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Developing a new production host from a blueprint: Bacillus pumilus as an industrial enzyme producer
(2014)
Therefore Fermat is right
(2014)
It was Fernat's idea to investigate how many numbers would fulfill the equation according to the Pythagorean Theorem if the exponent were increased to random, e.g. to a3 + b3 = c3. His question became therefore: are there two whole numbers the cubes of which add up to the volume of the cube of a third whole number? He posed this same question, of course, for all kinds of higher exponents, so that the equation could be generalized: is there an integral solution for the equation an + bn = cn, if the exponent n is higher than 2? Although in 1993, the English mathematician Andrew Wiles was able to produce an arithmetical proof for Fermat's famous theorem, I will show that there is a simple logical explanation which is also pragmatic and plausible and what is the result of a fundamental alternative idea how our world seems to be constructed.
This paper develops a new finite element method (FEM)-based upper bound algorithm for limit and shakedown analysis of hardening structures by a direct plasticity method. The hardening model is a simple two-surface model of plasticity with a fixed bounding surface. The initial yield surface can translate inside the bounding surface, and it is bounded by one of the two equivalent conditions: (1) it always stays inside the bounding surface or (2) its centre cannot move outside the back-stress surface. The algorithm gives an effective tool to analyze the problems with a very high number of degree of freedom. Our numerical results are very close to the analytical solutions and numerical solutions in literature.
Among the variety of transducer concepts proposed for label-free detection of biomolecules, the semiconductor field-effect device (FED) is one of the most attractive platforms. As medical techniques continue to progress towards diagnostic and therapies based on biomarkers, the ability of FEDs for a label-free, fast and real-time detection of multiple pathogenic and physiologically relevant molecules with high specificity and sensitivity offers very promising prospects for their application in point-of-care and personalized medicine for an early diagnosis and treatment of diseases. The presented paper reviews recent advances and current trends in research and development of different FEDs for label-free, direct electrical detection of charged biomolecules by their intrinsic molecular charge. The authors are mainly focusing on the detection of the DNA hybridization event, antibody-antigen affinity reaction as well as clinically relevant biomolecules such as cardiac and cancer biomarkers.
A multi-spot (4 × 4 spots) light-addressable potentiometric sensor (MLAPS) consisting of an Al–p-Si–SiO2 structure has been applied for the label-free electrical detection of DNA (deoxyribonucleic acid) immobilization and hybridization by the intrinsic molecular charge for the first time. Single-stranded probe ssDNA molecules (20 bases) were covalently immobilized onto the silanized SiO2 gate surface. The unspecific adsorption of mismatch ssDNA on the MLAPS gate surface was blocked by bovine serum albumin molecules. To reduce the screening effect and to achieve a high sensor signal, the measurements were performed in a low ionic-strength solution. The photocurrent–voltage (I–V) curves were simultaneously recorded on all 16 spots after each surface functionalization step. Large shifts of I–V curves of 25 mV were registered after the DNA immobilization and hybridization event. In contrast, a small potential shift (∼5 mV) was observed in case of mismatch ssDNA, revealing good specificity of the sensor. The obtained results demonstrate the potential of the MLAPS as promising transducer platform for the multi-spot label-free electrical detection of DNA molecules by their intrinsic molecular charge.
The metabolic activity of Chinese hamster ovary (CHO) cells was observed using a light-addressable potentiometric sensor (LAPS). The dependency toward different glucose concentrations (17–200 mM) follows a Michaelis–Menten kinetics trajectory with Kₘ = 32.8 mM, and the obtained Kₘ value in this experiment was compared with that found in literature. In addition, the pH shift induced by glucose metabolism of tumor cells transfected with the HPV-16 genome (C3 cells) was successfully observed. These results indicate the possibility to determine the tumor cells metabolism with a LAPS-based measurement device.
