Harnessing the Keratinolytic Activity of Bacillus licheniformis Through Random Mutagenesis Using Ultraviolet and Laser Irradiations

2021 ◽  
Author(s):  
Jamila Akter Tuly ◽  
Haile Ma ◽  
Hossain M. Zabed ◽  
Yating Dong ◽  
Quaisie Janet ◽  
...  
Keyword(s):  
Bacillus Licheniformis ◽  
Random Mutagenesis ◽  
Keratinolytic Activity

Keratinolytic potential ofBacillus licheniformisRG1: structural and biochemical mechanism of feather degradation

2005 ◽  
Vol 51 (3) ◽  
pp. 191-196 ◽  
Author(s):  
Priya Ramnani ◽  
Rajni Singh ◽  
Rani Gupta
Keyword(s):  
Bacillus Licheniformis ◽  
Bacterial Adhesion ◽  
Degradation Process ◽  
Bacterial Cells ◽  
Disulfide Bridges ◽  
Feather Degradation ◽  
Keratinolytic Activity ◽  
Disulfide Reductase ◽  
Live Bacterial Cells ◽  
Disulfide Reductases

Keratinolytic Bacillus licheniformis RG1 was used to study the mechanism of keratinolysis. Scanning electron microscopy studies revealed that bacterial cells grew closely adhered to the barbules of feathers, completely degrading them within 24 h. Biochemical studies indicated that the Bacillus strain produced an extracellular protease, which had keratinolytic potential. The extracellular keratinolytic activity (425 U) was synergistically enhanced by the addition of intracellular disulfide reductases (1712 U). However, these enzymes alone (keratinase and disulfide reductase), without live bacterial cells, failed to degrade the feather. Complete feather degradation was obtained only when living bacterial cells were present, emphasizing that bacterial adhesion plays a key role during the degradation process. The bacterial cells probably provide a continuous supply of reductant to break disulfide bridges. In addition, sulfite detected in the extracellular broth during feather degradation indicated that sulfitolysis may also play a role in feather degradation by the bacterium.Key words: Bacillus licheniformis, disulfide reductase, keratinase, sulfitolysis.

scholarly journals Unravelling the genetic basis for competence development of auxotrophic Bacillus licheniformis 9945A strains

Microbiology ◽  
2014 ◽  
Vol 160 (10) ◽  
pp. 2136-2147 ◽  
Author(s):  
Mareike Jakobs ◽  
Kerstin Hoffmann ◽  
Anja Grabke ◽  
Stefania Neuber ◽  
Heiko Liesegang ◽  
...  
Keyword(s):  
Bacillus Licheniformis ◽  
Extracellular Enzymes ◽  
Random Mutagenesis ◽  
Extracellular Enzyme ◽  
Competence Development ◽  
Colony Morphology ◽  
Enzyme Secretion ◽  
Dna Uptake ◽  
Genetic Competence ◽  
Regulator Genes

Bacterial natural genetic competence – well studied in Bacillus subtilis – enables cells to take up and integrate extracellularly supplied DNA into their own genome. However, little is known about competence development and its regulation in other members of the genus, although DNA uptake machineries are routinely encoded. Auxotrophic Bacillus licheniformis 9945A derivatives, obtained from repeated rounds of random mutagenesis, were long known to develop natural competence. Inspection of the colony morphology and extracellular enzyme secretion of two of these derivatives, M28 and M18, suggested that regulator genes are collaterally hit. M28 emerged as a 14 bp deletion mutant concomitantly displaying a shift in the reading frame of degS that encodes the sensor histidine kinase, which is part of the molecular switch that directs cells to genetic competence, the synthesis of extracellular enzymes or biofilm formation, while for M18, sequencing of the suspected gene revealed a 375 bp deletion in abrB, encoding the major transition state regulator. With respect to colony morphology, enzyme secretion and competence development, both of the mutations, when newly generated on the wild-type B. licheniformis 9945A genetic background, resulted in phenotypes resembling M28 and M18, respectively. All of the known naturally competent B. licheniformis representatives, hitherto thoroughly investigated in this regard, carry mutations in regulator genes, and hence genetic competence observed in domesticated strains supposedly results from deregulation.

scholarly journals High-Throughput Screening of Higher Α-Amylase Producing Bacillus Licheniformis by Fluorescence-Activated Droplet Sorting

2021 ◽  
Author(s):  
Huiling Yuan ◽  
Ran Tu ◽  
Xinwei Tong ◽  
Yuping Lin ◽  
Qinhong Wang
Keyword(s):  
Bacillus Licheniformis ◽  
High Throughput ◽  
High Throughput Screening ◽  
Single Cells ◽  
Random Mutagenesis ◽  
Strain Improvement ◽  
Industrial Applications ◽  
Cell Factory ◽  
Temperature Plasma ◽  
Droplet Sorting

