Effect of different genetic manipulations on the production of xylanase enzyme in Streptomyces strains

Cavendish banana peel can be used as a substitute for the expensive xylan, while molasses than as a source of carbon as well as nitrogen, minerals and nutrients needed for the growth of microbes that can produce the enzyme. Xylanase produced from Bacillus stearothermopillus DSM 22, using media cavendish banana peels with the addition of molasses 1%, 2%, and 3%. Fermentation is done in a shaker incubator at 550C temperature conditions, initial pH 8, and 250 rpm agitation. The result showed the highest enzyme activity of 4,14 ± 0,16 U/mL min., on the addition 2% molasses after 24 hours. Further fermentation carried out in the fermenter working volume of 3.5 liters, with the condition of temperature 550C, pH 8, aeration 1 vvm, agitation 250 rpm, the highest spesific enzyme of activity of 51,62 ± 0,16 U/mg after 24 hours. Partial purification of xylanase enzyme fermentation is done with the results of microfiltration, ultrafiltration, ammonium sulfate (0-80%) and dialysis. There is an increase in the purity of the enzyme at each stage of purification, the highest purity on dialysis 3.23 times of crude enzymes.Kulit buah pisang kapendis dapat digunakan sebagai pengganti xilan yang harganya mahal, sementara molases selain sebagai sumber karbon serta nitrogen, mineral dan nutrisi dibutuhkan untuk pertumbuhan mikroba yang dapat menghasilkan enzim. Xilanase yang dihasilkan dari Bacillus stearothermopillus DSM 22, menggunakan media kulit pisang kapendis dengan penambahan molase 1%, 2%, dan 3%. Fermentasi dilakukan dalam shaker inkubator pada temperatur 550C, pH awal 8, dan agitasi 250 rpm. Hasilnya menunjukkan aktivitas enzim tertinggi 4,14 ± 0,16 U/mL min., pada penambahan 2% molases setelah 24 jam. Selanjutnya fermentasi dilakukan di dalam fermentor, volume kerja dari 3,5 liter, dengan kondisi temperatur 550C, pH 8, aeration 1 vvm, agitasi 250 rpm, aktivitas spesifik tertinggi 51,62 ± 0,16 U/mg setelah 24 jam. Pemurnian parsial fermentasi enzim xilanase dilakukan dengan hasil mikrofiltrasi, ultrafiltrasi, amonium sulfat (0-80%) dan dialisis. Ada peningkatan kemurnian enzim pada setiap tahap pemurnian, kemurnian tertinggi pada dialisis 3,23 kali dari enzim kasar.Keywords: Xylanase, B. stearothermophillus DSM 22, Cavendish banana peel, molasses, enzyme activity

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Distribution of a Limited Sir2 Protein Pool Regulates the Strength of Yeast rDNA Silencing and Is Modulated by Sir4p

Genetics ◽

10.1093/genetics/149.3.1205 ◽

1998 ◽

Vol 149 (3) ◽

pp. 1205-1219 ◽

Cited By ~ 9

Author(s):

Jeffrey S Smith ◽

Carrie Baker Brachmann ◽

Lorraine Pillus ◽

Jef D Boeke

Keyword(s):

Saccharomyces Cerevisiae ◽

Simple Model ◽

Regulatory Mechanism ◽

Transcriptional Silencing ◽

Rdna Locus ◽

Genetic Manipulations ◽

Protein Levels ◽

Endogenous Level ◽

Protein Pool ◽

Negative Effect

Abstract Transcriptional silencing in Saccharomyces cerevisiae occurs at the silent mating-type loci HML and HMR, at telomeres, and at the ribosomal DNA (rDNA) locus RDN1. Silencing in the rDNA occurs by a novel mechanism that depends on a single Silent Information Regulator (SIR) gene, SIR2. SIR4, essential for other silenced loci, paradoxically inhibits rDNA silencing. In this study, we elucidate a regulatory mechanism for rDNA silencing based on the finding that rDNA silencing strength directly correlates with cellular Sir2 protein levels. The endogenous level of Sir2p was shown to be limiting for rDNA silencing. Furthermore, small changes in Sir2p levels altered rDNA silencing strength. In rDNA silencing phenotypes, sir2 mutations were shown to be epistatic to sir4 mutations, indicating that SIR4 inhibition of rDNA silencing is mediated through SIR2. Furthermore, rDNA silencing is insensitive to SIR3 overexpression, but is severely reduced by overexpression of full-length Sir4p or a fragment of Sir4p that interacts with Sir2p. This negative effect of SIR4 overexpression was overridden by co-overexpression of SIR2, suggesting that SIR4 directly inhibits the rDNA silencing function of SIR2. Finally, genetic manipulations of SIR4 previously shown to promote extended life span also resulted in enhanced rDNA silencing. We propose a simple model in which telomeres act as regulators of rDNA silencing by competing for limiting amounts of Sir2 protein.