The light-addressable potentiometric sensor (LAPS) is a semiconductor-based potentiometric sensor using a light probe with an ability of detecting the concentration of biochemical species in a spatially resolved manner. As an important biomedical sensor, research has been conducted to improve its performance, for instance, to realize high-speed measurement. In this work, the idea of facilitating the device-level simulation, instead of using an equivalent-circuit model, is presented for detailed analysis and optimization of the performance of the LAPS. Both carrier distribution and photocurrent response have been simulated to provide new insight into both amplitude-mode and phase-mode operations of the LAPS. Various device parameters can be examined to effectively design and optimize the LAPS structures and setups for enhanced performance.
Planar and three-dimensional (3D) interdigitated electrodes (IDE) with electrode digits separated by an insulating barrier of different heights were electrochemically characterized and compared in terms of their sensing properties. Due to the impact of the surface resistance, both types of IDE structures display a non-linear behavior in low-ionic strength solutions. The experimental data were fitted to an electrical equivalent circuit and interpreted taking into account the surface-charge-governed properties. The effect of a charged polyelectrolyte layer electrostatically assembled onto the sensor surface on the surface resistance in solutions with different KCl concentration is studied. In case of the same electrode footprint, 3D-IDEs show a larger cell constant and a higher sensitivity to molecular adsorption than that of planar IDEs. The obtained results demonstrate the potential of 3D-IDEs as a new transducer structure for a direct label-free sensing of charged molecules.
It is well known that biochemical and biotechnological processes are strongly dependent and affected by a variety of physico-chemical parameters such as pH value, temperature, pressure and electrolyte conductivity. Therefore, these quantities have to be monitored or controlled in order to guarantee a stable process operation, optimization and high yield. In this work, a sensor chip for the multiparameter detection of three physico-chemical parameters such as electrolyte conductivity, pH and temperature is realized using barium strontium titanate (BST) as multipurpose material. The chip integrates a capacitively coupled four-electrode electrolyte-conductivity sensor, a capacitive field-effect pH sensor and a thin-film Pt-temperature sensor. Due to the multifunctional properties of BST, it is utilized as final outermost coating layer of the processed sensor chip and serves as passivation and protection layer as well as pH-sensitive transducer material at the same time. The results of testing of the individual sensors of the developed multiparameter sensor chip are presented. In addition, a quasi-simultaneous multiparameter characterization of the sensor chip in buffer solutions with different pH value and electrolyte conductivity is performed. To study the sensor behavior and the suitability of BST as multifunctional material under harsh environmental conditions, the sensor chip was exemplarily tested in a biogas digestate.
Clostridium propionicum is the only organism known to ferment β-alanine, a constituent of coenzyme A (CoA) and the phosphopantetheinyl prosthetic group of holo-acyl carrier protein. The first step in the fermentation is a CoA-transfer to β-alanine. Subsequently, the resulting β-alanyl-CoA is deaminated by the enzyme β-alanyl-CoA:ammonia lyase (Acl) to reversibly form ammonia and acrylyl-CoA. We have determined the crystal structure of Acl in its apo-form at a resolution of 0.97 Å as well as in complex with CoA at a resolution of 1.59 Å. The structures reveal that the enyzme belongs to a superfamily of proteins exhibiting a so called “hot dog fold” which is characterized by a five-stranded antiparallel β-sheet with a long α-helix packed against it. The functional unit of all “hot dog fold” proteins is a homodimer containing two equivalent substrate binding sites which are established by the dimer interface. In the case of Acl, three functional dimers combine to a homohexamer strongly resembling the homohexamer formed by YciA-like acyl-CoA thioesterases. Here, we propose an enzymatic mechanism based on the crystal structure of the Acl·CoA complex and molecular docking. Proteins 2014; 82:2041–2053. © 2014 Wiley Periodicals, Inc.
