Alkaline Protease Production Research Articles

Integration of Ultrasound into the Development of Plant-Based Protein Hydrolysate and Its Bio-Stimulatory Effect for Growth of Wheat Grain Seedlings In Vivo.

This study was dedicated to increasing the efficiency of producing plant-based protein hydrolysate using traditional and non-traditional treatments. Low- and high frequency ultrasound (US) at different intensities were applied to corn steep liquor (CSL) at 50 °C for 30 min, and enzymatic hydrolysis was performed using industrially produced alkaline protease. The efficiency of US and enzymatic treatments was characterized by protein solubility (soluble protein (SP) content, hydrolyzed protein (HP) concentration, and free amino acid (FAA) profile) and kinetic parameters: Michaelis–Menten constant (KM) and apparent breakdown rate constant (kA). A significant effect of 37 kHz US pre-treatment for CSL enzymatic hydrolysis was found and resulted in the highest HP concentration (17.5 g/L) using the lowest enzyme concentration (2.1 g/L) and the shortest hydrolysis time (60 min). By using US pre-treatment, on average, a 2.2 times higher FAA content could be achieved compared to traditional hydrolysis. Additionally, results for the kinetic parameters kM and kA confirmed the potential of applying US treatment before hydrolysis. The effect of CSL protein hydrolysate on plant growth was tested in vivo on wheat grain seed germination and resulted in the significant increase in germination parameters compared to the control treatment. These findings indicate that by-products of starch industry could be a promising source for the production of low-cost sustainable biostimulants.

Production and characterisation of thermostable alkaline protease from Bacillus subtilis isolated from LA hot spring, Terengganu

This research aims to produce thermostable alkaline protease from Bacillus subtilis isolated from La Hot Spring, Terengganu, Malaysia. The study was also conducted to determine the optimum conditions for protease production and stability by considering several parameters including pH, temperature and salt concentration. All seven bacteria were screened on skim milk agar overnight at 37 °C. Three strains with the highest proteolytic activity were identified in protease specific medium. The thermostable alkaline protease had an optimum temperature of 60 °C which achieved 85.73, 82.90 and 83.05 U/mL of protease activity for the three strains respectively. Furthermore, the strains exhibited significant activity of more than 90% from their original activity. Meanwhile, the optimum pH for protease production was pH 9 with the protease activity of 76.76, 79.71 and 88.39 U/mL for TB4, TB6 and TB9 strains, respectively. Proteases were found stable at pH 9 where the loss did not exceed 30% of its original activity. Collectively, all of the data emphasised that proteases from B. subtilis were alkaline thermostable proteases in accordance with a recent report. The finding highlights the viability of the proteases for biotechnological and industrial applications.

Bio-production of alkaline protease by Trichoderma longibrachiatum and Penicillium rubidurum using different agro-industrial products

Alkaline protease being active in neutral to alkaline pH has huge demands in food, detergent, leather and pharmaceutical industries. Its production from agro-industrial wastes not only lowers the production costs but also reduces the environmental problems. Hence, the present study aimed to search for new potential microbes, which can produce alkaline protease enzyme, to meet the industrial demands. In this study, 13 fungal spp. were isolated on potato dextrose agar medium (PDA) from mangrove soil through serial dilution, and then were streaked on the skim milk agar medium for qualitative screening of protease production. Out of 13 fungal spp.; only 7 spp. were able to produce proteolytic zones through the proteolytic assay. The Relative enzymatic index (REI) value (Zone diameter/Colony diameter) of all the fungal isolates that produced proteolytic zones on skim milk agar medium was evaluated. Only 2 fungal isolates which showed maximum REI value were selected, and then identified morphologically and molecularly as Trichoderma longibrachiatum (Accession no. MF144551) and by Penicillium rubidurum (Accession no. MF144561). Submerged fermentation was carried out using different agro industrial substrates to quantify for protease production, where the supernatants obtained were used for alkaline protease estimation. Among the different tested substrates, soybean powder and wheat bran were the most suitable substrates for maximum protease production by T. longibrachiatum (233.78±7.12 U/ mg) and P. rubidurum (228.61±11.13 U/ mg), respectively. The partial purified enzyme from these fungi showed maximum proteolytic potentials at pH 8.0 (P. rubidurum) and pH 9.0 (T. longibrachiatum), with optima temperature of 40 °C. Among the tested heavy metals, only Mn2+ expressed marginal enhancement of the protease enzyme activity.

