Downstream process intensification for biotechnologically generated hyaluronic acid: Purification and characterization.

Hyaluronic acid (HA), an anionic, non-sulfated glycosaminoglycan, has several clinical applications. This study examines several downstream methods for purifying HA with maximum recovery and purity. Following the fermentation of Streptococcus zooepidemicus MTCC 3523 to produce HA, the broth was thoroughly purified to separate cell debris and insoluble impurities using a filtration procedure and a variety of adsorbents for soluble impurities. Nucleic acids, proteins with high molecular weight, were successfully removed from the broth using activated carbons and XAD-7 resins. In contrast, insoluble and low molecular weight impurities were removed using diafiltration, with HA recovery of 79.16% and purity close to 90%. Different analytical and characterization procedures such as Fourier transform-infrared spectroscopy, X-ray diffraction, nuclear magnetic resonance, and scanning electron microscopy validated the presence, purity, and structure of HA. Microbial HA showed activity in tests for 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) radical-scavenging (4.87 ± 0.45 kmol TE/g), total antioxidant capacity (13.32 ± 0.52%), hydroxyl radical-scavenging (32.03 ± 0.12%), and reducing power (24.85 ± 0.45%). The outcomes showed that the precipitation, adsorption, and diafiltration processes are suitable for extracting HA from a fermented broth under the chosen operating conditions. The HA produced was of pharmaceutical grade for non-injectable applications.

Hyaluronic acid production and purification techniques: a review.

Hyaluronic acid (HA) is an exopolysaccharide extracted from several sources such as rooster combs, umbilical cords and microorganisms. A system that controls temperature, agitation and aeration of bacterial cultures could make the HA production autonomous. Therefore, HA of microbial origin is set to take over alternative methods of production. Furthermore, the use of different nutrient sources in the culture medium and the purification stage applied in the process can cause physicochemical alterations on the bioproduct. For instance, structural modifications that change the molecular weight of HA may alter its elastic and viscoelastic properties. As a result, HA synthesized by microbes has applications in pharmacology, biotechnology, and tissue engineering. Our aim here, is to show the vast range of applications by compiling articles and patents on the culture media or genetic modifications of microorganisms that synthesize HA.

Current advances in the biosynthesis of hyaluronic acid with variable molecular weights.

Hyaluronic acid (HA) is a naturally formed acidic mucopolysaccharide, with excellent moisturising properties and used widely in the medicine, cosmetics, and food industries. The industrial production of specific molecular weight HA has become imperative. Different biological activities and physiological functions of HA mainly depend on the degree of polymerisation. This article reviews the research status and development prospects of the green biosynthesis and molecular weight regulation of HA. There is an application-based prerequisite of specific molecular weight of HA that could be regulated either during the fermentation process or via a controlled HA degradation process. This work provides an important theoretical basis for the downstream efficient production of diversified HA, which will further accelerate the research applications of HA and provide a good scientific basis and method reference for the study of the molecular weight regulation of similar biopolymers.

Chitosan/Hyaluronic acid/Alginate and an assorted polymers loaded with honey, plant, and marine compounds for progressive wound healing-Know-how.

Biomaterials are being extensively used in regenerative medicine including tissue engineering applications, as these enhance tissue development, repair, and help in the process of angiogenesis. Wound healing is a crucial biological process of regeneration of ruptured tissue after getting injury to the skin and other soft tissue in humans and animals. Besides, the accumulation of microbial biofilms around the wound surface can increase the risk and physically obstruct the wound healing activity, and may even lead to amputation. Hence, in both acute and chronic wounds, prominent biomaterials are required for wound healing along with antimicrobial agents. This review comprehensively addresses the antimicrobial and wound healing effects of chitosan, chitin, cellulose acetate, hyaluronic acid, pullulan, bacterial cellulose, fibrin, alginate, etc. based wound dressing biomaterials fabricated with natural resources such as honey, plant bioactive compounds, and marine-based polymers. Due to their excellent biocompatibility and biodegradability, bioactive compounds derived from honey, plants, and marine resources are commonly used in biomedical and tissue engineering applications. Different types of polymer-based biomaterials including hydrogel, film, scaffold, nanofiber, and sponge dressings fabricated with bioactive agents including honey, curcumin, tannin, quercetin, andrographolide, gelatin, carrageenan, etc., can exhibit significant wound healing process in, diabetic wounds, diabetic ulcers, and burns, and help in cartilage repair along with good biocompatibility and antimicrobial effects. Among the reviewed biomaterials, carbohydrate polymers such as chitosan-based biomaterials are prominent and widely used for wound healing applications followed by hyaluronic acid and alginate-based biomaterials loaded with honey, plant, and marine compounds. This review first provides an overview of the vast natural resources used to formulate different biomaterials for the treatment of antimicrobial, acute, and chronic wound healing processes.