In this work, the catalyst manganese(IV) oxide (MnO2), of calorimetric gas sensors (to monitor the sterilization agent vaporized hydrogen peroxide) has been investigated in more detail. Chemical analyses by means of X-ray-induced photoelectron spectroscopy have been performed to unravel the surface chemistry prior and after exposure to hydrogen peroxide vapor at elevated temperature, as applied in the sterilization processes of beverage cartons. The surface characterization reveals a change in oxidation states of the metal oxide catalyst after exposure to hydrogen peroxide. Additionally, a cleaning effect of the catalyst, which itself is attached to the sensor surface by means of a polymer interlayer, could be observed.
The Dry Low NOx (DLN) Micromix combustion principle with increased energy density is adapted for the industrial gas turbine APU GTCP 36-300 using hydrogen and hydrogen-rich syngas with a composition of 90%-Vol. hydrogen (H₂) and 10%-Vol. carbon-monoxide (CO). Experimental and numerical studies of several combustor geometries for hydrogen and syngas show the successful advance of the DLN Micromix combustion from pure hydrogen to hydrogen-rich syngas. The impact of the different fuel properties on the combustion principle and aerodynamic flame stabilization design laws, flow field, flame structure and emission characteristics is investigated by numerical analysis using a hybrid Eddy Break Up combustion model and validated against experimental results.
It is well known that the degradation environment can strongly influence the biodegradability and kinetics of biodegradation processes of polymers. Therefore, besides the monitoring of the degradation process, it is also necessary to control the medium in which the degradation takes place. In this work, a micromachined multi-parameter sensor chip for the control of the polymer-degradation medium has been developed. The chip combines a capacitive field-effect pH sensor, a four-electrode electrolyte-conductivity sensor and a thin-film Pt-temperature sensor. The results of characterization of individual sensors are presented. In addition, the multi-parameter sensor chip together with an impedimetric polymer-degradation sensor was simultaneously characterized in degradation solutions with different pH and electrolyte conductivity. The obtained results demonstrate the feasibility of the multi-parameter sensor chip for the control of the polymer-degradation medium.
Multimodal bioimage sensor
(2014)
To visualize the biochemical distribution two-dimensionally, we invented a solid-state-type ion image sensor that indicates the chemical activity of solutions and cells. The device, which consists of a CCD array covered with a functionalized membrane to detect charge accumulation, is highly sensitive to changes in the concentration and two-dimensional distribution of ions and biomaterials.
Background
True date palms (Phoenix dactylifera L.) are impressive trees and have served as an indispensable source of food for mankind in tropical and subtropical countries for centuries. The aim of this study is to differentiate date palm tree varieties by analysing leaflet cross sections with technical/optical methods and artificial neural networks (ANN).
Results
Fluorescence microscopy images of leaflet cross sections have been taken from a set of five date palm tree cultivars (Hewlat al Jouf, Khlas, Nabot Soltan, Shishi, Um Raheem). After features extraction from images, the obtained data have been fed in a multilayer perceptron ANN with backpropagation learning algorithm.
Conclusions
Overall, an accurate result in prediction and differentiation of date palm tree cultivars was achieved with average prediction in tenfold cross-validation is 89.1% and reached 100% in one of the best ANN.
The Gram-positive endospore-forming bacterium Bacillus licheniformis can be found widely in nature and it is exploited in industrial processes for the manufacturing of antibiotics, specialty chemicals, and enzymes. Both in its varied natural habitats and in industrial settings, B. licheniformis cells will be exposed to increases in the external osmolarity, conditions that trigger water efflux, impair turgor, cause the cessation of growth, and negatively affect the productivity of cell factories in biotechnological processes. We have taken here both systems-wide and targeted physiological approaches to unravel the core of the osmostress responses of B. licheniformis. Cells were suddenly subjected to an osmotic upshift of considerable magnitude (with 1 M NaCl), and their transcriptional profile was then recorded in a time-resolved fashion on a genome-wide scale. A bioinformatics cluster analysis was used to group the osmotically up-regulated genes into categories that are functionally associated with the synthesis and import of osmostress-relieving compounds (compatible solutes), the SigB-controlled general stress response, and genes whose functional annotation suggests that salt stress triggers secondary oxidative stress responses in B. licheniformis. The data set focusing on the transcriptional profile of B. licheniformis was enriched by proteomics aimed at identifying those proteins that were accumulated by the cells through increased biosynthesis in response to osmotic stress. Furthermore, these global approaches were augmented by a set of experiments that addressed the synthesis of the compatible solutes proline and glycine betaine and assessed the growth-enhancing effects of various osmoprotectants. Combined, our data provide a blueprint of the cellular adjustment processes of B. licheniformis to both sudden and sustained osmotic stress.