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An optogenetic method for investigating presynaptic molecular regulation

Scientific Reports ◽

10.1038/s41598-021-90244-0 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Yuni Kay ◽

Bruce E. Herring

Keyword(s):

Pyramidal Neurons ◽

Molecular Regulation ◽

Glutamatergic Synapses ◽

Experimental Conditions ◽

Regulatory Pathways ◽

Genetic Manipulations ◽

Postsynaptic Protein ◽

Presynaptic Function ◽

Synaptotagmin 1 ◽

Ca3 Pyramidal Neurons

AbstractWhile efficient methods are well established for studying postsynaptic protein regulation of glutamatergic synapses in the mammalian central nervous system, similarly efficient methods are lacking for studying proteins regulating presynaptic function. In the present study, we introduce an optical/electrophysiological method for investigating presynaptic molecular regulation. Here, using an optogenetic approach, we selectively stimulate genetically modified presynaptic CA3 pyramidal neurons in the hippocampus and measure optically-induced excitatory postsynaptic currents produced in unmodified postsynaptic CA1 pyramidal neurons. While such use of optogenetics is not novel, previous implementation methods do not allow basic quantification of the changes in synaptic strength produced by genetic manipulations. We find that incorporating simultaneous recordings of fiber volley amplitude provides a control for optical stimulation intensity and, as a result, creates a metric of synaptic efficacy that can be compared across experimental conditions. In the present study, we utilize our new method to demonstrate that inhibition of synaptotagmin 1 expression in CA3 pyramidal neurons leads to a significant reduction in Schaffer collateral synapse function, an effect that is masked with conventional electrical stimulation. Our hope is that this method will expedite our understanding of molecular regulatory pathways that govern presynaptic function.

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Health and longevity studies in C. elegans: the “healthy worm database” reveals strengths, weaknesses and gaps of test compound-based studies

Biogerontology ◽

10.1007/s10522-021-09913-2 ◽

2021 ◽

Vol 22 (2) ◽

pp. 215-236

Author(s):

Nadine Saul ◽

Steffen Möller ◽

Francesca Cirulli ◽

Alessandra Berry ◽

Walter Luyten ◽

...

Keyword(s):

Healthy Aging ◽

Model Organism ◽

Research Field ◽

Aging Research ◽

Genetic Manipulations ◽

C Elegans ◽

Starting Point ◽

Health Related ◽

Phenotype Measurement ◽

Or Gene

AbstractSeveral biogerontology databases exist that focus on genetic or gene expression data linked to health as well as survival, subsequent to compound treatments or genetic manipulations in animal models. However, none of these has yet collected experimental results of compound-related health changes. Since quality of life is often regarded as more valuable than length of life, we aim to fill this gap with the “Healthy Worm Database” (http://healthy-worm-database.eu). Literature describing health-related compound studies in the aging model Caenorhabditis elegans was screened, and data for 440 compounds collected. The database considers 189 publications describing 89 different phenotypes measured in 2995 different conditions. Besides enabling a targeted search for promising compounds for further investigations, this database also offers insights into the research field of studies on healthy aging based on a frequently used model organism. Some weaknesses of C. elegans-based aging studies, like underrepresented phenotypes, especially concerning cognitive functions, as well as the convenience-based use of young worms as the starting point for compound treatment or phenotype measurement are discussed. In conclusion, the database provides an anchor for the search for compounds affecting health, with a link to public databases, and it further highlights some potential shortcomings in current aging research.

