This research was supported by American Lung Association and American Thoracic Society awards to LS. Authors sincerely want to thank Dr. Jack Elias and Dr. Chun Geun Lee for providing transgenic mouse strains.

All animal procedures were performed under an IACUC-approved protocol at Yale University School of Medicine.
1. Mouse Sample Collection
<ol>
	<li>Anesthetization
	<ol>
		<li>Anesthetize mice using ketamine and xylazine (100 mg/kg of ketamine and 10 mg/kg of xylazine).</li>
	</ol>
	</li>
	<li>Blood sample collection
	<ol>
		<li>Confirm the depth of anesthesia by non-responsiveness to a toe pinch.</li>
		<li>Surgically open the chest cavity to expose the heart.</li>
		<li>Insert a 26.5 G needle into the left ventricle and collect blood into a syringe. 
		NOTE: Typically, about 0.5&#8211;1 mL of blood can be harvested from a healthy 20&#8211;25 g mouse.</li>
		<li>Collect the blood in heparinized tubes, for plasma collection, or non-heparinized tubes for serum collection.</li>
	</ol>
	</li>
	<li>Collection of bronchoalveolar lavage fluid (BAL)
	<ol>
		<li>To collect the BAL, make a vertical cut on the neck to expose the trachea.</li>
		<li>Cannulate the trachea with a 22 G catheter and secure it firmly using a string in order to prevent leakage out of the nose of the mouse.</li>
		<li>Slowly inject two aliquots of sterile phosphate-buffered saline (PBS, 0.75 mL each) into the lungs via the trachea and retrieve back.</li>
		<li>Pool aliquots and keep on ice for further analysis.</li>
	</ol>
	</li>
	<li>Processing the biological samples.
	<ol>
		<li>Blood sample: Either fresh (plasma) or after allowing coagulation for 4 h (serum), centrifuge at 600 x g for 10 min. Take the supernatant and either freeze for storage or use for experiment.</li>
		<li>BAL: Centrifuge the sample at 350 x g for 5 min. Take the supernatant and either freeze for storage or use for experiment.</li>
	</ol>
	</li>
</ol>
2. Chitinase Assay
NOTE: The science behind the assay is relatively simple. A non-fluorescent substrate is cleaved by enzymatically active chitinase to produce a fluorescent product, which is then measured as an indirect marker of chitinase activity. The chitinases within the samples break down the substrate 4-methylumbelliferyl-D-N, N&#8217;-diacetylchitobiose present in the 1x McIlvain buffer. A fluorescent molecule 4-methylumbelliferyl is released. By measuring the total fluorescence of each well, we obtain an accurate measurement of the active chitinase in each sample. Because the breakdown of chitin by chitinase is a hydrolytic reaction, the stop buffer, a mixture of 0.3 M glycine and NaOH (12.0 g/L at pH 10.6), ends the breakdown of chitin by creating an environment that is too basic for the enzyme to function.
<ol>
	<li>Prepare the substrates, standards, and solutions.
	<ol>
		<li>Prepare McIlvain buffer. McIlvain Buffer is comprised of 0.1 M citrate and 0.2 M phosphate at a pH of 5.2. Dilute at a ratio of 1:1 for 1x McIlvain buffer.</li>
		<li>Dissolve both 4-methylumbelliferyl-D-N, N&#8217;-diacetylchitobiose and 4-methylumbelliferyl &#223;-D-N, N&#8217;, N&#8221;-triacetylchitotriose in 1x McIlvain Buffer to a concentration of 500 &#181;M each. 
		NOTE: Stock solution can be stored at -20 &#176;C for further uses; best stored in aliquots.</li>
		<li>Dissolve standard, 4-methylumbelliferone, to a concentration of 0.1 mM in stop buffer (mixture of 0.3 M glycine and NaOH (12.0 g/L at pH 10.6)) to create the standard curve stock solution.</li>
	</ol>
	</li>
	<li>Create and plate the working solution.
	<ol>