The composition and physiochemical properties of aquatic-phase natural organic matter (NOM) are most important problems for both environmental studies and water industry. Laser desorption/ionization (LDI) mass spectrometry facilitated successful examinations of NOM, as humic and fulvic acids in NOM are readily ionized by the nitrogen laser. In this study, hydrophobic NOMs (HPO NOMs) from river, reservoir and waste water were characterized by this technique. The effect of analytical variables like concentration, solvent composition and laser energy was investigated. The exact masses of small molecular NOM moieties in the range of 200–1200 m/z were determined in reflectron mode. In addition, spectra of post-source-decay experiments in this range showed that some compounds from different natural NOMs had the same fragmental ions. In the large mass range of 1200–15 000 Da, macromolecules and their aggregates were found in HPO NOMs from natural waters. Highly humic HPO exhibited mass peaks larger than 8000 Da. On the other hand, the waste water and reservoir water mainly had relatively smaller molecules of about 2000 Da. The LDI-MS measurements indicated that highly humic river waters were able to form large aggregates and membrane foulants, while the HPO NOMs from waste water and reservoir water were unlikely to form large aggregates. Copyright © 2014 John Wiley & Sons, Ltd.
Knowledge-based productivity in “low-tech” industries: evidence from firms in developing countries
(2014)
Using firm-level data from five developing countries—Brazil, Ecuador, South Africa, Tanzania, and Bangladesh—and three industries—food processing, textiles, and the garments and leather products—this article examines the importance of various sources of knowledge for explaining productivity and formally tests whether sector- or country-specific characteristics dominate these relationships. Knowledge sources driving productivity appear mainly sector specific. Also differences in the level of development affect the effectiveness of knowledge sources. In the food processing sector, firms with higher educated managers are more productive, and in least-developed countries, additionally those with technology licenses and imported machinery and equipment. In the capital-intensive textiles sector, productivity is higher in firms that conduct R&D. In the garments and leather products sector, higher education of the managers, licensing, and R&D raise productivity.
An enzyme system organized in a flow device was used to mimic a reversible Controlled NOT (CNOT) gate with two input and two output signals. Reversible conversion of NAD⁺ and NADH cofactors was used to perform a XOR logic operation, while biocatalytic hydrolysis of p-nitrophenyl phosphate resulted in an Identity operation working in parallel. The first biomolecular realization of a CNOT gate is promising for integration into complex biomolecular networks and future biosensor/biomedical applications.
In any books about genetics it can still today be read that our genetic code is called “degenerate” because it is still believed that 43 = 64 triplets encode the 20 essential amino acids. Indeed we have to assume the inverse law, what means that 34 = 81 exact code positions are really effective for our genetic code and encode the amino acids, compiled to proteins. This very important discovery leads to two completely new results that are limits-overlooking: 1) 34 (=81) genetic code positions mean exactly the same number as there are stable and naturally existing chemical elements in our universe. This famous argument should now lead to some alternative, as well as new fundamental conclusions about our existence. 2) A genetic code positioning system shows that nature is much smarter than expected: mutations are made less dangerous than believed, because they won't be that easily able any more to cause severe damages in the protein-synthesis. This should also lead to some alternative views upon evolution of life.