Abstract Backgroundα-Amylases is one of the most important starch degrading enzymes and has the widest range of industrial applications. Bacillus licheniformis has been widely used as a cell factory for industrial production of amylase. However, difficulties in genetic modification of B. licheniformis have limited its widespread use. Directed evolution, based on the combination of random mutagenesis and high throughput screening (HTS), has been proven an effective strategy in strain improvement for increasing the productivity, but it requires a suitable HTS system to screen the desired mutants. Droplet-based microfluidics has emerged as a powerful tool for single-cell screening with ultra-high throughput, however, the accessibility of a droplet microfluidic HTS platform to users having no background in microfluidics is still an issue. ResultsHere, we first developed a microfluidic HTS platform based on fluorescence-activated droplet sorting (FADS) technology. This platform allowed (i) encapsulation of single cells in monodisperse water-in-oil droplets; (ii) cell growth and protein production in droplets; (iii) sorting of droplets based on their fluorescence intensities. To validate the platform, a model selection experiment of a binary mixture of Bacillus strains was performed and a 45.6-fold enrichment was achieved at a sorting rate of 300 droplets per second. Furthermore, we used the platform for the selection of higher α-amylase-producing strains from a library of B. licheniformis strains (a strain already used at industrial-scale). The B. licheniformis mutant library was generated by atmospheric and room temperature plasma (ARTP) mutagenesis. The clones displaying over 50% improvement in α-amylases productivity compared to the wild-type were isolated.ConclusionsWe established an efficient droplet microfluidic platform which consisted of droplet generation, droplet incubation, and sorting of droplets with a throughput of up to 1 × 106 droplets per h. The screening platform was demonstrated by successfully identifying B. licheniformis clones with improved α-amylase production. We believe that the droplet platform could be extended to the development of other industrially valuable strains.

Microanatomy of the Periplasm of Bacillus Licheniformis

1971 ◽  
Vol 29 ◽  
pp. 262-263
Author(s):  
B.K. Ghosh
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Bacillus Licheniformis ◽  
External Environment ◽  
Permeability Barrier ◽  
Periplasmic Space ◽  
Modified Technique ◽  
Gram Positive ◽  
Gram Negative ◽  
Gram Positive Bacteria

Periplasm of bacteria is the space outside the permeability barrier of plasma membrane but enclosed by the cell wall. The contents of this special milieu exterior could be regulated by the plasma membrane from the internal, and by the cell wall from the external environment of the cell. Unlike the gram-negative organism, the presence of this space in gram-positive bacteria is still controversial because it cannot be clearly demonstrated. We have shown the importance of some periplasmic bodies in the secretion of penicillinase from Bacillus licheniformis.In negatively stained specimens prepared by a modified technique (Figs. 1 and 2), periplasmic space (PS) contained two kinds of structures: (i) fibrils (F, 100 Å) running perpendicular to the cell wall from the protoplast and (ii) an array of vesicles of various sizes (V), which seem to have evaginated from the protoplast.

Interrelationship of the cellular distribution and secretion of regular structure (RS) protein in Bacillus licheniformis nm 105

1992 ◽  
Vol 50 (1) ◽  
pp. 712-713
Author(s):  
Xiaorong Zhu ◽  
Richard McVeigh ◽  
Bijan K. Ghosh
Keyword(s):  
Alkaline Phosphatase ◽  
Bacillus Licheniformis ◽  
Self Assembly ◽  
Gel Electrophoresis ◽  
Culture Supernatant ◽  
Regular Structure ◽  
The Self ◽  
Cellular Distribution ◽  
Structural Protein ◽  
Laboratory Cultures

A mutant of Bacillus licheniformis 749/C, NM 105 exhibits some notable properties, e.g., arrest of alkaline phosphatase secretion and overexpression and hypersecretion of RS protein. Although RS is known to be widely distributed in many microbes, it is rarely found, with a few exceptions, in laboratory cultures of microorganisms. RS protein is a structural protein and has the unusual properties to form aggregate. This characteristic may have been responsible for the self assembly of RS into regular tetragonal structures. Another uncommon characteristic of RS is that enhanced synthesis and secretion which occurs when the cells cease to grow. Assembled RS protein with a tetragonal structure is not seen inside cells at any stage of cell growth including cells in the stationary phase of growth. Gel electrophoresis of the culture supernatant shows a very large amount of RS protein in the stationary culture of the B. licheniformis. It seems, Therefore, that the RS protein is cotranslationally secreted and self assembled on the envelope surface.