		<li>Combine 1x McIlvain Buffer and chitin substrate 4-methylumbelliferyl-D-N, N&#8217;-diacetylchitobiose to create the working solution. For every 10 samples, mix 100 &#181;L of substrate with 2.17 mL of 1x McIlvain buffer. See Table 1 for additional calculations based on sample size. 
		NOTE: Use 4-methylumbelliferyl &#223;-D-N, N&#8217;, N&#8221;-triacetylchitotriose for human samples. While both substrates can be cleaved by either human or mouse enzymes, they have been assigned for either human or mouse samples based on efficiency of cleavage6.</li>
		<li>Pipette 95 &#181;L of working solution into each well of a 96-well plate.</li>
	</ol>
	</li>
	<li>Add biospecimen samples to the working solution.
	<ol>
		<li>Next, add 5 &#181;L of the test sample (blood/BAL/tissue lysate/other biological fluid) to each well.</li>
		<li>Mix the sample into the working solution for the most accurate and reliable results. 
		NOTE: Samples should be run in duplicates/triplicates with special attention paid to potential edge effects. All the samples should be pre-warmed to minimize the edge effect. As the assay involves a reaction between chitinases in the sample and substrate in the working solution, it is best to work relatively quickly to minimize the difference in time between when the first sample is plated and the last sample is plated. Multichannel pipette is recommended.</li>
	</ol>
	</li>
	<li>Incubate at 37 &#176;C.
	<ol>
		<li>Cover the plate and shake briefly (5 s).</li>
		<li>Incubate the plate at 37 &#176;C for 15 min. This allows the enzymatic reaction to take place.</li>
	</ol>
	</li>
	<li>Prepare the standard curve.
	<ol>
		<li>While the samples are incubating, prepare a serial dilution of 4-methylumbelliferone, a standard often used in the fluorimetric determination of enzyme activity.</li>
		<li>Dilute the stock of the standard solution in stop buffer to a concentration of 5 &#181;M.</li>
		<li>Perform a series of dilutions as indicated in Table 2. Add the components in the amount indicated and mix well. 
		NOTE: The standard curve helps to ensure that measured samples fall in the linear range of the standard curve.</li>
	</ol>
	</li>
	<li>Stop the reaction with stop buffer.
	<ol>
		<li>Add 200 &#181;L of stop buffer to each well to stop the reaction.</li>
	</ol>
	</li>
	<li>Plate the standards.
	<ol>
		<li>Add 300 &#181;L of each standard per well in duplicates on the same plate.</li>
	</ol>
	</li>
	<li>Read the plate using a fluorometric reader.
	<ol>
		<li>Read the plate at an excitation of 360 nm, and an emission of 455 nm.</li>
	</ol>
	</li>
</ol>
3. Data Analysis
<ol>
	<li>Subtract the blanks
	<ol>
		<li>Average the values of the duplicate standard dilutions that have no 4-methylumbelliferone. Subtract this value from all of the other recorded values.</li>
	</ol>
	</li>
	<li>Take average of technical replicates and plot the standards. 
	<ol>
		<li>Average the two readings for each concentration and graph the average reading by concentration. 
		NOTE: The resulting plot should look linear and have an R2 value close to 1. If it does not, it may be necessary to redo the standards and reread the plate to ensure quality of data analysis.</li>
	</ol>
	</li>
	<li>Standardize the read-out values.
	<ol>
		<li>Create a best-fit line for the plotted standards data. Divide the read-outs of the samples by the slope of the best-fit line.</li>
	</ol>
	</li>
	<li>Compare values from control group and variable group.
	<ol>
		<li>Use a t-test or ANOVA, for more than two groups, to determine if the difference between groups is statistically significant. Values will take the units nmol/mL&#183;h.</li>
	</ol>
	</li>
</ol>

