scholarly journals Cleavage of Rubber by the Latex Clearing Protein (Lcp) of Streptomyces sp. Strain K30: Molecular Insights

2016 ◽  
Vol 82 (22) ◽  
pp. 6593-6602 ◽  
Author(s):  
Wolf Röther ◽  
Stefanie Austen ◽  
Jakob Birke ◽  
Dieter Jendrossek
Keyword(s):  
Biochemical Characterization ◽  
Mutational Analysis ◽  
Axial Position ◽  
Amino Acid Residues ◽  
Content Type ◽  
Research Activities ◽  
Domain Of Unknown Function ◽  
Arginine Residues ◽  
Aldehyde Groups ◽  
Key Residues

ABSTRACTGram-positive rubber degraders such asStreptomycessp. strain K30 cleave rubber [poly(cis-1,4-isoprene)] to low-molecular-mass oligoisoprenoid products with terminal keto and aldehyde groups by the secretion of a latex clearing protein (Lcp) designated rubber oxygenase. LcpK30is a hemebcytochrome and has a domain of unknown function (DUF2236) that is characteristic of orthologous Lcps. Proteins with a DUF2236 domain are characterized by three highly conserved residues (R164, T168, and H198 in LcpK30). Exchange of R164 or T168 by alanine and characterization of the purified LcpK30muteins revealed that both were stable and contained a heme group (red color) but were inactive. This finding identifies both residues as key residues for the cleavage reaction. The purified H198A mutein was also inactive and stable but was colorless due to the absence of heme. We constructed and characterized alanine muteins of four additional histidine residues moderately conserved in 495 LcpK30homologous sequences (H203A, H232A, H259A, H266A). All muteins revealed wild-type properties, excluding any importance for activity and/or heme coordination. Since LcpK30has only eight histidines and the three remaining residues (H103, H184, and H296) were not conserved (<11%), H198 presumably is the only essential histidine, indicating its putative function as a heme ligand. The second axial position of the heme is likely occupied by a not yet identified molecule. Mutational analysis of three strictly conserved arginine residues (R195, R202, R328) showed that R195A and R202A muteins were colorless and instable, suggesting that these residues are important for the protein stability.IMPORTANCELarge amounts of rubber waste materials have been permanently released into the environment for more than a century, yet accumulation of rubber particles released, e.g., by abrasion of tires along highways has not been observed. This is indicative of the ubiquitous presence and activity of rubber-degrading microorganisms. Despite increasing research activities on rubber biodegradation during the last 2 decades, the knowledge of the enzymatic cleavage mechanism of rubber by latex clearing protein (Lcp) still is limited. In particular, the catalytic cleavage mechanism and the amino acids of Lcp proteins (Lcps) that are involved have not yet been identified for any Lcp. In this study, we investigated the importance of 10 amino acid residues of Lcp fromStreptomycessp. K30 (LcpK30) by mutagenesis, mutein purification, and biochemical characterization. We identified several essential residues, one of which most likely represents an axial heme ligand in Lcp ofStreptomycessp. K30.

scholarly journals Trehalose Degradation by Cellvibrio japonicus Exhibits No Functional Redundancy and Is Solely Dependent on the Tre37A Enzyme

2020 ◽  
Vol 86 (22) ◽  
Author(s):  
Cecelia A. Garcia ◽  
Jackson A. Narrett ◽  
Jeffrey G. Gardner
Keyword(s):  
Biochemical Characterization ◽  
Mutational Analysis ◽  
Glycoside Hydrolase Family ◽  
Gut Health ◽  
Phosphotransferase System ◽  
Carbohydrate Active Enzymes ◽  
Human Gut ◽  
Content Type ◽  
Degrading Bacteria ◽  
Cellvibrio Japonicus