Directed Evolution of P450 Fatty Acid Decarboxylases via High-Throughput Screening Towards Improved Catalytic Activity

2019 ◽  
Author(s):  
Huifang Xu ◽  
Weinan Liang ◽  
Linlin Ning ◽  
Yuanyuan Jiang ◽  
Wenxia Yang ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Fatty Acid ◽  
Directed Evolution ◽  
High Throughput ◽  
High Throughput Screening ◽  
Colorimetric Detection ◽  
Screening Method ◽  
Random Mutagenesis ◽  
Direct Production ◽  
Consumption Activity

P450 fatty acid decarboxylases (FADCs) have recently been attracting considerable attention owing to their one-step direct production of industrially important 1-alkenes from biologically abundant feedstock free fatty acids under mild conditions. However, attempts to improve the catalytic activity of FADCs have met with little success. Protein engineering has been limited to selected residues and small mutant libraries due to lack of an effective high-throughput screening (HTS) method. Here, we devise a catalase-deficient <i>Escherichia coli</i> host strain and report an HTS approach based on colorimetric detection of H<sub>2</sub>O<sub>2</sub>-consumption activity of FADCs. Directed evolution enabled by this method has led to effective identification for the first time of improved FADC variants for medium-chain 1-alkene production from both DNA shuffling and random mutagenesis libraries. Advantageously, this screening method can be extended to other enzymes that stoichiometrically utilize H<sub>2</sub>O<sub>2</sub> as co-substrate.

XYLANASE, AN ENVIRONMENTALLY FRIENDLY BLEACHING AGENT FOR PULP AND PAPER: Characterization And Stabilization Study Of Bacillus licheniformis I-5 CRUDE Xylanase

2018 ◽  
Vol 7 (3) ◽  
Author(s):  
Budiasih Wahyuntari., dkk
Keyword(s):  
Thermal Stability ◽  
Bacillus Licheniformis ◽  
Hot Spring ◽  
Sodium Dodecyl ◽  
Luria Broth ◽  
Crude Enzyme ◽  
Half Time ◽  
Triton X100 ◽  
Sds Page ◽  
Xylanolytic Enzymes

Isolate I-5 was isolated from Ciseeng hot spring, West Java and was identified as Bacillus licheniformis I-5. The isolate produces extracellular xylanolytic enzymes on Oatspelt containing Luria broth agar medium. Optimal activity of the crude enzyme was  observed at 50ºC and pH 7. The effect of sodium dodecyl sulphate, b-mercaptoethanol and Triton-X100 were observed. Incubating the crude enzyme in 1.5% SDS and 1.5% b-mercaptoethanol at 50oC for 90 minutes then adding Triton-X100 at final concentration of 3.5% for 45 minutes only reduced 5.75% of the initial enzyme activity. SDS/PAGE and zymogram analysis showed that at least two xylanolytic enzymes presence in the crude enzyme. The molecular weight of the enzyme was estimated about 127 and 20kD. The enzyme hydrolysed xylan into xylobiose, xylotriose and other longer xylooligosaccharides. Thermal stability of the crude enzyme was observed at 50, 60, and 70oC and pH 7 and 8. The results showed that the half time of the crude enzyme incubated at 50, 60, and 70oC pH 7 was 2 hours 55 minutes; 2 hours 33 minutes and 1 hour 15 minutes respectively. The half time at 50, 60 and 70oC, pH 8 was 2 hours 48 minutes; 1 hour 22 minutes and 1 hour 9 minutes respectively.keywords: Xilanase, Bacillus licheniformis I-5, thermal stability

Probiotics in the diets of laying quails

2020 ◽  
pp. 60-66
Author(s):  
O. Merzlyakova ◽  
V. Rogachyev ◽  
V. Chegodaev
Keyword(s):  
Bacillus Subtilis ◽  
Bacillus Licheniformis ◽  
Egg Production ◽  
Positive Impact ◽  
Hematological Parameters ◽  
Economic Effect ◽  
Egg Laying ◽  
Control Group ◽  
Egg Mass ◽  
The Cost

The efficiency of introducing probiotics based on strains of Bacillus subtilis, Bacillus licheniformis and their consortium in the amount of 150 g/t of feed into the diets of laying quails has been studied. The experiment lasting 182 days has been carried out on four groups of quails with 30 heads in each. The quails have been housed in the broiler battery in compliance with the required microclimate conditions. Quails of all groups have been received the main diet (compound feed) developed taking into account their age and physiological characteristics. The quails of the 1st, 2nd and 3rd experimental groups in addition to the main diet received probiotics (150 g/t compound feed) based on strains Bacillus subtilis, Bacillus licheniformis and their consortium, respectively. It has been found that feeding the laying quails of the consortium of strains Bacillus subtilis and Bacillus licheniformis had the most significant positive impact on their productive performance, it allowed to increase egg production by 7,81 %, egg laying intensity by 5,0 %, egg mass yield by 9,77 %, while reducing feed expenditures for 10 eggs by 13,35 %. The yield of hatching eggs has been increased by 7,03 %, hatchability of chickens from laid and fertilized eggs by 8,33 and 8,35 %, brooding waste decreased by 21,74 %. Hematological parameters of quails during the whole experiment were within the physiological norm. The economic effect calculated on the basis of data on the cost of compound feed, probiotics and the cost of sold eggs of quail laying was 14,56 % in the 3rd experimental group (in relation to the control group).

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