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Chitinase activity has emerged as an important biomarker for predicting disease severity, disease progression, therapeutic effectiveness and the presence of specific pathogens18. Although many of the long-postulated theories about the role of chitinases have not been experimentally proven19, new studies have provided important insights into the role of chitinases and chitinase like proteins in various diseases2,9<su...

Chitin is the second most abundant polysaccharide on the earth after cellulose, serving as a major structural component of a variety of organisms including the exoskeleton of insects, fungi, yeast, and algae; some vertebrates also have chitin1. Chitinases are a family of enzymes that are capable of breaking down chitin and are highly conserved throughout evolution in species ranging from bacteria to mammals2,3. In addition to chitinases, mammals also have chitinase-like proteins which are similar to chitinases in their ability to bind chitin but differ in that they lack enzymatic ability to cleave the chitin4.
Although chitin and chitinases have long been studied&#8212;with investigations dating back to the early 1900&#8217;s&#8212;the main focus has been on their role in insects and other invertebrates. In fact, it was not until the 1960s when vertebrates were found to have chitinases at all. Using a chitobiase-based assay, chitinases were shown to be found in the digestive tract of a number of vertebrates including lizards and blackbirds&#8212;an observation hypothesized to be due to the consumption of chitin-containing organisms such as insects5.
Mammals have two enzymatically active forms of chitinase: acidic mammalian chitinase (AMCase; also known as CHIT2 and CHIA) and chitotriosidase (CHIT1)4. Both of these proteins are capable of hydrolyzing chitin. However, CHIT1 is more enzymatically active in humans, where almost all of chitinase activity is derived from CHIT1.4 In mice, both Chit1 and AMCase almost equally contribute to the overall chitinase activity6. On the other hand, chitinase-like proteins lack chitinase activity.
Chit1 is mainly secreted by macrophages and is often considered to be an immune response to chitin-containing pathogens7. The enzyme has also been shown to be involved in the maturation of monocytes into both M1 and M2 macrophage subtypes, even without the presence of the substrate chitin8. Furthermore, it may also be involved in the maturation of other immune cells including T helper type 2 (Th2) cells and eosinophils&#8212;as was shown to be the case in cryptococcal lung infection9. These studies point to a complex role of chitinases in the immune system.
In recent years, Chit1 levels have been found to serve as an important biomarker of progression for over 40 different human diseases including lysosomal storage diseases, infectious diseases, respiratory diseases, endocrinological diseases, cardiovascular diseases, neurological diseases, and others (reviewed10). In many of these diseases, the levels of Chit1 are a strong predictor of disease severity and therapeutic effectiveness10.
As chitinases have gained a reputation as a biomarker for numerous medical conditions including Gaucher Disease, tools and assays have been developed to facilitate testing for chitinase presence. Older methods include Schales&#8217; procedure, a protocol adapted from a blood glucose test, and the 3,5 dinitrosalicylic acid (DNS) method. However, these methods are often time sensitive and technically difficult11. The procedure for both of these tests requires the reduction of inorganic oxidants, ferricyanide in the case of the Schales&#8217; procedure for example, producing a color change that can only be measured spectrophotometrically. Additionally, both tests involve a heating or boiling step that is both time consuming and necessary for the color to develop12,13.
Described here is a quick and simple fluorimetric assay to determine chitinase levels in mammalian samples14,15. Two of the samples used here include serum and bronchoalveolar lavage fluids (BAL); chitinase activity has also been measured in breast milk and urine samples, and the technique can be performed in those type of samples as well as in any other biological fluid16,17.

<table><tbody><tr><td>4-methylumbelliferone</td><td>Sigma</td><td>M1381</td><td>Standard: commonly used in flourimetric assays for determination of enzyme activity</td></tr><tr><td>4MU-GlcNAc2</td><td>Sigma</td><td>M9763</td><td>fluorescent chitinase substrate for use in mouse samples</td></tr><tr><td>4MU-GlcNAc3</td><td>Sigma</td><td>M5639</td><td>fluorescent chitinase substrate for use in human samples</td></tr><tr><td>Citric Acid-monohydrate</td><td></td><td></td><td>for use in McIlvain Buffer</td></tr><tr><td>Glycine</td><td></td><td></td><td>for use in Stop Buffer at a concentration of 0.3 M</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;xH&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td>for use in McIlvain Buffer</td></tr><tr><td>NaOH</td><td></td><td></td><td>for use in Stop Buffer at a concentration of 12 g/L</td></tr><tr><td>Vision Plate: Non-sterile, untreated black 96 well plate</td><td>4titude</td><td>4ti-0224</td><td></td></tr></tbody></table>

measurement of chitinase activity in biological samples

Chitinases are the enzymes that cleave chitin. Even in the absence of chitin, mammalians have significant amounts of chitinases present in the body including at baseline. The precise role of chitinase is not known, however it was believed to play important role in digestion and host defense against chitin-containing food and pathogens, respectively. Recent work, including ours, has shown an important role of chitinase and chitinase-like proteins in host immunity and allergic diseases. Importantly, chitinase activities serve as important biomarkers of disease severity in a wide-range of diseases including type 2 inflammatory diseases such as asthma and pulmonary fibrosis. Similarly, patients with genetic disorders like Gaucher disease have significantly elevated chitinase levels, which not only correlate with disease severity but also serve as a reliable biomarker for therapeutic effectiveness. The protocol outlined here describes a simple, quick, and straightforward way to measure chitinase activity in BAL or serum samples of mice and can be widely adapted to human subjects and other model organisms due to the highly conserved nature of the enzymes.