ABSTRACT The α-diglucoside trehalose has historically been known as a component of the bacterial stress response, though it more recently has been studied for its relevance in human gut health and biotechnology development. The utilization of trehalose as a nutrient source by bacteria relies on carbohydrate-active enzymes, specifically those of the glycoside hydrolase family 37 (GH37), to degrade the disaccharide into substituent glucose moieties for entry into metabolism. Environmental bacteria using oligosaccharides for nutrients often possess multiple carbohydrate-active enzymes predicted to have the same biochemical activity and therefore are thought to be functionally redundant. In this study, we characterized trehalose degradation by the biotechnologically important saprophytic bacterium Cellvibrio japonicus. This bacterium possesses two predicted α-α-trehalase genes, tre37A and tre37B, and our investigation using mutational analysis found that only the former is essential for trehalose utilization by C. japonicus. Heterologous expression experiments found that only the expression of the C. japonicus tre37A gene in an Escherichia coli treA mutant strain allowed for full utilization of trehalose. Biochemical characterization of C. japonicus GH37 activity determined that the tre37A gene product is solely responsible for cleaving trehalose and is an acidic α-α-trehalase. Bioinformatic and mutational analyses indicate that Tre37A directly cleaves trehalose to glucose in the periplasm, as C. japonicus does not possess a phosphotransferase system. This study facilitates the development of a comprehensive metabolic model for α-linked disaccharides in C. japonicus and more broadly expands our understanding of the strategies that saprophytic bacteria employ to capture diverse carbohydrates from the environment. IMPORTANCE The metabolism of trehalose is becoming increasingly important due to the inclusion of this α-diglucoside in a number of foods and its prevalence in the environment. Bacteria able to utilize trehalose in the human gut possess a competitive advantage, as do saprophytic microbes in terrestrial environments. While the biochemical mechanism of trehalose degradation is well understood, what is less clear is how bacteria acquire this metabolite from the environment. The significance of this report is that by using the model saprophyte Cellvibrio japonicus, we were able to functionally characterize the two predicted trehalase enzymes that the bacterium possesses and determined that the two enzymes are not equivalent and are not functionally redundant. The results and approaches used to understand the complex physiology of α-diglucoside metabolism from this study can be applied broadly to other polysaccharide-degrading bacteria.

scholarly journals Mutational Analysis of the Shigella flexneri O-Antigen Polymerase Wzy: Identification of Wzz-Dependent Wzy Mutants

2014 ◽  
Vol 197 (1) ◽  
pp. 108-119 ◽  
Author(s):  
Pratiti Nath ◽  
Elizabeth Ngoc Hoa Tran ◽  
Renato Morona
Keyword(s):  
Chain Length ◽  
Mutational Analysis ◽  
Shigella Flexneri ◽  
Random Mutagenesis ◽  
Amino Acid Residues ◽  
Transmembrane Region ◽  
Content Type ◽  
O Antigen ◽  
Important Amino Acid ◽  
Colicin E2

The O-antigen (Oag) component of lipopolysaccharide (LPS) is a major virulence determinant ofShigella flexneriand is synthesized by the O-antigen polymerase, WzySf. Oag chain length is regulated by chromosomally encoded WzzSfand pHS-2 plasmid-encoded WzzpHS2. To identify functionally important amino acid residues in WzySf, random mutagenesis was performed on thewzySfgene in a pWaldo-TEV-GFP plasmid, followed by screening with colicin E2. Analysis of the LPS conferred by mutated WzySfproteins in thewzySf-deficient (Δwzy) strain identified 4 different mutant classes, with mutations found in periplasmic loop 1 (PL1), PL2, PL3, and PL6, transmembrane region 2 (TM2), TM4, TM5, TM7, TM8, and TM9, and cytoplasmic loop 1 (CL1) and CL5. The association of WzySfand WzzSfwas investigated by transforming these mutatedwzySfplasmids into awzySf- andwzzSf-deficient (Δwzy Δwzz) strain. Comparison of the LPS profiles in the Δwzyand Δwzy Δwzzbackgrounds identified WzySfmutants whose polymerization activities were WzzSfdependent. Colicin E2 and bacteriophage Sf6c sensitivities were consistent with the LPS profiles. Analysis of the expression levels of the WzySf-GFP mutants in the Δwzyand Δwzy Δwzzbackgrounds identified a role for WzzSfin WzySfstability. Hence, in addition to its role in regulating Oag modal chain length, WzzSfalso affects WzySfactivity and stability.