Isolation and Identification of Alkaline Protease Producing Aspergills niger from Iraqi Soils

Twenty purified isolates were obtained by using different soil sources, only twelve isolates belonging to Aspergillus genera depending on cultural and morphological characterization. The isolates were used as alkaline protease producer. The highest proteolytic, enzymatic activity (95.83U/ml) was obtained from Aspergillus sp. ZE isolate. This isolate was identified by 5.8 rRNA gene sequencing as Aspergills niger (accuracy of 99%), which was matched with the sequence of Aspergills niger strain GM775228 recorded in Gene bank under the ID: GM 775228.1.

Isolation, production and optimization of endogenous alkaline protease from in-situ sludge and its evaluation as sludge hydrolysis enhancer.

Bioconversion (e.g. anaerobic fermentation and compost) is the common recycling method of waste activated sludge (WAS) and its hydrolysis, as the rate-limiting step of fermentation, could be accelerated by protease. However, the commercial protease was unstable in a sludge environment, which increased the cost. An endogenous alkaline protease stable in sludge environment was screened in this study and its suitability for treating the sludge was analyzed. The optimal production medium was determined by Response Surface Methodology as starch 20 g/L, KH2PO4 4 g/L, MgSO4·7H2O 1 g/L, sodium carboxy-methyl-cellulose 4 g/L, casein 4 g/L and initial pH 11.3, which elevated the yield of protease by up to 15 times (713.46 U/mL) compared with the basal medium. The obtained protease was active and stable at 35 °C-50 °C and pH 7.0-11.0. Furthermore, it was highly tolerant to sludge environment and maintained high efficiency of sludge hydrolysis for a long time. Thus, the obtained protease significantly hydrolyzed WAS and improved its bioavailability. Overall, this work provided a new insight for enzymatic treatment of WAS by isolating the endogenous and stable protease in a sludge environment, which would promote the resource utilization of WAS by further bioconversion.

Enhancing the Expression of the Substrate by the Extracellular Secreted Enzymes and Improving the Alkaline Protease Production in Bacillus licheniformis

Enhancing the Expression of the Substrate by the Extracellular Secreted Enzymes and Improving the Alkaline Protease Production in Bacillus licheniformis

Production of Alkaline Proteases using Aspergillus sp. Isolated from Injera: RSM-GA Based Process Optimization and Enzyme Kinetics Aspect.

Alkaline proteases are well known to be significant industrial enzymes. This study focused on isolating the fungus producing proteases from, a typical Ethiopian food, Injera. Further, the process optimization for protease production using response surface methodology (RSM) and the characterization of the acquired protease were investigated. The 18S rDNA gene sequence homology of the fungus isolate revealed that it was Aspergillus sp. Further, it was deposited in NCBI GenBank with accession number MK4262821. Using the isolate, owing to maximize the protease production, the independent process parameters, temperature, pH, and sucrose concentration were optimized using RSM followed by a genetic algorithm (GA). Based on the statistical approach by RSM-GA optimization, maximum enzyme activity (166.4221 U/ml) was found at 30.5°C, pH 8.24, and 0.316% sucrose concentration. Also, the crude cocktail of enzymes acquired from optimal condition was partially purified using ammonium which showed that the increased activity by 1.96-fold. Considerably, the partially purified enzyme exhibited stable performance during the temperature range 30-60°C, pH range 7-10, and NaCl concentration of 0.5-2mM. Also, the antioxidant activity, degree hydrolysis for the protein, Michaelis-Menten (M-M) kinetic parameters, and activation energy were determined for the obtained enzyme cocktail. It showed that the M-M kinetic parameters, Km (5.54mg/ml), and Vmax (24.44mg/ml min) values were observed. Using Arrhenius law, the value of activation energy for the enzyme cocktail was determined as 32.42kJ/mol.