Bacillus pumilus reveals a remarkably high resistance to hydrogen peroxide provoked oxidative stress
(2014)
Bacillus pumilus is characterized by a higher oxidative stress resistance than other comparable industrially relevant Bacilli such as B. subtilis or B. licheniformis. In this study the response of B. pumilus to oxidative stress was investigated during a treatment with high concentrations of hydrogen peroxide at the proteome, transcriptome and metabolome level. Genes/proteins belonging to regulons, which are known to have important functions in the oxidative stress response of other organisms, were found to be upregulated, such as the Fur, Spx, SOS or CtsR regulon. Strikingly, parts of the fundamental PerR regulon responding to peroxide stress in B. subtilis are not encoded in the B. pumilus genome. Thus, B. pumilus misses the catalase KatA, the DNA-protection protein MrgA or the alkyl hydroperoxide reductase AhpCF. Data of this study suggests that the catalase KatX2 takes over the function of the missing KatA in the oxidative stress response of B. pumilus. The genome-wide expression analysis revealed an induction of bacillithiol (Cys-GlcN-malate, BSH) relevant genes. An analysis of the intracellular metabolites detected high intracellular levels of this protective metabolite, which indicates the importance of bacillithiol in the peroxide stress resistance of B. pumilus.
In this study, a high-speed chemical imaging system was developed for visualization of the interior of a microfluidic channel. A microfluidic channel was constructed on the sensor surface of the light-addressable potentiometric sensor (LAPS), on which the ion concentrations could be measured in parallel at up to 64 points illuminated by optical fibers. The temporal change of pH distribution inside the microfluidic channel was recorded at a maximum rate of 100 frames per second (fps). The high frame rate allowed visualization of moving interfaces and plugs in the channel even at a flow velocity of 111 mm/s, which suggests the feasibility of plug-based microfluidic devices for flow-injection analysis (FIA).
Synthesis of derivatives of the peptide sequence L-pyroglutamyl-L-phenylalanyl-L-aspartyl-glycyl-L-lysyl-glycyl-glycyl-glycine as the antigenic determinant representing the N-terminal non-helical region of the α-2-chain of rabbit skin collagen, and conjugation to two different polypeptide carriers, are described.
Thermal synthesis of the optical pure pentapeptide derivative Z-(L)-Ala-(L)-Phe-Gly-(L)-Phe-Gly-OMe
(1980)
In this work the transient simulations of four hybrid solar tower power plant concepts with open-volumetric receiver technology for a location in Barstow-Daggett, USA, are presented. The open-volumetric receiver uses ambient air as heat transfer fluid and the hybridization is realized with a gas turbine. The Rankine cycle is heated by solar-heated air and/or by the gas turbine's flue gases. The plant can be operated in solar-only, hybrid parallel or combined cycle-only mode as well as in any intermediate load levels where the solar portion can vary between 0 to 100%.
The simulated plant is based on the configuration of a solar-hybrid power tower project, which is in planning for a site in Northern Algeria. The meteorological data for Barstow-Daggett was taken from the software meteonorm. The solar power tower simulation tool has been developed in the simulation environment MATLAB/Simulink and is validated.
This paper describes the modeling of a high-temperature storage system for an existing solar tower power plant with open volumetric receiver technology, which uses air as heat transfer medium (HTF). The storage system model has been developed in the simulation environment Matlab/Simulink®. The storage type under investigation is a packed bed thermal energy storage system which has the characteristics of a regenerator. Thermal energy can be stored and discharged as required via the HTF air. The air mass flow distribution is controlled by valves, and the mass flow by two blowers. The thermal storage operation strategy has a direct and significant impact on the energetic and economic efficiency of the solar tower power plants.