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Compliance with Good Manufacturing Practices for facilities engaged in vector production, cell isolation, and genetic manipulations

Current Opinion in Biotechnology ◽

10.1016/s0958-1669(99)80052-4 ◽

1999 ◽

Vol 10 (3) ◽

pp. 295-297 ◽

Cited By ~ 3

Author(s):

Michael P Steel ◽

Blake J Roessler

Keyword(s):

Cell Isolation ◽

Genetic Manipulations ◽

Good Manufacturing Practices ◽

Production Cell

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Production of xylanase enzyme through bioconversion of agricultural waste

2011 3rd International Symposium & Exhibition in Sustainable Energy & Environment (ISESEE) ◽

10.1109/isesee.2011.5977107 ◽

2011 ◽

Cited By ~ 2

Author(s):

I. Norazlina ◽

S. R. Puvanesvaran

Keyword(s):

Agricultural Waste ◽

Xylanase Enzyme

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Reconstruction ofmreBExpression in Staphylococcus aureus via a Collection of New Integrative Plasmids

Applied and Environmental Microbiology ◽

10.1128/aem.00759-14 ◽

2014 ◽

Vol 80 (13) ◽

pp. 3868-3878 ◽

Cited By ~ 11

Author(s):

Ana Yepes ◽

Gudrun Koch ◽

Andrea Waldvogel ◽

Juan-Carlos Garcia-Betancur ◽

Daniel Lopez

Keyword(s):

Staphylococcus Aureus ◽

Cell Wall ◽

Cell Biology ◽

Genetic Manipulation ◽

Protein Localization ◽

Antibiotic Resistance Genes ◽

Wall Material ◽

Content Type ◽

Genetic Manipulations ◽

Discrete Structures

ABSTRACTProtein localization has been traditionally explored in unicellular organisms, whose ease of genetic manipulation facilitates molecular characterization. The two rod-shaped bacterial modelsEscherichia coliandBacillus subtilishave been prominently used for this purpose and have displaced other bacteria whose challenges for genetic manipulation have complicated any study of cell biology. Among these bacteria is the spherical pathogenic bacteriumStaphylococcus aureus. In this report, we present a new molecular toolbox that facilitates gene deletion in staphylococci in a 1-step recombination process and additional vectors that facilitate the insertion of diverse reporter fusions into newly identified neutral loci of theS. aureuschromosome. Insertion of the reporters does not add any antibiotic resistance genes to the chromosomes of the resultant strains, thereby making them amenable for further genetic manipulations. We used this toolbox to reconstitute the expression ofmreBinS. aureus, a gene that encodes an actin-like cytoskeletal protein which is absent in coccal cells and is presumably lost during the course of speciation. We observed that inS. aureus, MreB is organized in discrete structures in association with the membrane, leading to an unusual redistribution of the cell wall material. The production of MreB also caused cell enlargement, but it did not revert staphylococcal shape. We present interactions of MreB with key staphylococcal cell wall-related proteins. This work facilitates the useS. aureusas a model system in exploring diverse aspects of cellular microbiology.

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Axon Regeneration in Peripheral Nerves

Oxford Research Encyclopedia of Neuroscience ◽

10.1093/acrefore/9780190264086.013.306 ◽

2021 ◽

Author(s):

Arthur English

Keyword(s):

Nerve Injury ◽

Peripheral Nerves ◽

Axon Regeneration ◽

Axon Growth ◽

Injury Site ◽

Genetic Manipulations ◽

Novel Treatments ◽

The Central Nervous System ◽

Experimental Treatments ◽

Axonal Protein

Despite the intrinsically greater capacity for axons to regenerate in injured peripheral nerves than after injury to the central nervous system, functional recovery after most nerve injuries is very poor. A need for novel treatments that will enhance axon regeneration and improve recovery is substantial. Several such experimental treatments have been studied, each based on part of the stereotypical cellular responses that follow a nerve injury. Genetic manipulations of Schwann cells that have transformed from a myelinating to a repair phenotype that either increase their production of axon growth-promoting molecules, decrease production of inhibitors, or both result in enhanced regeneration. Local or systemic application of these molecules or small molecule mimetics of them also will promote regeneration. The success of treatments that stimulate axonal protein synthesis at the site of the nerve injury and in the growing axons, an early and important response to axon injury, is significant, as is that of manipulations of the types of immune cells that migrate into the injury site or peripheral ganglia. Modifications of the extracellular matrix through which the regenerating axons course, including the stimulation of new blood vessel formation, promotes the navigation of nascent regenerating neurites past the injury site, resulting in greater axon regeneration. Experimental induction of expression of regeneration associated gene activity in the cell bodies of the injured neurons is especially useful when regenerating axons must regenerate over long distances to reinnervate targets. The consistently most effective experimental approach to improving axon regeneration in peripheral nerves has been to increase the activity of the injured neurons, either through electrical, optical, or chemogenetic stimulation or through exercise. These activity-dependent experimental therapies show greatest promise for translation to use in patients.

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