Effect of alkaline protease produced from fish waste as substrate by Bacillus clausii on destaining of blood stained fabric

Alkaline protease or peptidases are the largest groups of enzymes in the biological industry with a variety of application in manufacturing units used in the process of substrate stabilization, dehairing, diagnosis, extraction, food production, destaining, etc., where the pH of the environmental conditions remain above neutral pH. Because of these wider applications of alkaline proteases in industries, their demand is increasing to compete with their chemical counterpart. An alkaline tolerant bacterial strain Bacillus clausii wasisolated from fish waste and used for mass production of alkaline protease using fish waste homogenate as media. Preliminary study on optimization of conditions for the mass production carried out. The optimum temperature for production ranges between 25°C and 35°C and pH determined as 9. The mass production of extracellular alkaline protease carried out using mobilized and immobilized cells of B. clausii at optimized condition using production media, the mixture of production media and fish waste homogenate and in nutrient broth. The recorded results showed that the maximum enzyme production obtained with immobilized cells in nutrient broth media and followed by fish waste homogenate media of 8900 U/ml and 8600 U/ml. Purification protease enzyme yielded 0.35 g/ml from the production media . Bloodstained cloth treated with immobilized enzyme removed the stain completely compared to treatment with non-immobilized enzyme and commercially used detergent. So, the current study suggests the usage of microbial alkaline protease in household detergents to replace the usage of synthetic detergents and save the environment from chemical pollutants.

Genotypic and Phenotypic Characterisation of Isolated Marine Bacteria and its Potential to Produce Alkaline Protease

In several industries such as food and detergent, enzymes are commonly used out of which microorganisms' proteolytic enzymes are dominant. Microorganisms from marine sources have advantageous commercial characteristics when it comes to protease production. Protease producing bacterial strains were isolated from four different stations in the South China Sea, located at Kuantan, Pahang-Malaysia. The temperature was ranging from 29 to 36.2°C; salinity varied from 36,18 to 37,9 ppt, turbidity from 10.21 to 16.4 NTU. Dissolved Oxygen had an average of 4.44 mg/mL, and pH was around 8. All the bacterial strains were screened for protease activity using Skim Milk agar plates. Out of 18 isolates, ten strains formed a clear zone on skim milk agar plates. Molecular identification by 16S rRNA results revealed that Bacillus spp was the most dominant bacteria, four isolates belong to Bacillus cereus, another 04 identified as Bacillus licheniformis in addition of one isolate was Bacillus safensis. They were followed by one isolate Staphylococcus warneri. Furthermore, a phenotypic characterization is conducted using The Biolog GENIII MicroPlate with 94 phenotypic tests. These tests consist of 71 carbon source utilization assays and 23 chemical sensitivity assays. The proteolytic activity was confirmed through a quantitative protease assay. Where the strain B. cereus-MD152 was the highest yield, followed by B. licheniformis-ABN13. On the contrary, the lowest yield was secreted by B. cereus-IIUM6 with no more than 16.5 U/ml.

Production of Alkaline Protease Using Industrial Waste Effluent as Low-cost Fermentation Substrate

Alkaline proteases are the most important groups of commercial enzymes, which have been broadly used in industrial processes. In this study, Bacillus sp. PTCC 1538 was selected as a biological agent to produce alkaline protease. Enzyme production under submerge fermentation using industrial waste effluent was investigated. Since the costs of the raw material plays an important role in the cost of enzyme production, corn steep liquor (CSL) was selected as a low-cost substrate to reduce the cost of enzyme production. Various carbon sources were used as the auxiliary substrates to enhance enzyme production. Results showed that maximum enzyme activity was obtained when wheat bran was used as an auxiliary substrate. Optimal media composition and growth conditions for alkaline protease production were defined. The optimum conditions were found to be pH 8, incubation temperature of 37 °C, CSL inoculum size of 5 v/v %, yeast extract and wheat bran concentrations of 2 and 6 g/l, respectively. CaCl2 was used as an activator to enhance proteolytic activity of the enzyme. Under optimum condition, enzyme activity of 100.7 U/ml was obtained at CaCl2 concentration of 1.5 g/l.

