Enzymatic Hydrolysis Of Urea Research Articles

Starch-based C-dots from natural Gadong tuber as pH fluorescence label for optical biosensing of arginine

A fluorescence bi-enzyme optical arginine biosensor based on novel fluorophore carbon nanodots (C-dots) pH indicator derived from natural Gadong (Dioscorea hispida) tuber was developed. The brightly luminescent pH sensitive carbon-based quantum dots (QDs) exploited from exotic wild yam polysaccharides via aqueous-based synthesis method were immobilized on the acrylic-N-acryloxysuccinimidecopolymer microspheres. Recombinant Glaciozyma antarctica arginase (GaArg) cloned in E. coli expression system and commercial urease were grafted on the acrylic microspheres (AMS) via peptide covalent link. During fluorescence arginine detection, the immobilized arginase catalyzed hydrolysis conversion of arginine to ornithine and urea, and subsequent enzymatic hydrolysis of urea by immobilized urease rendering deprotonation of starch-based C-dots, resulted in the heightening of C-dots’ photoluminescence intensity due to elevated localized pH. Under 380 nm excitation, the C-dots fluorescence intensity at 420 nm increased linearly with arginine over the concentration range of 1–10 mM, with the limit of detection (LOD) estimated at 0.9 mM. The Gadong starch-based C-dots, which prepared from different natural tuber samples showed high reproducible physico-chemical properties, and gave reproducible fluorescence intensity at different pH values with relative standard deviation (RSD) values obtained at <1.0%, which suggests high reproducibility of the synthesis of starch-based luminecent C-dots. The optical enzymatic biosensor doped with C-dots pH label demonstrated high selectivity towards fluorescence biorecognition of arginine compared to some other amino acids normally present in packaged fruit juices. No significant difference was found between C-dots-labeled enzymatic fluorescence microsensor and HPLC standard method for arginine content detection in fruit juice samples of grapes, mangoes and oranges.

Alkaline dehydration of source-separated fresh human urine: Preliminary insights into using different dehydration temperature and media

For sanitation systems aiming at recycling nutrients, separately collecting urine at source is desirable as urine contains most of the nutrients in wastewater. However, reducing the volume of the collected urine and recovering majority of its nutrients is necessary, as this improves the transportability and the end-application of urine-based fertilisers. In this study, we present an innovative method, alkaline dehydration, for treating fresh human urine into a nutrient-rich dry solid. Our aim was to investigate whether fresh urine (pH < 7) added to five different alkaline media (pH > 11) could be dehydrated at elevated temperatures (50 and 60 °C) with minimal loss of urea, urine's principal nitrogen compound. We found that it was possible to concentrate urine 48 times, yielding dry end-products with high fertiliser value: approximately, 10% N, 1% P, and 4% K. We monitored the physicochemical properties and the composition of various dehydration media to provide useful insights into their suitability for dehydrating urine. We demonstrated that it is possible to recover >90% nitrogen when treating fresh urine by alkaline dehydration by inhibiting the enzymatic hydrolysis of urea at elevated pH and minimising the chemical hydrolysis of urea with high urine dehydration rates.

A correlation study on optimum conditions of microbial precipitation and prerequisites for self-healing concrete

Microbial induced CaCO3 precipitation (MICP) based upon enzymatic urea hydrolysis has been verified as an effective way for crack treatment, especially for self-healing of concrete cracks. This paper aimed at correlating optimum conditions of MICP with prerequisites for self-healing concrete. Orthogonal experiments on a combination of factors contributing to the MICP process were firstly performed. Initial cell density and Ca2+ concentration were highly significant factor and significant factor respectively. High initial cell density (1×108 cells·mL-1) together with relatively low Ca2+ concentration (50 mM) favored microbial precipitation. The second part of this study was associated with dissolution tests to simulate the dissolving behavior of urea and calcium, since the dissolving of healing agents in cracks is a prerequisite of self-healing. By an addition of urea and Ca(NO3)2 with constant mass ratio of 2:3 in concrete, the highest values of the estimated urea concentration (345 mM) and Ca2+ concentration (44 mM) dissolved in cracks were close to the optimal values found by orthogonal studies. Although the addition of urea and Ca(NO3)2 would not have a negative impact on the mechanical properties of concrete, direct mixing is not recommended due to the low utilization efficiency of incorporating healing agents for self-healing.