Preparation, purification, and characterization of low-molecular-weight hyaluronic acid.

OBJECTIVE: The use and commercial value of hyaluronic acid (HA) as an important element in the pharmaceutical, biomedical, and cosmetics industry is because of its purity. Four recombinant strains of Corynebacterium glutamicum containing different genes were used to produce HA. RESULTS: The production parameters were measured and strain 183.2, with the highest amount of HA (2.15 mg/ml), was selected for further experiments. HA was precipitated by different ratios of ethanol-isopropanol at 4 °C and - 20 °C. Active charcoal (1%) was added to the solvent precipitation mixture at pH 5 and 10. Finally, to achieve more purity and separation, gel filtration chromatography was used. The best result was obtained using an ethanol-isopropanol ratio of 1:1 of at - 20 °C, followed by active charcoal treatment at the acidic pH, and three fractions of the chromatography with molecular weights of 27, 27-110, and < 27 KDa were more analyzed with electrophoresis and FTIR. CONCLUSIONS: The present study described a simple, economical, and reproducible method resulting in a high yield for low-MW HA from C. glutamicum.

Improving physical and biological properties of nylon monofilament as suture by Chitosan/Hyaluronic acid.

One way to give some properties such as antibacterial and good frictional properties to sutures is the application of natural antibacterial and hydrophilic components on their surfaces through layer by layer assembly (LBL) technique. In this regard, Chitosan as an antibacterial polycationic natural polymer along with Hyaluronic acid (HA) as a polyanionic polysaccharide could be used to form a polyelectrolyte complex. In this study, HA was extracted from rooster comb using different solvents. Characterization of the extracted HA by FTIR and GPC analysis showed extracted HA with Mw = 2.53 × 105 Da had no cytotoxicity. Then, a nylon monofilament (NMy) was coated by the extracted HA and chitosan with different concentrations using bilayer coating technique. Two dyes also were loaded to coating layer to investigate the release behavior of these two drug models. The morphology of coated layer showed that coating NMy by chitosan (4% w/v) following by HA (8% w/v) with roughness of 164 ± 129 nm and friction coefficient of 0.26 had suitable interaction between two layers to prevent from exfoliation of coating layers. The antibacterial activity and controlled release of coated NMy indicated how a NMy coated by Chitosan and HA is a promising material for using as a suture.

Hyaluronic acid and Chondroitin sulfate from marine and terrestrial sources: Extraction and purification methods.

Hyaluronic acid (HA) and chondroitin sulfate (CS) are valuable bioactive polysaccharides that have been highly used in biomedical and pharmaceutical applications. Extensive research was done to ensure their efficient extraction from marine and terrestrial by-products at a high yield and purity, using specific techniques to isolate and purify them. In general, the cartilage is the most common source for CS, while the vitreous humor is main used source of HA. The developed methods were based in general on tissue hydrolysis, removal of proteins and purification of the target biopolymers. They differ in the extraction conditions, enzymes and/or solvents used and the purification technique. This leads to specific purity, molecular weight and sulfation pattern of the isolated HA and CS. This review focuses on the analysis and comparison of different extraction and purification methods developed to isolate these valuable biopolymers from marine and terrestrial animal by-products.

Innovative Visualization and Quantification of Extracellular Vesicles Interaction with and Incorporation in Target Cells in 3D Microenvironments.