Production, Purification, and Characterization of an Industrially Important Enzyme Alkaline Protease Produced from Locally Isolated <i>Bacillus</i> Bacteria

The worldwide demand for industrially essential enzymes is growing due to their eco-friendly and sustainable applications in broad industrial sectors. The main objective of this investigation is intended to isolate potent alkalophilic native bacteria from local habitats for the cost-effective production of an enzyme alkaline protease. For screening of alkalophilic bacteria, bacterial samples were taken from various natural alkaline habitats, and bacterial cultures were screened on agar plate having skimmed milk as protein substrate by using the protein hydrolysis method. The bacteria showing maximum proteolytic potential were identified by microscopic, biochemical, and 16S rDNA analysis. Additionally, the culture components and other medium parameters have been optimized for higher enzyme production. High yield alkaline protease was obtained with conditions of 1% inoculum, pH 9.0, and at 37°C temperature, with 1% sugarcane molasses as the best suitable carbon substrate. Partial pur¬ification of the enzyme was performed and characterized for their optimum activity on various parameters (temperatures and pH range). The approx. molecular weight of the enzyme was estimated to be ~35 kDa. This study demonstrates the production of an industrially important enzyme (alkaline protease) from a newly isolated native Bacillus spp. Economical production of this enzyme can be commercially applied in vast industrial sectors like agriculture, textile, food, detergent, and leather industries.

Screening, selection and development of Bacillus subtilis apr-IBL04 for hyper production of macromolecule alkaline protease

Bacillus subtilis microbe is commonly found in soil and produces proteases on nitrogen and carbon-containing sources and increases the fertility rate by degrading nitrogenous organic materials. The present study was aimed to develop hyper producing mutant strain of B. subtilis for the production of proteases, to improve the process variables by the response surface methodology (RSM) under central composite design (CCD) and the production of protease by the particular mutant strain in a liquid state fermentation media. The mutation of the strain was carried out using ethidium bromide. Pure B. subtilis strain was collected and screened for hyper-production of protease. The production of protease by mutant B. subtilis strain was optimized by varying temperature, inoculum size, pH and incubation time under liquid state fermentation. The CCD model were found to be reliable with r2 of 0.999. The maximum enzyme activity of B. subtilis IBL-04 mutant with 3 mL/100 mL inoculum size, 72 h fermentation time, pH 8, and 45 °C temperature was developed with enzyme activity 631.09 U/mL, indicates 1–7-fold increase in enzyme activity than the parent strain having 82.32 U/mL activity. These characteristics render its potential use in industries for pharmaceutical and dairy formulation.

Alkaline protease production by halophilic Bacillus sp. strain SP II-4 and characterization with special reference to contact lens cleansing

This study aims to screening of protease producing bacterial strain from saltpan located in Kanyakumari. Proteolytic bacterial strains were isolated by using skim milk agar plates. The potent bacterial isolate (SP II-4) was typically identified as Bacillus sp. SP II-4 by studying phenotypic characteristics. The purified enzyme fraction showed 591.04 (U/mg) of specific activity. The protease enzyme liberated from this strain illustrated better stability and activity in pH 10.0, 40 °C and stable upto 7.5% salt. The protease enzyme demonstrated better content lens cleansing effectiveness along with common chemical disinfectant. These above properties of the enzyme clearly stating that may use in various industrial processes which are performed even at extreme conditions.

Production, purification, and characterization of alkaline protease from Aspergillus flavus and its compatibility with commercial detergents

Aspergillus flavus was used to produce alkaline protease. Solid state fermentation (SSF) strategy was adopted to explore the most favorable physical and nutritional conditions for enzyme production. Maximum production was achieved at pH 6.0 and a temperature of 30 °C after 84 h of growth period. For the optimization of the chemical parameters, different carbon and nitrogen sources were used including glucose, fructose, sucrose, ammonium sulphate, and urea. Maximum production was observed with 0.3% concentration of all the compounds. Ammonium sulphate salting out and gel chromatography was used to purify the enzyme. The enzyme was completely precipitated out at 80% saturation. The value of Vmax was 3.9 U/mL, while the value of Km was 1.9 mg/mL. The enzyme was tested for its compatibility with a few famous detergents available on the market. With the alkaline protease under study, the enzyme displayed a maximum retention of its activity i.e. 80.8% in the presence of commercial detergent Surf excel. The activity dropped down to 61.5% when the enzyme was allowed to work in the presence of another locally used detergent, i.e., Bonus. Protease production from A. flavus was carried out on rice bran and wheat bran and the wheat bran gave better results.