The results shown here are from a study measuring the chitinase activity in serum and BAL samples of wild type (C57BL/6) and Chit1 transgenic mice (on C57BL/6 background) that overexpress the Chit1 gene on a doxycycline promoter. Our data show that both serum and BAL samples have detectable chitinase activity at baseline 698.2 &#177; 189.9 nmol/mL&#183;h and 485.7 &#177; 114 nmol/ml&#183;h, respectively. Overexpression of the Chit1 gene using doxycycline in drinking water for 4 weeks resulted in the elevation of chitinas...

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Measurement of Chitinase Activity in Biological Samples

Presented here is a simple method to measure chitinase activity in biological fluids such as bronchoalveolar lavage or serum.

Chitinases are the enzymes that cleave chitin. Even in the absence of chitin, mammalians have significant amounts of chitinases present in the body ...

measurement-of-chitinase-activity-in-biological-samples

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Immunology and Infection

10.1K Views.  Yale University School of Medicine. Deze methode maakt een effectievere manier om chitinase activiteit in biologische monsters te kwantificeren. Chitinase activiteit is aangetoond dat het dienen als een goede marker voor diagnose en therapeutische werkzaamheid in tal van aandoeningen en ziekten. Eerdere methoden voor het meten van chitinase activiteit waren vaak tijdrovend en moeilijk uit te voeren, beide problemen die zijn opgelost door de test hier getoond.Deze techniek zal helpen bij het vervullen van de potentiële c...

Video: Measurement of Chitinase Activity in Biological Samples

High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper and food industries. A deeper understanding of CAZymes is important from both fundamental biology and industrial standpoints. Vast numbers of CAZymes exist in nature (especially in microorganisms) and hundreds of thousands have been cataloged and described in the carbohydrate active enzyme database (CAZy). However, the rate of discovery of putative enzymes has outstripped our ability to biochemically characterize their activities. One reason for this is that advances in genome and transcriptome sequencing, together with associated bioinformatics tools allow for rapid identification of candidate CAZymes, but technology for determining an enzyme's biochemical characteristics has advanced more slowly. To address this technology gap, a novel high-throughput assay kit based on insoluble chromogenic substrates is described here. Two distinct substrate types were produced: Chromogenic Polymer Hydrogel (CPH) substrates (made from purified polysaccharides and proteins) and Insoluble Chromogenic Biomass (ICB) substrates (made from complex biomass materials). Both CPH and ICB substrates are provided in a 96-well high-throughput assay system. The CPH substrates can be made in four different colors, enabling them to be mixed together and thus increasing assay throughput. The protocol describes a 96-well plate assay and illustrates how this assay can be used for screening the activities of enzymes, enzyme cocktails, and broths.

A high-throughput assay for enzyme screening is described. This multiplexed ready-to-use assay kit comprises of pre-chosen Chromogenic Polymer Hydrogel (CPH) substrates and complex Insoluble Chromogenic Biomass (ICB) substrates. Target enzymes are polysaccharide degrading endo-enzymes and proteases.

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper ...

Biochemistry

Chromatin Immunoprecipitation (ChIP) of Histone Modifications from Saccharomyces cerevisiae

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes that add or remove these marks in response to signals received by the cell. These PTMS are key contributors to the regulation of processes such as gene expression control and DNA repair. Chromatin immunoprecipitation (chIP) has been an instrumental approach for dissecting the abundance and localization of many histone PTMs throughout the genome in response to diverse perturbations to the cell. Here, a versatile method for performing chIP of post-translationally modified histones from the budding yeast Saccharomyces cerevisiae (S. cerevisiae) is described. This method relies on crosslinking of proteins and DNA using formaldehyde treatment of yeast cultures, generation of yeast lysates by bead beating, solubilization of chromatin fragments by micrococcal nuclease, and immunoprecipitation of histone-DNA complexes. DNA associated with the histone mark of interest is purified and subjected to quantitative PCR analysis to evaluate its enrichment at multiple loci throughout the genome. Representative experiments probing the localization of the histone marks H3K4me2 and H4K16ac in wildtype and mutant yeast are discussed to demonstrate data analysis and interpretation. This method is suitable for a variety of histone PTMs and can be performed with different mutant strains or in the presence of diverse environmental stresses, making it an excellent tool for investigating changes in chromatin dynamics under different conditions.

Chromatin Immunoprecipitation ChIP of Histone Modifications from Saccharomyces cerevisiae

Here, we describe a protocol for chromatin immunoprecipitation of modified histones from the budding yeast Saccharomyces cerevisiae. Immunoprecipitated DNA is subsequently used for quantitative PCR to interrogate the abundance and localization of histone post-translational modifications throughout the genome.