scholarly journals Transfer of Plasmid DNA to Clinical Coagulase-Negative Staphylococcal Pathogens by Using a Unique Bacteriophage

2015 ◽  
Vol 81 (7) ◽  
pp. 2481-2488 ◽  
Author(s):  
Volker Winstel ◽  
Petra Kühner ◽  
Bernhard Krismer ◽  
Andreas Peschel ◽  
Holger Rohde
Keyword(s):  
Plasmid Dna ◽  
Genetic Manipulation ◽  
Current Knowledge ◽  
Virulence Determinants ◽  
Coagulase Negative Staphylococci ◽  
Reliable Technique ◽  
Content Type ◽  
Research Activities ◽  
Wide Range ◽  
Genetic Barriers

ABSTRACTGenetic manipulation of emerging bacterial pathogens, such as coagulase-negative staphylococci (CoNS), is a major hurdle in clinical and basic microbiological research. Strong genetic barriers, such as restriction modification systems or clustered regularly interspaced short palindromic repeats (CRISPR), usually interfere with available techniques for DNA transformation and therefore complicate manipulation of CoNS or render it impossible. Thus, current knowledge of pathogenicity and virulence determinants of CoNS is very limited. Here, a rapid, efficient, and highly reliable technique is presented to transfer plasmid DNA essential for genetic engineering to important CoNS pathogens from a uniqueStaphylococcus aureusstrain via a specificS. aureusbacteriophage, Φ187. Even strains refractory to electroporation can be transduced by this technique once donor and recipient strains share similar Φ187 receptor properties. As a proof of principle, this technique was used to delete the alternative transcription factor sigma B (SigB) via allelic replacement in nasal and clinicalStaphylococcus epidermidisisolates at high efficiencies. The described approach will allow the genetic manipulation of a wide range of CoNS pathogens and might inspire research activities to manipulate other important pathogens in a similar fashion.

Europe continues to lead the way in the collaborative robot business

2016 ◽  
Vol 43 (1) ◽  
pp. 6-11 ◽  
Author(s):  
Robert Bogue
Keyword(s):  
Automotive Industry ◽  
Market Development ◽  
Future Market ◽  
Collaborative Robots ◽  
Content Type ◽  
Production Methods ◽  
Research Activities ◽  
Timely Review ◽  
New Applications ◽  
Collaborative Robot

Purpose – This paper aims to provide a European perspective on the collaborative robot business and to consider the factors governing future market development. Design/methodology/approach – Following an introduction, this first describes the collaborative robots launched recently by European manufacturers and their applications. It then discusses major European research activities and finally considers the factors stimulating the market. Findings – This article shows that collaborative robots are being commercialised by the major European robot manufacturers as well as by several smaller specialists. Although most have low payload capacities they are inexpensive and offer a number of operational benefits, making them well suited to a range of existing and emerging applications. Europe has a strong research base and several EU-funded programmes aim to stimulate collaborative robot development and use. Rapid market development is anticipated, driven in the main by applications in electronic product manufacture and assembly; new applications in the automotive industry; uses by small to medium-sized manufacturers; and companies seeking robots to support agile production methods. Originality/value – This paper provides a timely review of the rapidly developing European collaborative robot industry.

Digital competences of the workforce – a research topic?