Aggregation-driven fluorescence quenching of imidazole-functionalized perylene diimide for urea sensing.

Stimuli-responsive self-assembly of functional amphiphilic molecules by specific chemical stimulants is a promising strategy for sensor application. Herein, we demonstrate a fast optical detection of urea in human urine by exploiting bolaform perylene diimide functionalized with imidazoles (PDI-Hm), whose aggregation is induced by urea hydrolysis. The hydroxides produced from the enzymatic urea hydrolysis deprotonate the imidazoles to reduce electrostatic repulsion between PDI-Hm molecules in a HCl-methanol mixture, thereby leading to aggregation and consequent fluorescence quenching. The molecular interaction of PDI-Hm was further scrutinized to understand the aggregation behavior driven by the screening of electrical repulsion. As an optical sensing probe, PDI-Hm displays a prompt response (<1 min) to hydroxide and detection limit of 0.4 mM for urea. PDI-Hm incorporating urease offers considerable selectivity toward urea among various components in human urine. The urea sensing accuracy of this PDI-Hm fluorescence chemosensor is comparable to that of a clinical method, showing 93.4% consistency. Furthermore, the PDI-Hm was fabricated into a gel film allowed for the fast screening of excessive urea in urine.

Layer‐by‐layer self‐assembly of multifunctional enzymatic UF membranes

ABSTRACTIn order to eliminate membrane fouling and to ensure enzymatic hydrolysis of urea, multifunctional biocatalytic membranes were prepared by using urease (URE) and trypsin (TRY) enzymes on the sulfonated polysulfone (SPSf) ultrafiltration membrane via layer‐by‐layer (LbL) self‐assembly method. The membrane architecture consisted of multilayer assembly with TRY and URE enzymes as the outer layer and inner sandwiched layer, respectively. Polyethyleneimine (PEI) and alginate (ALG) were used as cationic and anionic polyelectrolytes. Sulfonation and PEI deposition were successfully accomplished as confirmed by attenuated total reflectance Fourier transform infrared spectroscopy, differential scanning calorimetry (DSC) and scanning electron microscopy analysis, contact angle measurements, staining with toluidine blue and Congo red dyes and dead‐end filtration experiments. A characteristic value of SPSf membrane with a high water permeability (1000 L/m2.h.bar) and 95% bovine serum albumin (BSA) rejection was observed. In static conditions, URE activities of SPSf2‐PEI‐URE membrane were not affected by BSA fouling, while TRY immobilizations with increased concentrations (SPSf2‐PEI‐URE‐PEI‐ALG‐TRY) significantly lowered the activity of URE. In dynamic conditions, each deposited layer exhibited individual resistance to flow that can be considered as irreversible fouling and caused 90% of flux decline for the SPSf2‐PEI‐URE‐PEI‐ALG‐TRY membrane assembly. The recovery of the initial flux for the multilayered membrane at the end of six fouling and washing cycles was observed 85%. Moreover, at the end of 5 cycles, 78% of the URE initial activity of the multilayered membrane was preserved. © 2019 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2020, 137, 48750.