Extracellular vesicles (EVs) showed therapeutic properties in several applications, many in regenerative medicine. A clear example is in the treatment of osteoarthritis (OA), where adipose-derived mesenchymal stem cells (ASCs)-EVs were able to promote regeneration and reduce inflammation in both synovia and cartilage. A still obscure issue is the effective ability of EVs to be internalized by target cells, rather than simply bound to the extracellular matrix (ECM) or plasma membrane, since the current detection or imaging technologies cannot fully decipher it due to technical limitations. In the present study, human articular chondrocytes (ACHs) and fibroblast-like synoviocytes (FLSs) isolated from the same OA patients were cocultured in 2D as well as in 3D conditions with fluorescently labeled ASC-EVs, and analyzed by flow cytometry or confocal microscopy, respectively. In contrast with conventional 2D, in 3D cultures, confocal microscopy allowed a clear detection of the tridimensional morphology of the cells and thus an accurate discrimination of EV interaction with the external and/or internal cell environment. In both 2D and 3D conditions, FLSs were more efficient in interacting with ASC-EVs and 3D imaging demonstrated a faster uptake process. The removal of the hyaluronic acid component from the ECM of both cell types reduced their interaction with ASC-EVs only in the 2D system, showing that 2D and 3D conditions can yield different outcomes when investigating events where ECM plays a key role. These results indicate that studying EVs binding and uptake both in 2D and 3D guarantees a more precise and complementary characterization of the molecular mechanisms involved in the process. The implementation of this strategy can become a valuable tool not only for basic research, but also for release assays and potency prediction for clinical EV batches.

Naked mole-rat very-high-molecular-mass hyaluronan exhibits superior cytoprotective properties.

Naked mole-rat (NMR), the longest-living rodent, produces very-high-molecular-mass hyaluronan (vHMM-HA), compared to other mammalian species. However, it is unclear if exceptional polymer length of vHMM-HA is important for longevity. Here, we show that vHMM-HA (>6.1 MDa) has superior cytoprotective properties compared to the shorter HMM-HA. It protects not only NMR cells, but also mouse and human cells from stress-induced cell-cycle arrest and cell death in a polymer length-dependent manner. The cytoprotective effect is dependent on the major HA-receptor, CD44. We find that vHMM-HA suppresses CD44 protein-protein interactions, whereas HMM-HA promotes them. As a result, vHMM-HA and HMM-HA induce opposing effects on the expression of CD44-dependent genes, which are associated with the p53 pathway. Concomitantly, vHMM-HA partially attenuates p53 and protects cells from stress in a p53-dependent manner. Our results implicate vHMM-HA in anti-aging mechanisms and suggest the potential applications of vHMM-HA for enhancing cellular stress resistance.

Characterization of hyaluronic acid extracted from Liparis tessellatus eggs grafted with phenolic acids and nisin.

In polymer therapeutics, polymer-based carrier systems conjugated with antioxidants have been synthesized and studied to improve diagnosis and treatment of diseases, such as, in cancer and tumor. The natural bioactive compound hyaluronic acid (HA), which is essential in medical and pharmaceutical fields, is a linear polymer composed of repeating disaccharide units of ß-1,3-N-acetyl glucosamine and ß-1,4-glucuronic acid. In this study, HA fractions of enzyme-assisted glycosaminoglycans (GAGs) extract from Liparis tessellatus eggs were grafted with gallic acid (GA), caffeic acid (CA), and ferulic acid (FA) via a free radical-mediated method, and with nisin via amide bond formation. The modification has been confirmed through FTIR and 1H NMR spectroscopy and quantified by Folin-Ciocalteu and Bradford assay. FTIR spectra of grafted HA samples exhibited the typical phenolic characteristics within 1450-1650 cm-1, and the formation of amide bond in nisin-grafted HA was shown by absorption peak within 1545-1646 cm-1. 1H NMR spectra showed new peaks of phenyl protons at 6.3-7.7 ppm and new peaks at 0.9-2.9 ppm of amino acids residues protons. These results all confirmed the successful grafting of GA, CA, FA and nisin onto the HA backbone extracted from L. tessellatus eggs.

Hyaluronan biofilms reinforced with partially deacetylated chitin nanowhiskers: Extraction, fabrication, in-vitro and antibacterial properties of advanced nanocomposites.

Fabrication of nanocomposite biofilms with enhanced mechanical and antibacterial properties was successfully achieved from hyaluronan (HA) and partially deacetylated chitin nanowhiskers (ChNWs) by a casting-evaporation method. The hydrolysis process of chitin showed an important role in the dimensions, stability, and the crystallinity of extracted ChNWs in a time-dependent manner. The volume fraction of ChNWs nanofiller varying from (0.001 to 0.5) exhibited a great influence on the mechanical properties of the biofilms (young modulus, strength) was enhanced by the high load-bearing capacity of NWs compared with net HA film. The antibacterial activity of the nanocomposite biofilms exhibited significant bactericidal activity against different types of bacteria (-/+ gram). HA/ChNWs Nanocomposite biofilms did not show any toxicity against normal human dermal fibroblasts (NHDF) and human primary osteogenic sarcoma (Saos-2) cell lines. The new biofilms with unique properties like edibleness, environmental friendliness, high mechanical properties, antibacterial performance, and non-cytotoxicity that could be used in skin tissue regenerations, and drug delivery applications.