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes ...

Genetics

Measuring Volatile and Non-volatile Antifungal Activity of Biocontrol Products

The protocol described is based on a plug-transfer technique that allows accurate determination of microorganism quantities and their developmental stages. A specified number of spores are spread on an agar plate. This agar plate is incubated for a defined period to allow the fungi to reach the expected developmental stage, except for spores where incubation is not required. Agar plugs covered by spores, hyphae, or mycelium are next withdrawn and transferred onto agar media containing the antifungal compound to be tested either placed at a distance from the fungi or in contact. This method is applicable to test both liquid extracts and solid samples (powders). It is particularly well suited for quantifying the relative contributions of volatile and non-volatile agents in bioactive mixtures and for determining their effects, specifically on spores, early hyphae, and mycelium.
The method is highly relevant for the characterization of the antifungal activity of biocontrol products, notably plant-derived products. Indeed, for plant treatment, the results can be used to guide the choice of mode of application and to establish the trigger thresholds.

We describe a modified agar-based method designed to quantify the antifungal effects of plant-derived products. Both volatile and non-volatile contributions to the antifungal activity can be assessed through this protocol. In addition, efficacy against fungi can be measured at key developmental stages in a single experimental setup.

The protocol described is based on a plug-transfer technique that allows accurate determination of microorganism quantities and their developmental ...

Environment

Preparation of Expanded Chitin Foams and their Use in the Removal of Aqueous Copper

Chitin is an underexploited, naturally abundant, mechanically robust, and chemically resistant biopolymer. These qualities are desirable in an adsorbent, but chitin lacks the necessary specific surface area, and its modification involves specialized techniques and equipment. Herein is described a novel chemical procedure for expanding chitin flakes, derived from shrimp shell waste, into foams with higher surface area. The process relies on the evolution of H2 gas from the reaction of water with NaH trapped in a chitin gel. The preparation method requires no specialized equipment. Powder X-ray diffraction and N2-physisorption indicate that the crystallite size decreases from 6.6 nm to 4.4 nm and the specific surface area increases from 12.6 &#177; 2.1 m2/g to 73.9 &#177; 0.2 m2/g. However, infrared spectroscopy and thermogravimetric analysis indicate that the process does not change the chemical identity of the chitin. The specific Cu adsorption capacity of the expanded chitin increases in proportion to specific surface area from 13.8 &#177; 2.9 mg/g to 73.1 &#177; 2.0 mg/g. However, the Cu adsorption capacity as a surface density remains relatively constant at an average of 10.1 &#177; 0.8 atom/nm2, which again suggests no change in the chemical identity of the chitin. This method offers the means to transform chitin into a higher surface area material without sacrificing its desirable properties. Although the chitin foam is described here as an adsorbent, it can be envisioned as a catalyst support, thermal insulator, and structural material.

This study describes a method to expand chitin into a foam by chemical techniques that require no specialized equipment.

Chitin is an underexploited, naturally abundant, mechanically robust, and chemically resistant biopolymer. These qualities are desirable in an adsorbent, ...

Chemistry

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2. Of particular interest are fungal cellulases3-8, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium11-12. 
We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (&#x201C;halos&#x201D;) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness. 
Traditional bacterial whole cell CMC/CR activity assays13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

Biology

Process Evaluation and Kinetics of Recombinant Chitin Deacetylase Expression in E. coli Rosetta pLysS Cells Using a Statistical Technique

In recent years, the greener route of the deacetylation of chitin to chitosan using the enzyme chitin deacetylase has gained importance. Enzymatically converted chitosan with emulating characteristics has a broad range of applications, particularly in the biomedical field. Several recombinant chitin deacetylases from various environmental sources have been reported, but there are no studies on process optimization for the production of these recombinant chitin deacetylases. The present study used the central composite design of response surface methodology to maximize the recombinant bacterial chitin deacetylase (BaCDA) production in E. coli Rosetta pLysS. The optimized process conditions were 0.061% glucose concentration, 1% lactose concentration, an incubation temperature of 22 &#176;C, an agitation speed at 128 rpm, and 30 h of fermentation. At optimized conditions, the expression due to lactose induction was initiated after 16 h of fermentation. The maximum expression, biomass, and BaCDA activity were recorded 14 h post-induction. At the optimized condition, the BaCDA activity of expressed BaCDA was increased ~2.39-fold. The process optimization reduced the total fermentation cycle by 22 h and expression time by 10 h post-induction. This is the first study to report the process optimization of recombinant chitin deacetylase expression using a central composite design and its kinetic profiling. Adapting these optimal growth conditions could result in cost-effective, large-scale production of the lesser-explored moneran deacetylase, embarking on a greener route for biomedical-grade chitosan production.