Transcriptome based functional identification and application of regulator AbrB on alkaline protease synthesis in Bacillus licheniformis 2709

Bacillus licheniformis 2709 is the major alkaline protease producer, which has great potential value of industrial application, but how the high-producer can be regulated rationally is still not completely understood. It's meaningful to understand the metabolic processes during alkaline protease production in industrial fermentation medium. Here, we collected the transcription database at various enzyme-producing stages (preliminary stage, stable phase and decline phase) to specifically research the synthesized and regulatory mechanism of alkaline protease in B. licheniformis. The RNA-sequencing analysis showed differential expression of numerous genes related to several processes, among which genes correlated with regulators were concerned, especially the major differential gene abrB on enzyme (AprE) synthesis was investigated. It was further verified that AbrB is a repressor of AprE by plasmid-mediated over-expression due to the severely descending enzyme activity (11,300 U/mL to 2695 U/mL), but interestingly it is indispensable for alkaline protease production because the enzyme activity of the null abrB mutant was just about 2279 U/mL. Thus, we investigated the aprE transcription by eliminating the theoretical binding site (TGGAA) of AbrB protein predicated by computational strategy, which significantly improved the enzyme activity by 1.21-fold and gene transcription level by 1.77-fold in the mid-log phase at a cultivation time of 18 h. Taken together, it is of great significance to improve the production strategy, control the metabolic process and oriented engineering by rational molecular modification of regulatory network based on the high throughput sequencing and computational prediction.

Characterization of a Protease Hyper-Productive Mutant of Bacillus pumilus by Comparative Genomic and Transcriptomic Analysis

Bacillus pumilus BA06 has great potential for the production of alkaline proteases. To improve the protease yield, classical mutagenesis to combine the physical and chemical mutagens was performed to obtain a protease hyper-productive mutant SCU11. The full genome sequences of BA06 and SCU11 strains were assembled through DNA sequencing using the PacBio sequencing platform. By comparative genomics analysis, 147 SNPs and 15 InDels were found between these two genomes, which lead to alternation of coding sequence in 15 genes. Noticeable, the gene (kinA) encoding sporulation kinase A is interrupted by introducing a stop codon in its coding region in BA06. Interestedly, this gene is reversely corrected in SCU11. Furthermore, comparative transcriptome analysis revealed that kinA and two positive regulatory genes (DegU and Spo0A) were upregulated in transcription in SCU11. In terms of the transcriptional data, upregulation of a phosphorylation cascade starting with KinA may enhance Spo0A phosphorylation, and thus activate expression of the gene aprE (encoding major extracellular protease) through repression of AbrB (a repressor of aprE) and activation of SinI, an antagonist of SinR (a repressor of aprE). In addition, the other genes involved in various metabolic pathways, especially of membrane transport and sporulation, were altered in transcription between these two strains. Conclusively, our transcriptome data suggested that upregulation degU and spo0A, as well as kinA, may at least partially contribute to the high production of alkaline protease in SCU11.

Optimized production of extracellular alkaline protease from Aspergillus tamarii with natural by-products in a batch stirred tank bioreactor

Proteolytic enzymes are one of the significant commercially manufactured enzymes. The manufacture of extracellular alkaline protease by Aspergillus tamarii MTCC5152 was explored using several agricultural by-products as substrates viz., cottonseed meal, wheat bran, skimmed milk and soya flour in submerged fermentation, were found to be efficient for enzyme production and commercially significant. Response surface methodology (RSM) is a statistics-based experimental design, sourced to explore the impact of physical parameters on the manufacture of protease from A. tamarii in a batch stirred tank bioreactor (STBR). The four substantial variables (pH, temperature, inoculum size, and agitation) were carefully chosen for optimization analyses and the statistical pattern was created using a central composite design and the quadratic model has been developed. The optimum conditions for protease production (1.51 U mL−1) where: pH 6.4, temperature 27 °C, inoculum size 2.6%, and agitation 327 rpm. The analysis revealed that the anticipated values were in accord with trial data with a correlation coefficient of 0.969.

