Unoptimized Conditions Research Articles

Talaromyces verruculosus tannase production, characterization and application in fruit juices detannification

Abstract A potential tannase producing fungus S3-07 was isolated from the tannin-rich soil sample and screened as better tannase producer with 38 ± 3 mm diameter of hydrolysis zone formation. It was identified as T. verruculosus (accession number KX863699) on the basis of the internal transcribed spacer ribosomal DNA (ITS-rDNA) sequence analysis. Tannase was produced with 8.22 ± 0.35 Unit per gram dry substrate (U/gds) activity in the unoptimized condition through solid state fermentation (SSF) using Babul (Acacia nilotica) bark as a solid substrate. Central composite design (CCD) was used to maximize the production and achieved 3.91 fold increments (32.18 ± 1.31 U/gds). Ammonium sulfate precipitation with 70% saturation and DEAE fractionation obtained 13.289 specific activity, 47.489% yield and 6.896 purification fold. SDS-PAGE and zymographic analysis showed two bands of tannase with ∼75 kDa and 58 kDa pH optima of tannase was measured at 8.00, whereas pH stability was recorded between 4.0 and 8.0. Tannase had temperature optima at 60 °C, however, stability was observed between 30 and 60 °C. In presence of ethanol, SDS, Mn2+ ions and gallic acid increased tannase activity, whereas ethyl acetate, tween 20, Ba2+ ion and urea inhibited maximum activity. Most of tannin reduction (93.27%) was measured in case of orange juice after 15 min incubation and 89.36% reduction in pomegranate juice after 30 min, whereas aonla juice was measured with 75.49% tannin reduction after 45 min incubation.

Optimization of production and partial characterization of cellulase and protease enzymes from Aeromonas hydrophila ASM-S32

Microbial hydrolytic enzymes, especially cellulase and protease, are widely used in industrial processes due to their low cost, large productivity, chemical stability, environmental protection, plasticity and vast availability. But existing cellulase and protease enzymes are not capable enough to fulfil the industrial demands due to the lack of adapting operational conditions. So, the present study focused on extracting enzymes from microbial sources to overcome the problems. 260 isolated bacterial strains from different areas of Sylhet, Bangladesh, were screened for their cellulolytic and proteolytic activities using Carboxymethylcellulose (CMC) and skim milk agar respectively. Among them 6 cellulolytic and 15 proteolytic bacteria were initially identified. Based on the capability to degrade CMC and skim milk, strain S32 was found to be the most potential among the isolates. Biochemical tests and molecular identification revealed that S32 is a strain of Aeromonas hydrophila and was later named as Aeromonas hydrophila ASM-S32. Maximum cellulase production by the strain A. hydrophila ASM-S32 was obtained after 18 h of incubation in a fermentation medium with an initial pH of 6.5 at 37°C and that was 4-fold higher as compared to unoptimized conditions. Maximum cellulase activity was observed at 60°C with a pH of 6.5 in presence of Ca2+ metal ion. In case of protease, optimum enzyme production was observed after 24 h of incubation with an initial pH of 8.5 at 37°C and protease production was increased by 1.2-folds when optimized conditions were used. Maximum protease activity resulted from pH 6.0, at 70°C and in presence of Cu2+ ion.

Optimization of Expression Condition, Two Dimensional And Melting Point-Based Characterization of Recombinant Human Interferon Alpha-2a Fusion and Non Fusion Forms

Recombinant Human Interferon Alpha-2a (rhIFNα-2a) is a therapeutic protein that used in hepatitis and cancer treatments. In our previous research, we developed higher molecular weight of the protein through human serum albumin fusion. The fusion and non fusion form of rhIFNα-2a were produced in Pichia pastoriswith 86 kDa and 19 kDa in size respectively. In previous research, protein yield was not reproducible due to unoptimized expression conditions. This reseach was aimed to optimize expression condition process and to characterize the fusion and non fusion forms of rhIFNα-2a. The parameters to observe in overproduction include nutrient (media and methanol concentration) and non nutrient (temperature andincubation period). Affinity and size exclusion cromatographicwere compared in protein purification. BCA assay was used to determine quantity of protein. Protein characterization was conducted using two-dimensional SDS PAGE and denaturation analyses. The optimal condition of expression was achieved using complex media with 1% of methanol for 3 day incubation period at 25°C. The protein yield was reproducible and higher comparing to previous research. Affinity chromatography resulted in higher purity of the proteins comparing to size exclusions. Characterization using two dimensional gel analysis revealed that isoelectric point of rhIFNα-2a is 6.5 for fusion form and 6.0 for non fusion form. The melting points of fusion protein were 56°C and 62°C whilst that of non fusion was 56°C.

Bacterial indoleacetic acid-induced synthesis of colloidal Ag2O nanocrystals and their biological activities.