Urea Biosensor Based on Electrochromic Properties of Prussian Blue

AbstractPrussian blue (PB) is an electrochromic material, which can be used as a signal transducer in the formation of optical urea biosensors. The previous researches in electrochromic properties of PB demonstrated the optical PB response to ammonium ions, which occurs when ammonium ions are interacting with PB layer at a constant 0.2 V vs Ag|AgCl|KClsat potential. In this work PB optical dependence on ammonium ions concentration was applied in the formation of electrochromic urea biosensor. Biosensor was formed by modifying the optically transparent indium tin oxide (ITO) coated glass electrode (glass/ITO) with Prussian blue layer and immobilizing urease (glass/ITO/PB‐urease). Calibration curve showed the linear dependency (R2=0.995) between the change of maximal absorbance (ΔA) and urea concentration in concentration range varying from 3 mM to 30 mM. The highest sensitivity (4 ΔA M−1) of glass/ITO/PB‐urease biosensor is in the concentration range from 7 mM to 30 mM. It was determined that working principle of the glass/ITO/PB‐urease biosensor is not related to pH changes occurring during enzymatic hydrolysis of urea.

Soyuretox, an Intrinsically Disordered Polypeptide Derived from Soybean (Glycine Max) Ubiquitous Urease with Potential Use as a Biopesticide.

Ureases from different biological sources display non-ureolytic properties that contribute to plant defense, in addition to their classical enzymatic urea hydrolysis. Antifungal and entomotoxic effects were demonstrated for Jaburetox, an intrinsically disordered polypeptide derived from jack bean (Canavalia ensiformis) urease. Here we describe the properties of Soyuretox, a polypeptide derived from soybean (Glycine max) ubiquitous urease. Soyuretox was fungitoxic to Candida albicans, leading to the production of reactive oxygen species. Soyuretox further induced aggregation of Rhodnius prolixus hemocytes, indicating an interference on the insect immune response. No relevant toxicity of Soyuretox to zebrafish larvae was observed. These data suggest the presence of antifungal and entomotoxic portions of the amino acid sequences encompassing both Soyuretox and Jaburetox, despite their small sequence identity. Nuclear Magnetic Resonance (NMR) and circular dichroism (CD) spectroscopic data revealed that Soyuretox, in analogy with Jaburetox, possesses an intrinsic and largely disordered nature. Some folding is observed upon interaction of Soyuretox with sodium dodecyl sulfate (SDS) micelles, taken here as models for membranes. This observation suggests the possibility for this protein to modify its secondary structure upon interaction with the cells of the affected organisms, leading to alterations of membrane integrity. Altogether, Soyuretox can be considered a promising biopesticide for use in plant protection.

Urease immobilization on magnetic micro/nano-cellulose dialdehydes: Urease inhibitory of Biginelli product in Hantzsch reaction by urea.

Micro/nano celluloses (MC)/NC) were magnetized by nanoγ-Fe2O3 into the nanoγ-Fe2O3@MC (NMMC) and nanoγ-Fe2O3@NC (NMMC) which oxidized to NMMCD and NMNCD dialdehydes for Schiff-base immobilization of urease as NMMCD/urease and NMMCD/urease. The relative enzyme-activity of the immobilized ureases were comparable with the free-urease, although 75%-80% of the enzyme activity preserved for NMMCD/urease and NMNCD/urease after six cycles. The compared catalytic activities of the NMMCD/urease and NMMCD/urease in Biginelli/Hantzsch reactions in water at 60 °C surprised us by 100% selectivity for the Biginelli product 3,4-dihydropyrimidin-2(1H)-one (DHPM1). With the superiority of NMNCD/urease, this high selectivity using immobilized ureases is owing to the admirable urease inhibitory of the formed Biginelli product DHPM1 by urea condensation instead of urea hydrolysis. The robust enzyme inhibitory of the DHPM1 for free urease was also confirmed by phenol red test to show the deactivation of enzyme for enzymatic hydrolysis of urea and ammonia production in water.