Selective laser melting of metals: desktop machines open up new chances even for small companies
(2012)
Additive manufacturing (AM) of metal parts by using Selective Laser Melting (SLM) has become a powerful tool mostly in the area of automotive, aerospace engineering and others. Especially in the field of dentistry, jewelry and related branches that require individualized or even one-of-a-kind products, the direct digital manufacturing process opens up new ways of design and manufacturing. In these fields, mostly small and medium sized businesses (SME) are operating which do not have sufficient human and economic resources to invest in this technology. But to stay competitive, the application of AM can be regarded as a necessity. In this situation a new desktop machine (Realizer SLM 50) was introduced that cost about 1/3 of a shop floor SLM machine and promises small quality parts. To find out whether the machine really is an alternative for SMEs the University of Applied Science, Aachen, Germany, designed, build and optimized typical parts from the dentistry and the jewelry branches using CoCr and silver material, the latter being new with this application. The paper describes the SLM procedure and how to find and optimize the most important parameters. The test is accompanied by digital simulation in order to verify the build parameters and to plan future builds. The procedure is shown as well as the resulting parts made from CoCr and silver material.
Optical coherence tomography : a potential tool to predict premature rupture of fetal membranes
(2013)
Since several decades, dam-break waves have been of main research interest. Mathematical approaches have been developed by analytical, physical and numerical models within the past 120 years. During the past 10 years, the number of research investigations has increased due to improved measurement techniques as well as significantly increased computer memories and performances. In this context, the present research deals with the initial stage of two-dimensional dam-break waves by comparing physical and numerical model results as well as analytical approaches. High-speed images and resulting particle image velocimetry calculations are thereby compared with the numerical volume-of-fluid (VOF) method, included in the commercial code FLOW-3D. Wave profiles and drag forces on placed obstacles are analysed in detail. Generally, a good agreement between the laboratory and VOF results is found.
Risk factors for cardiovascular calcifications in non-diabetic Caucasian haemodialysis patients
(2009)
In this study we observed courses of micturition symptoms and differentiated degrees of symptoms for each point in time while also considering the impact of bothersomeness. Our data show that not only significantly more patients who have undergone BT suffer from OAB than those who have undergone RP, but also that those affected show significantly higher values for severity of OAB symptoms throughout the whole observation period of 36 months. Our data analysis further shows that variability of OAB symptoms as well as fluctuation of severity of OAB symptoms vary to a significantly higher degree after BT than after RP. Looking only at mean figures at a given point in time clearly underestimates the underlying problem. This fact is not reflected in the literature.
Poly(vinyl acetate), PVAc, with a degree of polymerization Xn = 10 was prepared by chain-transfer radical polymerization using carbon tetrachloride and used as oligomeric plasticizer for commercial PVAc. However, the chlorinated chain ends cause a low thermal stability requiring mild Cl/H substitution. The product exhibits high thermal stability and excellent melt-compounding properties. Blends of oligomeric and commercial PVAc show single glass transition temperatures which decrease with higher oligomer content and exhibit small negative deviations from Fox' linear additivity rule. This indicates plasticization and miscibility being mainly due to entropic effects. Injection-moulded thick specimens show ductile behaviour at oligomer contents >10 wt %, while sheets with a thickness of 0.2–0.5 mm appear flexible already at 7.5 wt %. The oxygen permeability coefficients are an order of magnitude lower than those of low-density polyethylene. Due to the sum of their properties, the plasticized sheets present a promising alternative in the preparation of barrier materials.
We study the estimation of some linear functionals which are based on an unknown lifetime distribution. The observations are assumed to be generated under the semi-parametric random censorship model (SRCM), that is, a random censorship model where the conditional expectation of the censoring indicator given the observation belongs to a parametric family. Under this setup a semi-parametric estimator of the survival function was introduced by the author. If the parametric model assumption is correct, it is known that the estimated functional which is based on this semi-parametric estimator is asymptotically at least as efficient as the corresponding one which rests on the nonparametric Kaplan–Meier estimator.
