A versatile ceramic capillary membrane reactor system for continuous enzyme-catalyzed hydrolysis.

As an alternative to classical batch processes, enzyme-catalyzed hydrolysis can also be carried out continuously. To facilitate this, a continuous ceramic capillary membrane reactor system (CCCMRS) was developed which can be operated with various proteolytic enzymes immobilized on the porous ceramic capillary membranes. This system has several advantages over common batch processes regarding stability, reproducibility and controllability and can easily be adapted to optimal reaction conditions and individual preferences. Two exemplary applications utilizing the CCCMRS were carried out and investigated in long-term stability studies. In the first application the continuous enzymatic cleavage of human IgG into the antibody fragments Fab and Fc by immobilized papain was performed. A total volume of 22 mL of 1 mg mL-1 IgG-solution was enzymatically cleaved over a period of 33.3 h. The antibody cleavage products could be detected in an SEC-HPLC over the whole process time thus indicating long-term stability of the continuous hydrolysis process. The second application investigated the continuous digestion of pea and almond protein isolates by immobilized Alcalase resulting in the generation of a large variety of different peptides. This peptide fingerprint remains constant over a long period of time enabling fractionation and thus making the peptides accessible for further bioactivity studies in sufficient quantities. The constant peptide fingerprint could be shown in the RP-HPLC analysis for all 30 samples with a total volume of 29.7 mL collected over a period of 45 h.

Whole-Cell Production of Patchouli Oil Sesquiterpenes in Escherichia coli: Metabolic Engineering and Fermentation Optimization in Solid-Liquid Phase Partitioning Cultivation.

Patchouli oil is a major ingredient in perfumery, granting a dark-woody scent due to its main constituent (-)-patchoulol. The growing demand for patchouli oil has raised interest in the development of a biotechnological process to assure a reliable supply. Herein, we report the production of patchouli oil sesquiterpenes by metabolically engineered Escherichia coli strains, using solid-liquid phase partitioning cultivation. The (-)-patchoulol production was possible using the endogenous methylerythritol phosphate pathway and overexpressing a (-)-patchoulol synthase isoform from Pogostemon cablin but at low titers. To improve the (-)-patchoulol production, the exogenous mevalonate pathway was overexpressed in the multi-plasmid PTS + Mev strain, which increased the (-)-patchoulol titer 5-fold. Fermentation was improved further by evaluating several defined media, and optimizing the pH and temperature of culture broth, enhancing the (-)-patchoulol titer 3-fold. To augment the (-)-patchoulol recovery from fermentation, the solid-liquid phase partitioning cultivation was analyzed by screening polymeric adsorbers, where the Diaion HP20 adsorber demonstrated the highest (-)-patchoulol recovery from all tests. Fermentation was scaled-up to fed-batch bioreactors, reaching a (-)-patchoulol titer of 40.2 mg L-1 and productivity of 20.1 mg L-1 d-1. The terpene profile and aroma produced from the PTS + Mev strain were similar to the patchouli oil, comprising (-)-patchoulol as the main product, and α-bulnesene, trans-ß-caryophyllene, ß-patchoulene, and guaia-5,11-diene as side products. This investigation represents the first study of (-)-patchoulol production in E. coli by solid-liquid phase partitioning cultivation, which provides new insights for the development of sustainable bioprocesses for the microbial production of fragrant terpenes.

Comparison of different three dimensional-printed resorbable materials: In vitro biocompatibility, In vitro degradation rate, and cell differentiation support.