In vitro synthesis of bio-brick using locally isolated marine ureolytic bacteria, a comparison with natural calcareous rock

Microbially Induced Calcite Precipitation (MICP) using ureolytic bacteria has emerged as a promising technology with several applications in geotechnical engineering. In the current study, bacterial isolates from marine environment (coastal sandstones) have been investigated for their calcification potential. The isolated ureolytic bacterial strains NIOT-1 exhibited high urease activity and identified as Sporsarcina pausterii, were selected for further studies. The laboratory tests revealed that Sporsarcina pausterii NIOT-1 precipitated calcite crystals between the sand particles through the enzymatic hydrolysis of urea, which increased the ammonium concentration to the maximum of 366.54 mM and pH of 8.32. The petrological analysis of the samples indicated that the dominant carbonate phase was calcite. This precipitation of calcite crystals between the sand particles resulted in the physical stabilization of sand leading to the unconfined compressive strength to 3950 kpa. The strength of the laboratory made calcareous sandstone was comparatively higher when compared with the naturally formed calcareous sand rocks (3100 kpa (Uvari) and 1000 kpa (Pudumadam)) along the coasts, but the naturally formed ferrugineous sandstone belongs to Vepalodai was higher compared to all (30500 kpa). This has been envisaged by adopting MICP techniques in preventing the beach sand movement to protect eroding shoreline and to protect coastal populace at critical pockets of the coast as a nonconventional coastal protection measure.

Rapid and quantitative determination of urea in milk by reaction headspace gas chromatography

In this paper, an efficient and robust technique based on reaction headspace gas chromatographic analysis for the quantification of urea in milk is presented, which is realized by measuring formed carbon dioxide from the enzymatic hydrolysis of urea under the catalysis of enzyme urease. It was observed that the headspace equilibration was finished in 40 min at 35 °C. The new technique was proved to be accurate (relative errors ≤7.17%) and precise (relative standard deviations ≤2.93%). In addition, compared with the reference spectrophotometric technique, the new technique has a higher sample throughput. The proposed approach is simple, fast and has the potential to be a practical tool for the batch analysis of urea in milk production and processing.

Environmental safety and biosafety in construction biotechnology.

The topics of Construction Biotechnology are the development of construction biomaterials and construction biotechnologies for soil biocementation, biogrouting, biodesaturation, bioaggregation and biocoating. There are known different biochemical types of these biotechnologies. The most popular construction biotechnology is based on precipitation of calcium carbonate initiated by enzymatic hydrolysis of urea which follows with release of ammonia and ammonium to environment. This review focuses on the hazards and remedies for construction biotechnologies and on the novel environmentally friendly biotechnologies based on precipitation of hydroxyapatite, decay of calcium bicarbonate, and aerobic oxidation of calcium salts of organic acids. The use of enzymes or not living bacteria are the best options to ensure biosafety of construction biotechnologies. Only environmentally safe construction biotechnologies should be used for such environmental and geotechnical engineering works as control of the seepage in dams, channels, landfills or tunnels, sealing of the channels and the ponds, prevention of soil erosion and soil dust emission, mitigation of soil liquefaction, and immobilization of soil pollutants.

A mechanized urease activity assay

Two fully mechanized flow analysis systems for urease activity assays have been developed, characterized and compared. Both of them are based on almost the same compact system of solenoid micropumps and microvalves controlled and actuated by highly effective, low-power and economic Arduino microcontroller. For photometric detection of ammonia formed in the course of enzymatic hydrolysis of urea, the Berthelot method and the Nessler reaction have been examined. For both these detection schemes very simple dedicated optoelectronic flow-through detectors made of paired light emitting diodes have been developed. In both systems single enzyme assay lasting a few minutes allows determination of urease in activity range 0.02–5.3 U mL−1 with detection limit 0.02 U mL−1 and in 1.3–5.3 U mL−1 range with 0.75 U mL−1 detection limit for Nessler reaction and Berthelot method based systems, respectively. When compared with mechanized Berthelot method, the bioanalytical system based on Nessler reaction offers higher sensitivity, lower detection/determination limits, better selectivity and lower cost of the assay. It has been demonstrated that the developed bioanalytical flow systems could be useful for urease determination in complex biological matrix like plant extracts and media for microbial cultures as well as for inhibitive determination of heavy metals at sub-ppm levels.

