Improving Product Specificity of Whole-Cell Alkane Oxidation in Nonconventional Media: A Multivariate Analysis Approach.

Two-liquid-phase reaction media have long been used in bioconversions to supply or remove hydrophobic organic reaction substrates and products to reduce inhibitory and toxic effects on biocatalysts. In case of the terminal oxyfunctionalization of linear alkanes by the AlkBGT monooxygenase the excess alkane substrate is often used as a second phase to extract the alcohol, aldehyde, and acid products. However, the selection of other carrier phases or surfactants is complex due to a large number of parameters that are involved, such as biocompatibility, substrate bioavailability, and product extraction selectivity. This study combines systematic high-throughput screening with chemometrics to correlate physicochemical parameters of a range of cosolvents to product specificity and yield using a multivariate regression model. Partial least-squares regression shows that the defining factor for product specificity is the solubility properties of the reaction substrate and product in the cosolvent, as measured by Hansen solubility parameters. Thus the polarity of cosolvents determines the accumulation of either alcohol or acid products. Whereas usually the acid product accumulates during the reaction, by choosing a more polar cosolvent the 1-alcohol product can be accumulated. Especially with Tergitol as a cosolvent, a 3.2-fold improvement in the 1-octanol yield to 18.3 mmol L-1 is achieved relative to the control reaction without cosolvents.

Customized microscale approach for optimizing two-phase bio-oxidations of alkanes with high reproducibility.

BACKGROUND: Numerous challenges remain to achieve industrially competitive space-time yields for bio-oxidations. The ability to rapidly screen bioconversion reactions for characterization and optimization is of major importance in bioprocess development and biocatalyst selection; studies at conventional lab scale are time consuming and labor intensive with low experimental throughput. The direct ω-oxyfunctionalization of aliphatic alkanes in a regio- and chemoselective manner is efficiently catalyzed by monooxygenases such as the AlkBGT enzyme complex from Pseudomonas putida under mild conditions. However, the adoption of microscale tools for these highly volatile substrates has been hindered by excessive evaporation and material incompatibility. RESULTS: This study developed and validated a robust high-throughput microwell platform for whole-cell two-liquid phase bio-oxidations of highly volatile n-alkanes. Using microwell plates machined from polytetrafluoroethylene and a sealing clamp, highly reproducible results were achieved with no significant variability such as edge effects determined. A design of experiment approach using a response surface methodology was adopted to systematically characterize the system and identify non-limiting conditions for a whole cell bioconversion of dodecane. Using resting E. coli cells to control cell concentration and reducing the fill volume it is possible to operate in non-limiting conditions with respect to oxygen and glucose whilst achieving relevant total product yields (combining 1-dodecanol, dodecanal and dodecanoic acid) of up to 1.5 mmol g DCW-1 . CONCLUSIONS: Overall, the developed microwell plate greatly improves experimental throughput, accelerating the screening procedures specifically for biocatalytic processes in non-conventional media. Its simplicity, robustness and standardization ensure high reliability of results.

Scalable preparation of silCoat-biocatalysts by use of a fluidized-bed reactor.

SilCoat-biocatalysts are immobilized enzyme preparations with an outstanding robustness against leaching and mechanical stress and therefore promising tools for technical synthesis. They consist of a composite material made from a solid enzyme carrier and silicone. In this study, a method has been found to enable provision of these catalysts in large scale. It makes use of easily scalable fluidized-bed technology and, in contrast to the original method, works in almost complete absence of organic solvent. Thus, it is both a fast and safe method. When the Pt-catalyst required for silicone formation is cast on the solid enzyme carrier before coating, resulting composites resemble the original preparations in morphology, catalytic activity, and stability against leaching and mechanical forces. Only the maximum total content of silicone in the composites lies about 10% w/w lower resulting in an overall leaching stability below the theoretical maximum. When the Pt-catalyst is mixed with cooled siloxane solution before coating, surficial coating of the enzyme carriers is achieved, which provides maximum leaching stability at very low silicone consumption. Thus, the technology offers the possibility to produce both composite and for the first time also core-shell silCoat-particles, and optimize leaching stability over mechanical strength according to process requirements.

The Power of Biocatalysis: A One-Pot Total Synthesis of Rhamnolipids from Butane as the Sole Carbon and Energy Source.

Microbially derived surfactants, so-called biosurfactants, have drawn much attention in recent years and are expected to replace current petrochemical surfactants, owing to their environmental and toxicological benefits. One strategy to support that goal is to reduce production costs by replacing relatively expensive sugars with cheaper raw materials, such as short-chain alkanes. Herein, we report the successful one-pot total synthesis of rhamnolipids, a class of biosurfactants with 12 stereocenters, from butane as sole carbon and energy source through the design of a tailored whole-cell biocatalyst.