2017 ◽  
Vol 23 (3) ◽  
pp. 721-734 ◽  
Author(s):  
Matthias Murawski ◽  
Markus Bick
Keyword(s):  
Research Topic ◽  
Future Research ◽  
Digital Competence ◽  
Content Type ◽  
Research Activities ◽  
Starting Point ◽  
Pros And Cons ◽  
Definition Of ◽  
The Individual ◽  
Practical Implications

Purpose Considering working in the digital age, questions on the consequences for the individual workers are, so far, often neglected. The purpose of this paper is to deal with the question of whether the digital competences of the workforce is a research topic. The authors argue for the thesis that it is indeed a research topic. Design/methodology/approach In addition to a literature analysis of the top IS, HR, and learning publications, non-scientific sources, as well as the opinions of the authors, are included. The authors’ thesis is challenged through a debate of corresponding pros and cons. Findings The definition of digital competences lacks scientific depth. Focussing on the workforce is valid, as a “lifelong” perspective is not mandatory for research. Digital competence research is a multidisciplinary task to which the IS field can make a valuable contribution. Research limitations/implications Although relevant references are included, some aspects are mainly driven by the opinions of the authors. The theoretical implications encompass a call for a scientific definition of digital competences. Furthermore, scholars should focus on the competences of the workforce, including occupations, roles, or industries. The authors conclude by providing a first proposal of a research agenda. Practical implications The practical implications include the alignment of multiple stakeholders for the design of “digital” curricula and the integration by HR departments of the construct of digital competences, e.g. for compensation matters and job requirements. Originality/value This paper is one of very few contributions in the area of the digital competences of the workforce, and it presents a starting point for future research activities.

scholarly journals Genetic Analysis of Pathway Specificity during Posttranslational Protein Translocation across the Escherichia coli Plasma Membrane

2003 ◽  
Vol 185 (9) ◽  
pp. 2811-2819 ◽  
Author(s):  
Natascha Blaudeck ◽  
Peter Kreutzenbeck ◽  
Roland Freudl ◽  
Georg A. Sprenger
Keyword(s):  
Escherichia Coli ◽  
Signal Peptide ◽  
Protein Translocation ◽  
Alternative Possibilities ◽  
Amino Acid Residues ◽  
Sec Pathway ◽  
Twin Arginine Translocation ◽  
Tat Pathway ◽  
Arginine Residues ◽  
Dependent Protein

ABSTRACT In Escherichia coli, the SecB/SecA branch of the Sec pathway and the twin-arginine translocation (Tat) pathway represent two alternative possibilities for posttranslational translocation of proteins across the cytoplasmic membrane. Maintenance of pathway specificity was analyzed using a model precursor consisting of the mature part of the SecB-dependent maltose-binding protein (MalE) fused to the signal peptide of the Tat-dependent TorA protein. The TorA signal peptide selectively and specifically directed MalE into the Tat pathway. The characterization of a spontaneous TorA signal peptide mutant (TorA*), in which the two arginine residues in the c-region had been replaced by one leucine residue, showed that the TorA*-MalE mutant precursor had acquired the ability for efficiently using the SecB/SecA pathway. Despite the lack of the “Sec avoidance signal,” the mutant precursor was still capable of using the Tat pathway, provided that the kinetically favored Sec pathway was blocked. These results show that the h-region of the TorA signal peptide is, in principle, sufficiently hydrophobic for Sec-dependent protein translocation, and therefore, the positively charged amino acid residues in the c-region represent a major determinant for Tat pathway specificity. Tat-dependent export of TorA-MalE was significantly slower in the presence of SecB than in its absence, showing that SecB can bind to this precursor despite the presence of the Sec avoidance signal in the c-region of the TorA signal peptide, strongly suggesting that the function of the Sec avoidance signal is not the prevention of SecB binding; rather, it must be exerted at a later step in the Sec pathway.