A hyaluronan-based polysaccharide peptide generated by a genetically modified Streptococcus zooepidemicus.

Polysaccharide peptides (or protein-bound polysaccharides, PSPs) are commonly found in mushrooms and plants and possess important nutritional properties and health benefits. The pathogenic bacterium Streptococcus zooepidemicus does not inherently produce PSPs but secretes the capsular polysaccharide hyaluronan. However, in a previous investigation of the catalytic mechanism of UDP-glucose dehydrogenase (UGDH), a PSP of peptide-bound hyaluronan was found to be produced by S. zooepidemicus through the in vivo expression of a mutant of the gene encoding UGDH. In the present study, this hyaluronan-derived PSP was structurally characterized by FT-IR, NMR, and high-performance liquid chromatography-mass spectrometry (HPLC-MS), and the data confirmed that the polysaccharide backbone, hyaluronan, is covalently bound to the side-chain peptides via an amide linkage. More importantly, the bacterial production of a PSP via this genetic modification method should inspire further research on the in vitro enzymatic synthesis of PSPs or even naturally occurring polysaccharide derivatives and may provide a theoretical foundation for investigating the in vivo synthetic mechanism of PSPs.

Methods for Hyaluronan Molecular Mass Determination by Agarose Gel Electrophoresis.

The average molecular mass of hyaluronan (HA) in most healthy biological fluids and tissues is usually about 6000-8000 kDa, but the biosynthetic mechanism results in a polydisperse mixture of sizes. Subsequent enzymatic degradation, or the action of reactive oxygen and nitrogen species, can further increase polydispersity and decrease the average size. Fragmented HA can be a biomarker of inflammation. In addition, reductions in HA size are associated with tissue remodeling and repair processes. Some cell-surface receptor proteins have been reported to have HA-binding affinities that are size specific, and participate in activation of signaling cascades controlling multiple aspects of cell behavior. Here we describe simple agarose gel electrophoresis protocols for the determination of the molecular mass distribution of HA isolated from tissues and fluids.

Recovery and Purity of High Molar Mass Bio-hyaluronic Acid Via Precipitation Strategies Modulated by pH and Sodium Chloride.

The effects of ethanol/broth proportions and the number of steps at varying pH in the presence or absence of sodium chloride (NaCl) were studied as precipitation strategies for the recovery and purification of high molar mass bio-hyaluronic acid (Bio-HA). Bio-HA was synthesized by Streptococcus zooepidemicus in a culture medium containing glucose and soy peptones. A single-step precipitation was more attractive than multistep precipitation in terms of recovery and purity as well as decreased use of ethanol. The best conditions in the absence and presence of salt were 2:1 ethanol/broth (v/v) at pH 4 (55.0 ± 0.2% purity and 85.0 ± 0.7% recovery) and 2:1 ethanol/broth (v/v) at pH 7 + 2 mol L-1 NaCl (59.0 ± 0.9% purity and 82.0 ± 4.3% recovery). Dynamic light scattering (DLS) spectra showed different particle sizes as a consequence of the changes in the molecular structure of HA, mainly with changes in pH. Although slight changes in distribution were observed, the average HA molar mass was not affected by the precipitation strategy, remaining on the order of 105 Da. Therefore, pH and NaCl modulated the precipitation performance of HA. These findings are relevant to further optimizing the precipitation step, thus minimizing costs in the later stages of HA purification.

Efecto del extracto de cresta de gallo, rico en ácido hialurónico, sobre los parámetros isocinéticos en personas con gonalgia leve / Effect of rooster comb extract, rich in hyaluronic acid, on isokinetic parameters in adults with mild knee pain