Immobilised lipases in the cosmetics industry.

Commercial products for personal care, generally perceived as cosmetics, have an important impact on everyday life worldwide. Accordingly, the market for both consumer products and specialty chemicals comprising their ingredients is considerable. Lipases have started to play a minor role as active ingredients in so-called 'functional cosmetics' as well as a major role as catalysts for the industrial production of various specialty esters, aroma compounds and active agents. Interestingly, both applications almost always require preparation by appropriate immobilisation techniques. In addition, for catalytic use special reactor concepts often have to be employed due to the mostly limited stability of these preparations. Nevertheless, these processes show distinct advantages based on process simplification, product quality and environmental footprint and are therefore apt to more and more replace traditional chemical processes. Here, for the first time a review on the various aspects of using immobilised lipases in the cosmetics industry is given.

The metagenome-derived enzymes LipS and LipT increase the diversity of known lipases.

Triacylglycerol lipases (EC 3.1.1.3) catalyze both hydrolysis and synthesis reactions with a broad spectrum of substrates rendering them especially suitable for many biotechnological applications. Most lipases used today originate from mesophilic organisms and are susceptible to thermal denaturation whereas only few possess high thermotolerance. Here, we report on the identification and characterization of two novel thermostable bacterial lipases identified by functional metagenomic screenings. Metagenomic libraries were constructed from enrichment cultures maintained at 65 to 75 °C and screened resulting in the identification of initially 10 clones with lipolytic activities. Subsequently, two ORFs were identified encoding lipases, LipS and LipT. Comparative sequence analyses suggested that both enzymes are members of novel lipase families. LipS is a 30.2 kDa protein and revealed a half-life of 48 h at 70 °C. The lipT gene encoded for a multimeric enzyme with a half-life of 3 h at 70 °C. LipS had an optimum temperature at 70 °C and LipT at 75 °C. Both enzymes catalyzed hydrolysis of long-chain (C(12) and C(14)) fatty acid esters and additionally hydrolyzed a number of industry-relevant substrates. LipS was highly specific for (R)-ibuprofen-phenyl ester with an enantiomeric excess (ee) of 99%. Furthermore, LipS was able to synthesize 1-propyl laurate and 1-tetradecyl myristate at 70 °C with rates similar to those of the lipase CalB from Candida antarctica. LipS represents the first example of a thermostable metagenome-derived lipase with significant synthesis activities. Its X-ray structure was solved with a resolution of 1.99 Å revealing an unusually compact lid structure.

Simultaneous determination of mono-, di-, and triglycerides in multiphase systems by online Fourier transform infrared spectroscopy.

Glycerides are of significant value for industry as ingredients with different purposes in food or cosmetics. The analysis of glycerides is mainly performed by gas chromatography (GC) or high-pressure liquid chromatography (HPLC), which demonstrate limitations in dealing with multiphase systems. In this article, an in situ differentiation between mono-, di-, and triglycerides in multiphase systems by Fourier transform infrared (FT-IR) spectroscopy is demonstrated. The enzymatic esterification of glycerol with lauric acid was analyzed as a model system. The reaction was carried out in a bubble column reactor containing four phases (two liquid phases of glycerol and lauric acid, air as gaseous phase, and a heterogeneous catalyst as solid phase). As a feasibility study, a chemometric model was generated for the pure components only. The quantities of lauric acid and the three products (mono-, di-, and trilaurin) were simultaneously determined over the course of the reaction with acceptable errors (1.8-12.5%) with regard to the calibration effort. This technology has the potential to give accurate results, particularly in unstable emulsion systems containing fats, oils, or emulsifiers, which are currently afflicted by analytical errors caused by the challenge of accurate sampling.

Online monitoring of biotransformations in high viscous multiphase systems by means of FT-IR and chemometrics.

In unstable emulsion systems, the determination of concentrations is a challenge. The use of standard methods like GC, HPLC, or titration is highly inaccurate and makes the acquisition of precise data for these systems complex. In addition, the handicap of high viscosity often comes into play. To overcome these fundamental limitations, the online FT-IR technique was identified in combination with chemometric modeling in order to improve accuracy. The reactor type used in this study is a bubble column reactor with up to four dispersed phases (solid catalyst, two liquid immiscible substrates, and a gaseous phase). The investigated reactions are solvent free enzymatic esterifications yielding myristyl myristate (10 mPa s) and high viscous polyglycerol-3-laurate (300-1500 mPa s), representative industrial products for cosmetic applications. For both reactions, chemometric models were successfully set up and reproducibly applied in the prediction of progress curves of a new set of experiments. This allows the automated determination of sensitive kinetic and thermodynamic data as well as reaction velocities in high viscous multiphase (bio)chemical systems.