Organizational knowledge documentation in project-based institutes

2017 ◽  
Vol 35 (5) ◽  
pp. 994-1012 ◽  
Author(s):  
Fatemeh Navidi ◽  
Mohammad Hassanzadeh ◽  
Ali Zolghadr Shojai
Keyword(s):  
Tacit Knowledge ◽  
Explicit Knowledge ◽  
Structured Interview ◽  
Organizational Knowledge ◽  
Technical Knowledge ◽  
Content Type ◽  
Junior Staff ◽  
Research Activities ◽  
Space Agency ◽  
Research Institute

Purpose Employees, as the most important assets of an organization, acquire a great deal of experience, skills and knowledge throughout the time period they work for the organization. If their skills and technical knowledge are not documented properly, these will be lost once the employees leave the organization. Therefore, documentation is necessary for preserving this invaluable knowledge, avoiding duplication and preventing repeated mistakes that occurred in the past and, providing the junior staff with experiences gained by their predecessors. Thus, this research aims to elaborate on the role of organizational knowledge management (KM) as an essential tool for turning tacit knowledge into explicit knowledge and sharing the gained experiences with others. Design/methodology/approach This research is developmental applied research with qualitative approach and it was conducted using thematic analysis method. This method includes a semi-structured interview with 18 researchers conducting research projects at the Satellite Research Institute under the supervision of the Iran Space Agency. Findings The projects contain knowledge that is a combination of “know why”, “know what”, “know who” and “know how”. A large amount of this knowledge is, indeed, the tacit knowledge. Most of this tacit knowledge is not reflected in the project documents. Generally, the documents contain results only and they do not include experience, technical details, methodology, analysis and mistakes that were made during research activities. Documentation challenges fall into three major types: technical, human resources and administrative. Originality value Considering the necessity of documentation within the knowledge transfer process and its important role in KM; and, with respect to the lack of technical knowledge and experience transfer observed in the documents of Satellite Research Institute, this research proposes some steps that need to be taken to turn the knowledge sharing into an organizational culture.

The long-term development of Russian biotech sector

foresight ◽  
2017 ◽  
Vol 19 (5) ◽  
pp. 491-500 ◽  
Author(s):  
Anna Grebenyuk ◽  
Nikolai Ravin
Keyword(s):  
High Performance ◽  
Large Scale ◽  
Technological Development ◽  
Science And Technology ◽  
Plant Biotechnology ◽  
High Tech ◽  
Content Type ◽  
Research Areas ◽  
Research Activities ◽  
The Usa

Purpose To define strategic directions for the Russia’s social, economic, scientific and technological development in 2011-2013, a large-scale foresight study including the deep analysis of prospects of biotechnology development there was undertaken (Russia 2030: Science and Technology Foresight). This paper aims to present results of this research. Design/methodology/approach The study was based on a combination of technology-push and market-pull approaches that aimed not only to identify most promising science and technology (S&T) areas but also to understand how they can be realized in practice. Representatives from federal authorities, science and business were involved in the project to create future visions of technological directions; analyze grand challenges, weak signals and wild cards; and set research and development (R&D) priorities. Findings According to results of the study, Russia has a potential for biotech sector development, although the level of R&D in the majority of areas is lagging behind that in the USA and leading EU countries. However, there are several advanced applied research areas where efforts can be focused. Among them are high-performance genomics and post-genomics research platforms, systems and structural biology, microbial metabolic engineering, plant biotechnology and microbial strains and consortia for development of symbiotic plant–microbial communities. Originality/value Concentration of available resources of government and business on biotechnological sector development can help to find answers for challenges that Russia faces today or will face tomorrow. It will help to pick up on the current level of research activities, improve the quality of personnel training, make this area the engine of the economy and carry out the so-called new industrialization of the country, building a new, high-tech device industry.