Process Evaluation and Kinetics of Recombinant Chitin Deacetylase Expression in E. coli Rosetta pLysS Cells Using a Statistical Technique

In the current protocol, a statistical technique, central composite design (CCD), was applied to optimize the process conditions for the expression of recombinant bacterial chitin deacetylase (BaCDA) in E. coli Rosetta pLysS cells. The employment of CCD resulted in a ~2.39-fold increase in the expression and activity of BaCDA.

In recent years, the greener route of the deacetylation of chitin to chitosan using the enzyme chitin deacetylase has gained importance. Enzymatically ...

Bioengineering

Quantifying the Antifungal Activity of Peptides Against Candida albicans

Traditional methods for performing antifungal susceptibility testing for Candida albicans are time-consuming and lack quantitative results. For example, a common approach relies on plating cells treated with different concentrations of antifungal molecules on agar plates and then counting the colonies to determine the relationship between molecule concentration and growth inhibition. This method requires many plates and substantial time to count the colonies. Another common approach eliminates the plates and counting of colonies by visually inspecting cultures treated with antifungal agents to identify the minimum concentration required to inhibit growth; however, visual inspection produces only qualitative results, and information on growth at subinhibitory concentrations is lost. This protocol describes a method for measuring the susceptibility of C. albicans to antifungal peptides. By relying on optical density measurements of cultures, the method reduces the time and materials needed to obtain quantitative results on culture growth at different peptide concentrations. The incubation of the fungus with peptides is performed in a 96-well plate using an appropriate buffer, with controls representing no growth inhibition and complete growth inhibition. Following the incubation with the peptide, the resulting cell suspensions are diluted to reduce peptide activity and then grown overnight. After overnight growth, the optical density of each well is measured and compared to the positive and negative controls to calculate the resulting growth inhibition at each peptide concentration. The results using this assay are comparable to the results using the traditional method of plating the cultures on agar plates, but this protocol reduces plastic waste and the time spent on counting colonies. Although the applications of this protocol have focused on antifungal peptides, the method will also be applicable to testing other molecules with known or suspected antifungal activity.

Quantifying the Antifungal Activity of Peptides Against Candida albicans

This protocol describes a method for obtaining quantitative data on the antifungal activity of peptides and other compounds, such as small-molecule antifungal agents, against Candida albicans. Its use of optical density rather than counting colony-forming units to quantify growth inhibition saves time and resources.

Traditional methods for performing antifungal susceptibility testing for Candida albicans are time-consuming and lack quantitative results. For example, a ...

An In Vitro Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilm

Source: Chandley, P., et al. <a href="https://app.jove.com/v/64425">A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms.</a> J. Vis. Exp. (2022).
This video demonstrates an in vitro technique to study the effect of antibodies on fungal biofilms. The pathogenic fungus Candida tropicalis secretes a virulence factor secreted aspartyl proteinase 2 (Sap2), facilitating biofilm formation. Upon introducing a heat-inactivated serum containing Sap2-specific antibodies that inhibit biofilm maturation, the viability of the fungal cells is assessed using a chromogenic assay.

1. C. tropicalis&#160;biofilm formation

	Prepare&#160;Candida&#160;biofilms in a 96-well flat-bottomed polystyrene microtiter plate as described earlier ...

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Characterization and Functional Analysis

In Vitro Measurement of &alpha;-Galactosidase A and Acid &alpha;-Glucosidase Enzyme Activity

Source: Lukas, J., et al. <a href="https://app.jove.com/v/56550">In Vitro Enzyme Measurement to Test Pharmacological Chaperone Responsiveness in Fabry and Pompe Disease.</a> J. Vis. Exp. (2017)
This video demonstrates the in vitro measurement of enzyme activity for &#945;-galactosidase A and acid &#945;-glucosidase in samples using synthetic 4-Methylumbelliferyl substrates.

1. Cell Harvest and &#945;-galactosidase A or Acid &#945;-glucosidase Activity Measurement

	Cell harvest&#8203;
	
		On the day of the harvest, remove the ...

Measurement of Chitinase Activity in Biological Samples

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