Effects of different feeding frequencies on growth, feed utilisation, digestive enzyme activities and plasma biochemistry of gilthead sea bream (Sparus aurata) fed with different fishmeal and fish oil dietary levels

In the context of Mediterranean aquaculture many efforts have been made in terms of reducing marine-derived ingredients in aquafeed formulation. On the other hand, little attention has been paid to the manipulation of feeding frequency at the on-growing phase, where the high costs related to feeding procedures and the optimisation of feed efficiency and fish health are key aspects for the economic and environmental sustainability of the production cycle. The effects of different feeding frequencies (F) (1F) one meal day−1, (2F) two meals day−1, (3F) three meals day−1 on growth, digestive enzyme activity, feed digestibility and plasma biochemistry were studied in gilthead sea bream (Sparus aurata, L. 1758) fed with high (FM30/FO15, 30% fishmeal FM, 15% fish oil, FO) and low (FM10/FO3; 10% FM and 3% FO) FM and FO levels. Isonitrogenous and isolipidic extruded diets were fed to triplicate fish groups (initial weight: 88.3 ± 2.4 g) by a fixed ration over 109 days. No significant effects of feeding frequency on overall performance, feed efficiency and feed digestibility during the on-growing of gilthead sea bream fed high or low fishmeal and fish oil dietary level were observed. Pepsin activity showed an apparent decrease in fish receiving more than one meal a day which was not compensated by an increased production of alkaline proteases (either trypsin or chymotrypsin), particularly in fish fed on low FM. Although there were no effects on growth and feed utilisation at increasing feeding frequency, trypsin decreased significantly with an increasing number of meals only under low FMFO diet. Thus, it seemed that consecutive meals could have amplified the potential trypsin inhibitor effect of the vegetable meal-based diet adopted. Most of the plasma parameters related to nutritional and physiological conditions were not affected by feeding frequency, however an effect on electrolytes (Na+, Cl), cortisol and creatinine was observed. The higher level of plasma creatinine detected in fish fed a single daily meal with high FMFO level seems to be within physiological values in relation to the higher protein efficiency observed with this diet. However, it will require further attention to exclude a possible overload of kidney functionality. According to the results, gilthead sea bream seems able to maximize feed utilisation regardless of the number of meals, and this could be a useful indicator for planning feeding activity at farm level to optimise growth of fish and costs of feeding procedures.

Enhanced catalytic activity of Bacillus aryabhattai P1 protease by modulation with nanoactivator

In the developing area of modern nanobiotechnology, the research is being focused on enhancement of catalytic performance in terms of efficiency and stability of enzymes to fulfill the industrial demand. In the context of this interdisciplinary era, we isolated and identified alkaline protease producer Bacillus aryabhattai P1 by polyphasic approach and then followed one variable at a time approach to optimize protease production from P1. The modified components of fermentation medium (g/L) were wheat bran 10, soybean flour 10, yeast extract 5, NaCl 10, KH2PO4 1, K2HPO4 1 and MgSO4·7H2O 0.2 (pH 9). The optimum alkaline protease production from P1 was recorded 75 ± 3 U/mg at 35 °C and pH 9 after 96 h of fermentation period. Molecular weight of partially purified P1 alkaline protease was 26 KDa as revealed by SDS-PAGE. Calcium based nanoceramic material was prepared by wet chemical precipitation method and doped in native P1 protease for catalytic activity enhancement. Catalytic activity of modified P1 protease was attained by nanoactivator mediated modulation was more by 5.58 fold at pH 10 and 30 °C temperature. The nanoceramic material named as nanoactivator, with grain size of 40–60 nm was suitable to redesign the active site of P1 protease. Such types of modified proteases can be used in different nanobiotechnological applications.

Immobilization of Pseudomonas Aeruginosa in Different Matrices for the Production of Alkaline Protease

Numerous enzymes are used in various types of industries, and one such enzyme used in several of these industries is proteases. Aforementioned, industries such as dairy, detergent, leather, fermentation and several other industries are benefitted with protease enzyme. In the present study, the efficiency of protease production was studied by enriching and immobilizing several matrices by a gram negative bacteria known as Pseudomonas aeruginosa .The immobilization was carried out by four different matrices under two different concentrations of 3% and 4%.Yielded results revealed highest enzymatic activity of 240 (U/ml) in 3% calcium alginate. Still, second highest enzymatic activity of 230 (U/ml) was seen in 4% calcium alginate. On the contrary, free microbial cells showed an enzymatic activity of 100 (U/ml). The peak activities for other methods area as follows: 4% calcium alginate - 133 (U/ml), 3% agar-agar - 100 (U/ml), 4% agar-agar - 91 (U/ml), 3% Gelatin - 85 (U/ml), 4% Gelatin - 88 (U/ml) and Polyacrylamide – 104 (U/ml). The most optimum matrix for the cellular entrapment of Pseudomonas aeruginosa is seen in 3% calcium alginate for alkaline protease production.