Business process model patterns: state-of-the-art, research classification and taxonomy

2019 ◽  
Vol 25 (5) ◽  
pp. 972-994 ◽  
Author(s):  
Michael Fellmann ◽  
Agnes Koschmider ◽  
Ralf Laue ◽  
Andreas Schoknecht ◽  
Arthur Vetter
Keyword(s):  
Business Process ◽  
Process Model ◽  
Pattern Language ◽  
Future Research ◽  
Business Process Model ◽  
Content Type ◽  
Research Activities ◽  
Starting Point ◽  
Research Classification

Purpose Patterns have proven to be useful for documenting general reusable solutions to a commonly occurring problem. In recent years, several different business process management (BPM)-related patterns have been published. Despite the large number of publications on this subject, there is no work that provides a comprehensive overview and categorization of the published business process model patterns. The purpose of this paper is to close this gap by providing a taxonomy of patterns as well as a classification of 89 research works. Design/methodology/approach The authors analyzed 280 research articles following a structured iterative procedure inspired by the method for taxonomy development from Nickerson et al. (2013). Using deductive and inductive reasoning processes embedded in concurrent as well as joint research activities, the authors created a taxonomy of patterns as well as a classification of 89 research works. Findings In general, the findings extend the current understanding of BPM patterns. The authors identify pattern categories that are highly populated with research works as well as categories that have received far less attention such as risk and security, the ecological perspective and process architecture. Further, the analysis shows that there is not yet an overarching pattern language for business process model patterns. The insights can be used as starting point for developing such a pattern language. Originality/value Up to now, no comprehensive pattern taxonomy and research classification exists. The taxonomy and classification are useful for searching pattern works which is also supported by an accompanying website complementing the work. In regard to future research and publications on patterns, the authors derive recommendations regarding the content and structure of pattern publications.

scholarly journals N-Glycosylation Improves the Pepsin Resistance of Histidine Acid Phosphatase Phytases by Enhancing Their Stability at Acidic pHs and Reducing Pepsin's Accessibility to Its Cleavage Sites

2015 ◽  
Vol 82 (4) ◽  
pp. 1004-1014 ◽  
Author(s):  
Canfang Niu ◽  
Huiying Luo ◽  
Pengjun Shi ◽  
Huoqing Huang ◽  
Yaru Wang ◽  
...  
Keyword(s):  
Acid Phosphatase ◽  
Biochemical Characterization ◽  
Animal Feed ◽  
Glycosylation Site ◽  
Site Directed Mutagenesis ◽  
Acidic Ph ◽  
Cleavage Sites ◽  
Ph Optimum ◽  
Content Type ◽  
Histidine Acid Phosphatase

ABSTRACTN-Glycosylation can modulate enzyme structure and function. In this study, we identified two pepsin-resistant histidine acid phosphatase (HAP) phytases fromYersinia kristensenii(YkAPPA) andYersinia rohdei(YrAPPA), each having anN-glycosylation motif, and one pepsin-sensitive HAP phytase fromYersinia enterocolitica(YeAPPA) that lacked anN-glycosylation site. Site-directed mutagenesis was employed to construct mutants by altering theN-glycosylation status of each enzyme, and the mutant and wild-type enzymes were expressed inPichia pastorisfor biochemical characterization. Compared with those of theN-glycosylation site deletion mutants andN-deglycosylated enzymes, allN-glycosylated counterparts exhibited enhanced pepsin resistance. Introduction of theN-glycosylation site into YeAPPA as YkAPPA and YrAPPA conferred pepsin resistance, shifted the pH optimum (0.5 and 1.5 pH units downward, respectively) and improved stability at acidic pH (83.2 and 98.8% residual activities at pH 2.0 for 1 h). Replacing the pepsin cleavage sites L197 and L396 in the immediate vicinity of theN-glycosylation motifs of YkAPPA and YrAPPA with V promoted their resistance to pepsin digestion when produced inEscherichia colibut had no effect on the pepsin resistance ofN-glycosylated enzymes produced inP. pastoris. Thus,N-glycosylation may improve pepsin resistance by enhancing the stability at acidic pH and reducing pepsin's accessibility to peptic cleavage sites. This study provides a strategy, namely, the manipulation ofN-glycosylation, for improvement of phytase properties for use in animal feed.

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