Introducción: La artrosis de rodilla se manifiesta en una primera fase como una gonalgia leve y produce una disminución de la fuerza muscular, que puede ser valorada objetivamente con la prueba isocinética. Dentro de su tratamiento se debe considerar el ácido hialurónico por vía oral. Objetivo: Valorar la evolución de los parámetros isocinéticos de fuerza muscular en la articulación de la rodilla con gonalgia leve antes y después del consumo de un extracto de cresta de gallo (ECG), rico en ácido hialurónico, por vía oral. Metodología: Ensayo de intervención nutricional, doble ciego, aleatorizado, controlado con placebo y en paralelo con dos grupos de tratamiento: grupo activo con ingesta de un yogur bajo en grasa con 80 mg de ECG rico en ácido hialurónico y grupo control con ingesta de un yogur bajo en grasa sin ECG. Se valoraron los parámetros isocinéticos de pico torque (PT), trabajo total (TT) y potencia media (PM) a las velocidades de 180º/seg y 240º/seg para los movimientos de flexión y extensión de la rodilla. Resultados: Después de 12 semanas del consumo del yogur, los hombres del grupo activo, en comparación con los del grupo control, obtuvieron diferencias estadísticamente significativas (p<0,05) y mejora clínica (>10%) a la velocidad de 180º/seg en el movimiento de extensión en la variable PT (p=0,048) (19,33%), TT (p=0,020) (37,97%) y PM (p=0,029) (47,25%), y en el movimiento de flexión en la variable PT (p=0,007) (25,41%), TT (p=0,014) (42,98%) y PM (p=0,022) (48,90%). Conclusión: La ingesta de un yogur bajo en graso con extracto de cresta de gallo rico en ácido hialurónico mejora la fuerza muscular de la rodilla en hombres con gonalgia leve

Background: Knee osteoarthritis manifests itself in a first phase as a mild gonalgia and produces a decrease in muscle strength, which can be objectively assessed with isokinetic testing. In the treatment of knee osteoarthritis, should be considered the hyaluronic acid orally. Objective: Assess the evolution of isokinetic parameters of muscle strength in the knee joint with mild gonalgia before and after the consumption of RCE, rich in hyaluronic acid, orally. Methodology: Nutritional intervention trial, double-blind, randomized, controlled with placebo and in parallel with two treatment groups: the active group with a low-fat yogurt with 80 mg of roster comb extract (RCE) and the control group with a low-fat yogurt without RCE. The main variables of the study were the peak torque, total work and mean power of the isokinetic valuation at the speed of 180 º/seg and 240 º/seg by the movements of flexion and extension of the knee joint. Results: After 12 weeks of consumption of yogurt, men of the active group, compared with those in the control group, obtained statistically significant differences (p <0.05) and clinical improvement (>10%) at the speed of 180º/seg in the movement extension in the PT variable (p=0.048) (19.33%), TT (p=0.020) (37.97%) and PM (p=0.029) (47.25%), and in the flexion movement in the variable PT (p=0.007) (25.41%), TT (p=0.014) (42.98%) and PM (p=0.022) (48.90%). Conclusions: The intake of a low-fat yogurt with RCE rich in hyaluronic acid improves the muscle strength of the knee in men with mild gonalgia

Separation of hydrophobically modified hyaluronic acid according to the degree of substitution by gradient elution high performance liquid chromatography.

Amphiphilic hyaluronic acid (HA), synthesised by modifying HA to varying extents with acrylate groups, was successfully separated according to degree of substitution (DS) using solvent gradient high performance liquid chromatography (HPLC). Two HPLC methods based on the amphiphilic structure of the HA were developed. In the first approach, normal phase gradient HPLC was explored, and separation was based on the interactions of HA's polar hydroxyl groups with a polar cyano stationary phase. In the second approach, separation was based on the interaction of the hydrophobic acrylate moieties with a non-polar C8 stationary phase (reversed phase gradient HPLC). The separation was optimised by using an electrolyte in the sample solvent to suppress non-covalent interactions and improve the selectivity of the developed method. The photolytic stability of the modified and unmodified HA was also investigated in order to optimise the sample preparation procedure. Furthermore, an alternative method to NMR spectroscopy was developed for determining the DS of HA. Graphical abstract ᅟ.

Glycosaminoglycans and glycolipids as potential biomarkers in lung cancer.

