:1, 22:6, and others) were released only in a single phase at a slow rate. The time course for the rapid and specific liberation of 20:4 coincided with the time course for the decrease in brain ATP concentration during ischemia. Pretreatment of the animals with nizofenone resulted in a marked suppression of both FFA liberation and ATP depletion during ischemia. This suppression was particularly noteworthy with 20:4 and 18:0. The present study indicates that there is a specific and rapid liberation of 20:4 and 18:0 in a very early stage of ischemia and that this liberation seems to depend on availability of ATP in the brain. The physiological role of this transient 20:4 liberation during ischemia is discussed.     

非常规介质中细胞生化反应研究进展

对完整细胞在非常规介质中的生物催化反应进行了回顾,分别总结了产物为醇,甾体,有机酸,生物大分子及其它各类反应的研究进展。并从溶剂和细胞两种角度对主要的研究方法进行了阐述 。     

Biphasic nitrogenase activity in Azospirillum brasilense in long lasting batch cultures

Long lasting batch cultures of Azospirillum brasilense SP 7 ATCC 29145 grown in liquid malate medium for 8–14 days without any fixed nitrogen source exhibited a biphasic nitrogenase activity, when incubated under gas atmospheres of 99.0% N₂ and 1.0% O₂ or 99.5% N₂ and 0.5% O₂ respectively. Maximum specific nitrogenase activity was 1100 nmol C₂H₄·mg protein^-1·h^-1. Poly-3-hydroxybutanoic acid (PHBA) synthesis and growth of the cells also showed two phases. Maxima and minima of glutamine synthetase activity developed synchronously with nitrogenase activity, whereas those of glutamate dehydrogenase and alanine aminotransferase were reverse. During a 192 h period of growth protein increased 3–4-fold and PHBA 25 fold. At maximum accumulation of the polymer the PHBA-nitrogen ratio was 6:1 or 8:1. Azospirillum brasilense was also able to fix nitrogen on agar surfaces exposed to air, but nitrogen fixation was monophasic under these conditions during a 14 d period. Specific nitrogenase activity was dependent on the type and concentration of the source of fixed nitrogen (leucine, ammonia) in solidified media. With 1 mM leucine maximum specific nitrogenase activity was 110 nmol C₂H₄·mg protein^-1·h^-1.Non-Standard Abbreviations PHBA poly-3-hydroxybutanoic acid - TAPS tris(hydroxymethyl)methylaminopropane sulfonic acid - TES N-tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid - TRICINE N-tris(hydroxymethyl)methylglycine - TRIS tris(hydroxymethyl)aminomethane     

Medium-Engineering For Bio-Organic Synthesis

An account is given on the state of the art of medium-engineering for bio-organic synthesis. Medium-engineering means designing (micro)environments for the biocatalyst with the objective of reaching maximal activity, stability and hence productivity. Medium-engineering therefore involves aspects such as stabilizing the essential water layer around the biocatalyst, controlling electrostatic and hydrophobic interactions between matrix, reactants and biocatalyst, as well as optimizing substrate and product fluxes through the entire biocatalytic system. Where possible, some general rules have been derived, which might be of help in designing media for bio-organic synthesis.     

Biocatalysis in organic media

The potentials of using organic reaction media in biotechnological conversions have already been demonstrated in several experimental studies. Examples of possible advantages are: possibility of higher substrate and/or product concentrations, favorable shift of reaction equilibria, reduced substrate and/or product inhibition, and facilitated product recovery. Especially water/organic solvent two-phase systems seem to possess several of these advantages. The solvent type will highly affect kinetics and stability of the (immobilized) biocatalyst, solubility and partitioning of reactants/products, and product recovery. Therefore the solvent choice can have a large influence on the economics of the two-liquid-phase biocatalytic process. Immobilization of the biocatalyst may be useful to provide protection against denaturating solvent effects. The polarity of the employed support material will also be decisive for the partitioning of substrates and products among the various phases.

A classification of biphasic systems, which is based on the possible types of theoretical concentration profiles and aqueous phase configurations, is discussed. Reversed micelles and aqueous two-liquid-phase systems can be considered as special cases. The design of two-liquid-phase bioreactors is dependent on the state of the biocatalyst, free or immobilized, and on the necessity for emulsification of one of the two liquid phases in the other. Many mass-transfer resistances, e.g. across the liquid/liquid interface, in the aqueous phase, across the liquid/solid interface, and in the biocatalyst phase, can limit the overall reaction rate. The epoxidation of alkenes in water/solvent two-phase systems is discussed to give an example of the scope of biotechnological processes that is obtained by using organic media. Finally, a design calculation of a packed-bed organic-liquid-phasel immobilized-biocatalyst reactor for the epoxidation of propene is given to illustrate some of the above aspects.     

Synthesis of pyridoxamine 5′-phosphate using an MBA:pyruvate transaminase as biocatalyst

Transaminases (TAs) have useful applications as biocatalysts because of their capability of introducing amino groups into ketones and keto acids with high enantioselectivity, regioselectivity and broad substrate specificity. In this study we have shown that purified His-tagged omega-TA CV2025 from Chromobacterium violaceum is capable of complete conversion of pyridoxal 5′-phosphate (PLP) to pyridoxamine 5′-phosphate (PMP) in the presence of (S)-α-methylbenzylamine (MBA) as the amine donor. Conversions of 5 mM PLP with at least 0.8 mg/ml CV2025 TA (5.8 U/ml) were complete within 24 h. The fastest completion was achieved with an enzyme concentration of 3 mg/ml (22 U/ml): Within 4 h 5 mM PLP/MBA were converted to 100% and 10 mM PLP/MBA to 70%. PLP amination was only partially inhibited in the presence of 0.5 mM gabaculine, whereas the MBA:pyruvate transamination was shown to be inhibited completely. PMP formation of comparable efficiency could not be achieved with equivalent units of porcine α-TA. This represents the first example of a PLP-converting TA with an attributed gene and the first demonstration of quantitative biocatalytic PMP synthesis.