Preliminary tests on a microfluidic device to study pore clogging during biocementation

Soil improvement using ureolytic bacteria or other biological agents is a promising technique currently under investigation. It is based on the precipitation of calcium carbonate (biocement) due to the enzymatic hydrolysis of urea. The biocement produced clogs the soil pores, consequently bonding the soil grains and increasing overall strength and stiffness while reducing permeability. This study focused mainly on pore clogging effects. The effect of the enzyme and feeding solution concentrations was studied in small test tubes to find the maximum amount of precipitate found when changing the concentrations of both. Based on it, selected concentrations of enzyme and feeding solution were tested in a microfluidic device conceived to mimic a two-dimensional uniform porous size medium. Qualitatively, the amount of precipitate was proportional to that of the concentrations used. The location of the precipitate was clearly related with the direction of fluid flow during inoculation. These preliminary results highlight the fact that the use of alternative testing devices such as the one developed is a potential tool for the study of clogging phenomena occurring during this treatment.

A liquid crystal based method for detection of urease activity and heavy metal ions by using stimulus-responsive surfactant-encapsulated phosphotungstate clusters.

A liquid crystal (LC) based method is described for the sensitive determination of the activity of urease and of heavy metal ions which acts as inhibitors. Stimulus-responsive surfactant-encapsulated phosphotungstate clusters (SECs) were fabricated and deposited onto octadecyltrichlorosilane-coated glass. A copper TEM grid filled with LCs was placed on the substrate to construct the LC optical cell. Upon addition of water to the LC interface, the optical appearance of LCs on the glass undergoes a bright-to-dark shift due to an orientational transition of the LCs from a planar to a homeotropic state. However, the LCs display a bright appearance if they are pretreated with an aqueous solution containing urea and urease. This is caused by the disassemby of the SECs from the glass surface due to an increase of the pH value that is induced by the enzymatic hydrolysis of urea by urease. The method is highly sensitive and can detect urease activities as low as 0.03mU/mL. It can also be applied to the determination of heavy metal ions which exert an inhibitory effect on the activity of urease. For example, Cu(II) can be quantified via urease inhibition in 1nM concentration. Graphical abstract Schematic presentation of a liquid crystal-based sensor for detection of urease and heavy metal ions by using stimulus-responsive surfactant-encapsulated phosphotungstate clusters.

Enzyme Multilayers on Graphene-Based FETs for Biosensing Applications.

Electrochemical sensors represent a powerful tool for real-time measurement of a variety of analytes of much significance to different areas, ranging from clinical diagnostics to food technology. Point-of-care devices which can be used at patient bedside or for online monitoring of critical parameters are of great importance in clinical daily routine. In this work, portable, low-cost electrochemical sensors for a fast and reliable detection of the clinically relevant analyte urea have been developed. The intrinsic pH sensitivity of reduced graphene oxide (rGO)-based field-effect transistors (FETs) was exploited to monitor the enzymatic hydrolysis of urea. The functionalization of the sensor platform using the layer-by-layer technique is especially advantageous for the immobilization of the biorecognition element provided that this approach preserves the enzyme integrity as well as the rGO surface. The great selectivity of the enzyme (urease) combined with the high sensitivity of rGO-based FETs result in the construction of urea biosensors with a limit of detection (LOD) of 1μM and a linear range up to 1mM. Quantification of Cu2+ with a LOD down to 10nM was performed by taking advantage of the specific inhibition of urease in the presence of heavy metals. These versatile biosensors offer great possibilities for further development of highly sensitive enzyme-based FETs for real-time, label-free detection of a wide variety of clinically relevant analytes.