Biodegradable materials play a crucial role in both material and medical sciences and are frequently used as a primary commodity for implants generation. Due to their material inherent properties, they are supposed to be entirely resorbed by the patients' body after fulfilling their task as a scaffold. This makes a second intervention (e.g. for implant removal) redundant and significantly enhances a patient's post-operative life quality. At the moment, materials for resorbable and biodegradable implants (e.g. polylactic acid or poly-caprolactone polymers) are still intensively studied. They are able to provide mandatory demands such as mechanical strength and attributes needed for high-quality implants. Implants, however, not only need to be made of adequate material, but must also to be personalized in order to meet the customers' needs. Combining three dimensional-printing and high-resolution imaging technologies a new age of implant production comes into sight. Three dimensional images (e.g. magnetic resonance imaging or computed tomography) of tissue defects can be utilized as digital blueprints for personalized implants. Modern additive manufacturing devices are able to use a variety of materials to fabricate custom parts within short periods of time. The combination of high-quality resorbable materials and personalized three dimensional-printing for the custom application will provide the patients with the best suitable and sustainable implants. In this study, we evaluated and compared four resorbable and three dimensional printable materials for their in vitro biocompatibility, in vitro rate of degradation, cell adherence and behavior on these materials as well as support of osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells. The tests were conducted with model constructs of 1 cm2 surface area fabricated with fused deposition modeling three dimensional-printing technology.

Bioproduction of α-humulene in metabolically engineered Escherichia coli and application in zerumbone synthesis.

Zerumbone is a sesquiterpene ketone with potent anti-cancerogenic activities, produced in several ginger species of the Zingiberaceae familiy. We have investigated the biotechnological production of α-humulene, a precursor of zerumbone. By implementing a heterologous mevalonate pathway in combination with the α-humulene synthase expression, we effectively synthesized α-humulene from glucose in Escherichia coli. In this study, we developed a practical and efficient in situ separation method for α-humulene by comparison of extractive and adsorptive strategies. By the in situ adsorption of the product to the hydrophobic resin Amberlite® XAD4 we were able to increase α-humulene yield by 2310% to 60.2 mg/L. Furthermore we present an easy applicable, short subsequent chemical process for the conversion of α-humulene to zerumbone by using transition metal catalysis. To reduce process steps, the chemical reaction was carried out in the same solvent as the eluting solvent that was used to elute α-humulene from the adsorbent resin. By allylic oxidation of α-humulene with manganeseII chloride as a catalyst and tert.-butylhydroperoxide as an oxidizing agent we were able to synthetize zerumbone with a selectivity of 51.6%. Product and byproducts of the oxidation reaction were identified by GC-MS.

Online analysis of protein inclusion bodies produced in E. coli by monitoring alterations in scattered and reflected light.

The online monitoring of recombinant protein aggregate inclusion bodies during microbial cultivation is an immense challenge. Measurement of scattered and reflected light offers a versatile and non-invasive measurement technique. Therefore, we investigated two methods to detect the formation of inclusion bodies and monitor their production: (1) online 180° scattered light measurement (λ = 625 nm) using a sensor platform during cultivation in shake flask and (2) online measurement of the light reflective interference using a porous Si-based optical biosensor (SiPA). It could be shown that 180° scattered light measurement allows monitoring of alterations in the optical properties of Escherichia coli BL21 cells, associated with the formation of inclusion bodies during cultivation. A reproducible linear correlation between the inclusion body concentration of the non-fluorescent protein human leukemia inhibitory factor (hLIF) carrying a thioredoxin tag and the shift ("Δamp") in scattered light signal intensity was observed. This was also observed for the glutathione-S-transferase-tagged green fluorescent protein (GFP-GST). Continuous online monitoring of reflective interference spectra reveals a significant increase in the bacterium refractive index during hLIF production in comparison to a non-induced reference that coincide with the formation of inclusion bodies. These online monitoring techniques could be applied for fast and cost-effective screening of different protein expression systems.

A new set up for multi-analyte sensing: at-line bio-process monitoring.