In this paper we show that the estimated functional which is based on this semi-parametric estimator is asymptotically efficient with respect to the class of all regular estimators under this semi-parametric model.
Design of enzyme reactors as chromatographic columns for racemic resolution of amino acid esters
(1989)
This article describes the fabrication, characterization and application of an epidermal temporary-transfer tattoo-based potentiometric sensor, coupled with a miniaturized wearable wireless transceiver, for real-time monitoring of sodium in the human perspiration. Sodium excreted during perspiration is an excellent marker for electrolyte imbalance and provides valuable information regarding an individual's physical and mental wellbeing. The realization of the new skin-worn non-invasive tattoo-like sensing device has been realized by amalgamating several state-of-the-art thick film, laser printing, solid-state potentiometry, fluidics and wireless technologies. The resulting tattoo-based potentiometric sodium sensor displays a rapid near-Nernstian response with negligible carryover effects, and good resiliency against various mechanical deformations experienced by the human epidermis. On-body testing of the tattoo sensor coupled to a wireless transceiver during exercise activity demonstrated its ability to continuously monitor sweat sodium dynamics. The real-time sweat sodium concentration was transmitted wirelessly via a body-worn transceiver from the sodium tattoo sensor to a notebook while the subjects perspired on a stationary cycle. The favorable analytical performance along with the wearable nature of the wireless transceiver makes the new epidermal potentiometric sensing system attractive for continuous monitoring the sodium dynamics in human perspiration during diverse activities relevant to the healthcare, fitness, military, healthcare and skin-care domains.
Size unlimited markerless deletions by a transconjugative plasmid-system in Bacillus licheniformis
(2013)
The response of Bacillus licheniformis to heat and ethanol stress and the role of the SigB regulon
(2013)
Disruption experiments targeted at the Bacillus licheniformis degSU operon and GFP-reporter analysis provided evidence for promoter activity immediately upstream of degU. pMutin mediated concomitant introduction of the degU32 allele – known to cause hypersecretion in Bacillus subtilis – resulted in a marked increase in protease activity. Application of 5-fluorouracil based counterselection through establishment of a phosphoribosyltransferase deficient Δupp strain eventually facilitated the marker-free introduction of degU32 leading to further protease enhancement achieving levels as for hypersecreting wild strains in which degU was overexpressed. Surprisingly, deletion of rapG – known to interfere with DegU DNA-binding in B. subtilis – did not enhance protease production neither in the wild type nor in the degU32 strain. The combination of degU32 and Δupp counterselection in the type strain is not only equally effective as in hypersecreting wild strains with respect to protease production but furthermore facilitates genetic strain improvement aiming at biological containment and effectiveness of biotechnological processes.
Bacillus subtilis and Bacillus licheniformis are widely used for the large-scale industrial production of proteins. These strains can efficiently secrete proteins into the culture medium using the general secretion (Sec) pathway. A characteristic feature of all secreted proteins is their N-terminal signal peptides, which are recognized by the secretion machinery. Here, we have studied the production of an industrially important secreted protease, namely, subtilisin BPN′ from Bacillus amyloliquefaciens. One hundred seventy-three signal peptides originating from B. subtilis and 220 signal peptides from the B. licheniformis type strain were fused to this secretion target and expressed in B. subtilis, and the resulting library was analyzed by high-throughput screening for extracellular proteolytic activity. We have identified a number of signal peptides originating from both organisms which produced significantly increased yield of the secreted protease. Interestingly, we observed that levels of extracellular protease were improved not only in B. subtilis, which was used as the screening host, but also in two different B. licheniformis strains. To date, it is impossible to predict which signal peptide will result in better secretion and thus an improved yield of a given extracellular target protein. Our data show that screening a library consisting of homologous and heterologous signal peptides fused to a target protein can identify more-effective signal peptides, resulting in improved protein export not only in the original screening host but also in different production strains.