The urea biosensor based on luminescence of Eu(III) ternary complex of DO3A ligand

The Eu(III) ternary complex with 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (H3DO3A) and picolinate “antenna” ligand ([Eu(DO3A)(L)]–) reacts with carbonate anions to form the more stable non-fluorescent [Eu(DO3A)(CO3)]2− complex. This substitution reaction was employed for indirect determination of carbonate formed in course of urea hydrolysis catalyzed by the urease enzyme. This new analytical procedure was proposed for the determination of both analytes (urea, urease) and this sensitive luminescence-based biosensor can be used in both kinetic time-dependent and equilibrium end-point modes. This method, with analytical figures of merit (limit of detection 0.05 mM, dynamic range 0.05–5.00 mM), does not show any systematic error and therefore can be utilized for urea analysis in biological samples (e.g., urine). It was also demonstrated that some metal ions (e.g., Zn(II), Pb(II), Cd(II), Ag(I), etc.) inhibit the enzymatic hydrolysis of urea and this effect can be utilized for their determination; therefore, it is recommended to add EDTA as masking agent prior to enzymatic urea analysis.

Rheology and gel point of the enzymatic hydrolysis of urea in the presence of urease

This study reports on the rheology of the gelation kinetics of raw chitosan (CTS) solutions (2% w/v) produced by enzymatic hydrolysis of urea at different urea concentrations (40, 50, 60, 80, and 100 mM) in the presence of urease at 1 U/mL. Viscoelastic parameters and pH values were evaluated during gelation process and the rheological properties of CTS hydrogels produced were monitored after 24 h at 37°C to simulate human body temperatures. pH measurements suggest that above some critical urea concentration (50 mM) the time required (tgel) to reach the critical pH gelation shows no dependence on urea concentration (tgel was ca. 70 minutes). Above 50 mM of urea concentration, CTS hydrogels exhibit an elastic modulus G′ higher than the viscous modulus G″ with no frequency dependence characteristic of a gel behavior. Gelation kinetics analyzed by rheology suggest that the G′ (i.e., structure) development depends on urea concentration during solution neutralization.

Fluorescence bioanalytical method for urea determination based on water soluble ZnS quantum dots

A new bioanalytical method for urea determination by using the immobilized urease on the highly luminescent ZnS quantum dots (QDs), which functioned as the pH fluorescent label has been developed. Bioconjugation of ZnS QDs to urease via amide bond was performed to obtain the ZnS QDs-urease bioconjugate. Upon enzymatic hydrolysis of urea by the immobilized urease, a pH change to a more alkaline condition has led to the deprotonation of ZnS QDs, and an increase in the fluorescence intensity can be observed. The fluorescence intensity of the urease conjugated ZnS QDs changed linearly with the urea concentrations between 4×10−9M and 4×10−3M (R2=0.992) at pH 6 with a calibration sensitivity of 179.46 intensity/decade. No noticeable influence by the Ca2+, Mg2+, K+ and Na+ ions on the response of the fluorescent pH bioprobe. Urea determination in soil sample by using the urea bioprobe was in good agreement with the standard DMAB (p-dimethylaminobenzaldehyde) UV–vis spectrophotometric method. By employing the strategy of ZnS QDs-urease bioconjugation, the stable covalent link between the two substances has appeared to widen the dynamic range and lower the detection limit for urea compared to free enzyme and QDs in solution for bioassay of urea concentration.