In this report, we used liquid chromatography-mass spectrometry and Western blotting to analyze the content and structure of glycosaminoglycans, glycolipids and selected proteins to compare differences between patient-matched normal and cancerous lung tissues obtained from lung cancer patients. The cancer tissue samples contained over twice as much chondroitin sulfate (CS)/dermatan sulfate (DS) as did the normal tissue samples, while the amount of heparan sulfate (HS) and hyaluronan (HA) in normal and cancer tissues were not significantly different. In HS, several minor disaccharide components, including NS6S, NS2S and 2S were significantly lower in cancer tissues, while the levels of major disaccharides, TriS, NS and 0S disaccharides were not significantly different in normal and cancer tissues. In regards to CS/DS, the level of 4S disaccharide (the major component of CS-type A and DS) decreased and the level of 6S disaccharide (the major component of CS- type C) increased in cancer tissues. We also compared the content and structure of GAGs in lung tissues from smoking and non-smoking patients. Analysis of the glycolipids showed all lipids present in these lung tissues, with the exception of sphingomyelin were higher in cancer tissues than in normal tissues. Western analysis showed that syndecan 1 and 2 proteoglycans displayed much higher expression in cancer tissue/biopsy samples. This investigation begins to provide an understanding of patho-physiological roles on glycosaminoglycans and glycolipids and might be useful in identifying potential biomarkers in lung cancer.

Quantification and comparison of acidic polysaccharides in edible fish intestines and livers using HPLC-MS/MS.

Fish intestines and livers are usually considered as delicious and nutritious food in China. Acidic polysaccharides are important nutrients in these food of animal origin, but there is currently little information regarding their quantitative distributions. The present study demonstrated a method to quantify acidic polysaccharides simultaneously by analyzing their disaccharides produced from the acid hydrolysis using high-performance liquid chromatography (HPLC) coupled with triple quadrupole mass spectrometry. The recoveries for these acidic polysaccharides were all 97%-115% with relative standard deviation of 3.0%-9.0%. All of the acidic polysaccharides had good linearities. Then this method was applied to determine the composition of acidic polysaccharides in 5 edible fish livers and intestines. Besides well-known glycosaminoglycans (GAGs) including hyaluronic acid (HA), Chondroitin sulfate (CS), dermatan sulfate (DS) and heparin (HP), 4 novel acidic polysaccharides including 2 GAGs and 2 non-GAGs comprised of hexose-hexuronic acid repeating units were also found. CS and HP were the major acidic polysaccharides components in fish intestines and livers, respectively. The absolute amounts of acidic polysaccharides differed greatly in these fish tissues, but their proportions showed similarity in the same type of tissues. The present study demonstrated an effective method for acidic polysaccharides quantification, and revealed acidic polysaccharides compositions of edible fish livers and intestines.

Targeting the Tumor Stroma: the Biology and Clinical Development of Pegylated Recombinant Human Hyaluronidase (PEGPH20).

The tumor stroma is increasingly recognized as a key player in tumorigenesis through its effects on cell signaling, immune responses, and access of therapeutic agents. A major component of the extracellular matrix is hyaluronic acid (HA), which raises the interstitial gel fluid pressure within tumors and reduces drug delivery to malignant cells, and has been most extensively studied in pancreatic ductal adenocarcinoma (PDA). Pegylated recombinant human hyaluronidase (PEGPH20) is a novel agent that degrades HA and normalizes IFP to enhance the delivery of cytotoxic agents. It has demonstrated promising preclinical results and early clinical evidence of efficacy in the first-line treatment of metastatic PDA with acceptable tolerability. Moreover, intratumoral HA content appears to be a predictive biomarker of response. Phase 2 and 3 trials of PEGPH20 plus chemotherapy are ongoing in metastatic PDA, and it is also being evaluated in other malignancies and in combination with radiation and immunotherapy.

A simple, rapid and sensitive plate assay for detection of microbial hyaluronidase activity.

Hyaluronidase (hyase) is a glycosidase enzyme that predominantly degrades hyaluronic acid (HA) having important applications in many biotechnological processes and therapeutics. Several assay methods have been proposed to screen hyase producing microorganisms; however, they rely on unique reagents and sophisticated instruments, which are expensive and could be unavailable in general laboratories. In the present studies, a rapid, simple, sensitive, highly reproducible, and cost-effective qualitative plate assay has been developed for the screening of hyase producing microorganisms. The routinely used plate assay method of Richman and Baer requires a special chemical cetylpyridinium chloride and long incubation period of 20 h; but still, the zones of clearance are not very clear and distinct. While, the present method requires an incubation period of only 1 h and the distinct zones of clearance appear with Gram's iodine within 1 min of time. This method does not require any special medium, unlike previously reported methods. Moreover, use of commonly available Gram's iodine makes this method suitable for many researchers. The results of the assay method were validated by TLC, zymographic analysis and determining the growth of isolates in minimal medium containing HA as a sole carbon source.