Increasing the synthesis/hydrolysis ratio of aminoacylase 1 by site-directed mutagenesis.

Aminoacylase-1 from pig kidney (pAcy1) catalyzes the highly stereoselective acylation of amino acids, a useful conversion for the preparation of optically pure N-acyl-l-amino acids. The kinetic of this thermodynamically controlled conversion is determined by maximal velocities for synthesis (V(mS)) and hydrolysis (V(mH)) of the N-acyl-l-amino acid. To investigate which parameter affects maximal velocities, we focused on the proton acceptor potential of the catalytic base, E146, and studied the influence of the active site architecture on its contribution to the pKa of residue E146. The modeled structure of pAcy1 identified residue D346 as having the strongest impact on the electrostatic features of the catalytic base. Substitutions of D346 generally decreased enzymatic activities but also altered both the pH-dependency of hydrolytic activity and the V(mS)/V(mH) ratio of pAcy1. A reduced theoretical pKa value and a lowered experimental pH optimum of hydrolytic rates for the D346A mutant were associated with a 9-fold increase in V(mS)/V(mH). This supports the importance of electrostatic contributions of D346 to the acid-base properties of E146 and demonstrates for the first time the possibility of engineering the V(mS)/V(mH) ratio of pAcy1.

Functional expression of porcine aminoacylase 1 in E. coli using a codon optimized synthetic gene and molecular chaperones.

Efficient recombinant expression of N-acyl-L-aminoacylase 1 from pig kidney (pAcy1) was achieved in the prokaryotic host Escherichia coli. An optimized nucleotide sequence (codon adaptation index 0.95 for E. coli), was cloned into vector pET-52(b) yielding an E. coli-expressible pAcy1 gene. Formation of inclusion bodies was alleviated by co-expression of molecular chaperones resulting in 2.7- and 4.2-fold increased recovery of active pAcy1 using trigger factor or GroEL-GroES, respectively. Facile purification was achieved via StrepTag affinity chromatography. Overall, more than 80 mg highly active pAcy1 (94 U/mg) was obtained per liter of cultivation broth. The protein was analyzed for structural and functional identity, and the performances of further described expression and purification systems for pAcy1 were compared.

A versatile toolbox for variable DNA functionalization at high density.

To broaden the applicability of chemically modified DNAs in nano- and biotechnology, material science, sensor development, and molecular recognition, strategies are required for introducing a large variety of different modifications into the same nucleic acid sequence at once. Here, we investigate the scope and limits for obtaining functionalized dsDNA by primer extension and PCR, using a broad variety of chemically modified deoxynucleotide triphosphates (dNTPs), DNA polymerases, and templates. All natural nucleobases in each strand were substituted with up to four different base-modified analogues. We studied the sequence dependence of enzymatic amplification to yield high-density functionalized DNA (fDNA) from modified dNTPs, and of fDNA templates, and found that GC-rich sequences are amplified with decreased efficiency as compared to AT-rich ones. There is also a strong dependence on the polymerase used. While family A polymerases generally performed poorly on "demanding" templates containing consecutive stretches of a particular base, family B polymerases were better suited for this purpose, in particular Pwo and Vent (exo-) DNA polymerase. A systematic analysis of fDNAs modified at increasing densities by CD spectroscopy revealed that single modified bases do not alter the overall B-type DNA structure, regardless of their chemical nature. A density of three modified bases induces conformational changes in the double helix, reflected by an inversion of the CD spectra. Our study provides a basis for establishing a generally applicable toolbox of enzymes, templates, and monomers for generating high-density functionalized DNAs for a broad range of applications.

Functional tuning of nucleic acids by chemical modifications: tailored oligonucleotides as drugs, devices, and diagnostics.

Chemical modifications of nucleic acids present vast opportunities for extending the functions and properties of these biomolecules. In general, efforts invested in this direction pertain to the introduction of reactive functional groups for further derivatizations of oligonucleotides with numerous reporter groups and for equipping nucleic acids with catalytic chemical moieties. This review deals with representative chemical modifications in the nucleobases, sugars, and the phosphate ester backbone and their application from novel catalytic RNA selection to nucleic acid-based biosensors.

Functionalized DNA: A New Replicable Biopolymer.

New DNA: By enzymatic polymerization of base-modified nucleoside triphosphates, a functionalized DNA (fDNA; see picture) was generated in which every base bears an additional amino acid like residue, thus mimicking the functional group repertoire of peptides on a nucleic acid backbone. These modified oligonucleotides can in turn serve as templates for polymerase chain reaction amplification, thus utilizing fDNA as a novel class of biopolymers for in vitro selection techniques.