A novel approach for stabilizing fresh urine by calcium hydroxide addition

In this study, we investigated the prevention of enzymatic urea hydrolysis in fresh urine by increasing the pH with calcium hydroxide (Ca(OH)2) powder. The amount of Ca(OH)2 dissolving in fresh urine depends significantly on the composition of the urine. The different urine compositions used in our simulations showed that between 4.3 and 5.8 g Ca(OH)2 dissolved in 1 L of urine at 25 °C. At this temperature, the pH at saturation is 12.5 and is far above the pH of 11, which we identified as the upper limit for enzymatic urea hydrolysis. However, temperature has a strong effect on the saturation pH, with higher values being achieved at lower temperatures. Based on our results, we recommend a dosage of 10 g Ca(OH)2 L−1 of fresh urine to ensure solid Ca(OH)2 always remains in the urine reactor which ensures sufficiently high pH values. Besides providing sufficient Ca(OH)2, the temperature has to be kept in a certain range to prevent chemical urea hydrolysis. At temperatures below 14 °C, the saturation pH is higher than 13, which favors chemical urea hydrolysis. We chose a precautionary upper temperature of 40 °C because the rate of chemical urea hydrolysis increases at higher temperatures but this should be confirmed with kinetic studies. By considering the boundaries for pH and temperature developed in this study, urine can be stabilized effectively with Ca(OH)2 thereby simplifying later treatment processes or making direct use easier.

Enzymatic control of chitosan gelation for cell delivery vehicle

Event Abstract Back to Event Enzymatic control of chitosan gelation for cell delivery vehicle Carla Tim1, Cintia C. Martignago2, Lia M. Neves3, Nivaldo A. Parizotto2 and Ana Claudia Rennó1 1 Federal University of São Paulo, Bioscience, Brazil 2 Federal University of São Carlos, Physiotherapy, Brazil 3 Federal University of São Carlos, Physiologic, Brazil Osteoarthritis (OA) is a common chronic disease among older people, which leads to a loss of articular cartilage. Due to the very limited cartilage regenerative capacity and consequently the limited efficacy of the standard treatments, the investigation of strategic innovative approaches to prevent the development of the clinical condition of OA is of great interest. Thus, development in strategies for cartilage repair have increasingly focused on cell-assisted repair, proposing to use autologous chondrocytes or other cell types to regenerate articular cartilage in situ. One promising cell delivery vehicle is chitosan hydrogels, mainly due to its several advantages including easy handling, high cell seeding efficiency and good formability to irregular defects in a minimally invasive manner. Chitosan hydrogels can be formed homogeneously by neutralization of chitosan solutions using ammonia produced in situ from enzymatic hydrolysis of urea. Therefore, this study aimed to investigate the possibility to control chitosan gelation using enzymatic hydrolysis of urea for the delivery of chodrocytes. Chitosan (Sigma–Aldrich) was dissolved at a concentration of 2.5 w/v% in 0.100M HCl. Urease (Sigma–Aldrich) was dissolved in phosphate buffered saline and Urea (Sigma–Aldrich) was dissolved in MilliQ water. To prepare 1.5 w/v% chitosan hydrogels, 1mL of chitosan was mixed with 0.67mL urease. Subsequently, 10 ul of the urea solution was added. The vials were inverted every 30 sec and the gelation time was recorded as the one at which the solution did not flow in 5 different conditions, i.e., urease concentration of 30 U/mL and urea concentrations of 7.5, 8.5, 9.5,10.5 and11.5 M. The speed at which the pH changed under different gel compositions was investigated and recorded with the use of a pHmeter electrode placed in a freshly prepared mixture of starting solutions immediately after addition of the urea. The results revealed that the gelation time of the chitosan hydrogels can be precisely controlled by varying the urea concentrations as shown in table 1. The pH during gelation of the chitosan hydrogels is displayed, which confirmed that the pH increased from initial values of about 5.7–6.8 to final values of 6.9–7.3 within 5–28 min after addition of urea. Final pH values were controlled by the concentration of urea as main source for alkaline ammonia groups, but did not depend on the concentration of urease, which allows for independent control over final pH and gelation kinetics by varying the concentrations of urea and urease, respectively. Thus, it is possible to set the desired gelation time and final pH of the gel for various applications. The results demonstrated that physically crosslinked chitosan hydrogels for biomedical purpose can be easily fabricated via enzymatic control, and such control over the gelation of chitosan hydrogels could be an option for the delivery of chondrocytes via an injectable system. We would like to acknowledge the contributions of Brazilian funding agency Fapesp (FAPESP project#2014/13702-6) for the financial support of this research.