A multi-analyte sensing device is described, for simultaneous at-line monitoring of glucose, ethanol, pO2-value and cell density. It consists of a dual biosensor, a modified microscope and a fiber optical pO2-sensor that are integrated into a flow analysis (FA) system. The biosensor is based on a conventional thin layer flow-through cell equipped with a gold (Au) dual electrode (serial configuration). The biosensors with no cross-talking were produced by modifying the electrochemical transducers. Each Au surface was initially modified by self-assembled monolayer (SAM) of cysteamine. Alcohol oxidase (AOx) and pyranose oxidase (PyOx) were immobilized each onto a gold surface by means of PAMAM (polyamidoamine) dendrimer via glutaraldehyde cross-linking. The responses for glucose and ethanol were linear up to 0.5 mM. The operational stability of the biosensors was very promising, after 11 h continuous operation, only 6.0% of the initial activity was lost. The potential of the described biosensor was demonstrated by parallel determination of ethanol and glucose in yeast fermentation process. Simultaneously the cell density of the culture was monitored with an in situ microscope (ISM), which was integrated into the FA system. Both the used in situ microscope and the image processing algorithm used for the analysis of the acquired image data are described. Furthermore the pO2-value was monitored using a fiber optical sensor, which was embedded in a flow cell. The multi-sensor device allows the at-line monitoring of several process values without the need for further sampling or time consuming offline measurements.

Offline glucose biomonitoring in yeast culture by polyamidoamine/cysteamine-modified gold electrodes.

This article deals with the use of pyranose oxidase (PyOx) and glucose oxidase (GOx) enzymes in amperometric biosensor design and their application in monitoring fermentation processes with the combination of flow injection analysis (FIA). The amperometric studies were carried out at -0.7 V by following the oxygen consumption due to the enzymatic reactions for both batch and FIA modes. Optimization studies (enzyme amounts and pH) and analytical parameters such as linearity, repeatability, effect of interference, storage, and operational stabilities have been studied. Under optimized conditions, for the PyOx-based biosensor, linear graph was obtained from 0.025 to 0.5 mM glucose in phosphate buffer (50 mM) at pH 7.0 with the equation of y = 3.358x + 0.028 and R(2) = 0.998. Linearity was found to be 0.01-1.0 mM in citrate buffer (50 mM and pH 4.0) with the equation of y = 1.539x + 0.181 and R(2) = 0.992 for the GOx biosensor. Finally, these biosensor configurations were further evaluated in a conventional flow injection system. Results from batch experiments provide a guide to design sensitive, stable, and interference-free biosensors for FIA mode. Biosensor stability, dynamic range, and repeatability were also studied in FIA conditions, and the applicability for the determination of glucose in fermentation medium could be successfully demonstrated. The FIA-combined glucose biosensor was used for the offline monitoring of yeast fermentation. The obtained results correlated well with HPLC measurements.

Alcohol biosensing by polyamidoamine (PAMAM)/cysteamine/alcohol oxidase-modified gold electrode.

A highly stable and sensitive amperometric alcohol biosensor was developed by immobilizing alcohol oxidase (AOX) through Polyamidoamine (PAMAM) dendrimers on a cysteamine-modified gold electrode surface. Ethanol determination is based on the consumption of dissolved oxygen content due to the enzymatic reaction. The decrease in oxygen level was monitored at -0.7 V vs. Ag/AgCl and correlated with ethanol concentration. Optimization of variables affecting the system was performed. The optimized ethanol biosensor showed a wide linearity from 0.025 to 1.0 mM with 100 s response time and detection limit of (LOD) 0.016 mM. In the characterization studies, besides linearity some parameters such as operational and storage stability, reproducibility, repeatability, and substrate specificity were studied in detail. Stability studies showed a good preservation of the bioanalytical properties of the sensor, 67% of its initial sensitivity was kept after 1 month storage at 4 degrees C. The analytical characteristics of the system were also evaluated for alcohol determination in flow injection analysis (FIA) mode. Finally, proposed biosensor was applied for ethanol analysis in various alcoholic beverage as well as offline monitoring of alcohol production through the yeast cultivation.

Development of a fast spectroscopic enzyme assay for on-site measurement of total polyphenol content in grapes and wine.

In recent years interest in polyphenols as a nutrient in vegetables and fruits has increased because of polyphenols' positive effects on human health. The interest focuses on the sensory properties of polyphenols and their influence on the taste of fruits and derived products. This article presents the development of a bioanalytical measurement technique enabling the determination of the total polyphenol content (TPC) of fresh grapes within a few minutes. Furthermore this technique allows the control of TPC during production processes, e. g. fermentation of wine.