The biosynthesis and biological activity of colloidal Ag2O nanocrystals have not been well studied, although they have potential applications in many fields. For the first time, we developed a reducing agent free, cost-effective technique for Ag2O biosynthesis using Xanthomonas sp. P5. The optimal conditions for Ag2O synthesis were 50°C, pH 8, and 2.5mM AgNO3. Using these conditions the yield of Ag2O obtained at 10h was about five times higher than that obtained at 12h under unoptimized conditions. Ag2O was characterized by FESEM-EDS, TEM, dynamic light scattering, XRD, and UV-Visible spectroscopy. Indoleacetic acid produced by the strain P2 was involved in the synthesis of Ag2O. Ag2O exhibited a broad antimicrobial spectrum against several human pathogens. Furthermore, Ag2O exhibited 1,1-diphenyl-2-picrylhydrazyl (IC50 = 25.1µg/ml) and 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonate (IC50 = 16.8µg/ml) radical scavenging activities, and inhibited collagenase (IC50 = 27.9mg/ml). Cytotoxicity of Ag2O was tested in fibroblast cells and found to be non-toxic, demonstrating biocompatibility.

Production, purification and characterization of an acid/alkali and thermo tolerant cellulase from Schizophyllum commune NAIMCC-F-03379 and its application in hydrolysis of lignocellulosic wastes

A cellulase producing fungus Schizophyllum commune NAIMCC-F-03379 was isolated from decomposed leaf sample of Lantana camera. The nutritional components (wheat bran, magnesium sulphate and calcium chloride concentrations) and physical parameters (temperature and pH) were optimised by response surface methodology for enhanced cellulase production by S. commune NAIMCC-F-03379. The optimized medium contained: 1% (w/v) wheat bran, 0.3 g/L MgSO4, 0.8–1.0 g/L CaCl2, optimum temperature and pH were 25 °C and 5 respectively. Under optimum condition, 5.35-fold increase in CMCase and 6.62-fold increase in FPase activity was obtained as compared to un-optimized condition. Crude cellulase enzyme was subjected to different purification techniques and comparative evaluation of their efficiency was performed. The aqueous two-phase system using polyethylene glycol 8000/MnSO4 system showed maximum purification with 10.4-fold increase in activity, 79.5% yield and 0.5 partition coefficient. The cellulase enzyme obtained from S. commune NAIMCC-F-03379 has shown high stability i.e. more than 55% relative activity after 12 h of incubation over wide range of temperature (25–65 °C) and pH (3–10). The molecular weight of the cellulase enzyme was estimated as ~ 60 kDa by using sodium dodecyl sulphate-polyacrylamide electrophoresis (SDS-PAGE) and zymography. Km and Vmax value of cellulase on carboxy-methyl cellulose were obtained as 0.0909 mg/mL and 45.45 μmol/min mg respectively. Rice straw and wheat bran were subjected to hydrolysis using cellulase and cellulase–xylanase cocktail and analysed by thin layer chromatography and high performance liquid chromatography (HPLC). The HPLC analysis showed glucose concentration of 1.162 mg/mL after enzymatic hydrolysis of rice straw.

Isolation of bacteria capable of hydrogen production in dark fermentation and intensification of anaerobic granular sludge activity

In the anaerobic biological treatment of pulp and papermaking wastewater, the gradual deposition of CaCO3 eventually leads to the inhibition of the activity of anaerobic granular sludge. In this study, a hydrogen production bacterial Raoultella DW01 was isolated from domesticated anaerobic granular sludge. The fermentation conditions were designed using central composite design, and the optimum conditions obtained by response surface analysis encompassed an initial pH 5.77, 4.13 g/L l-glutamic acid and an inoculation amount of 15%. The H2 production yield represented a 29.5% increase over the unoptimized conditions. Finally, the effect of adding DW01 on the biogas production in anaerobic granular sludge with different sludge ages was investigated. The cumulative biogas yield and the max biogas production rate increased by 27.8% and 53.5% after adding DW01 to a sludge with an age of 335 days compared with the on-intensified sludge. This paper provides a way to alleviate the CaCO3 deposition by intensifying the activity of H2 and acid-producing bacteria via improving the activity of granular sludge.

Optimization of suspension system of heavy off-road vehicle for stability enhancement using integrated anti-roll bar and coiling spring mechanism

Short suspension system has an indispensable effect on vehicle handling and ride, so, optimization of vehicle suspension system is one of the most effective methods, which could considerably enhance the vehicle stability and controllability. Motion control, stability maintenance and ride comfort improvement are fundamental issues in design of suspension system of off-road vehicles. In this work, a dependent suspension system mostly used in off-road vehicles is modeled using Trucksim software. Then, geometric parameters of suspension system are optimized using integrated anti-roll bar and coiling spring in a way that ride comfort, handling and stability of vehicle are improved. The simulation results of suspension system and variations of geometric parameters due to road roughness and different steering angles are presented in Trucksim and effects of optimization of suspension system during various driving maneuvers in both optimized and un-optimized conditions are compared. The simulation results indicate that the type of suspension system and geometric parameters have significant effect on vehicle performance.

Isolation, Identification and Medium Optimization for Tyrosinase Production by a Newly Isolated Bacillus subtilis NA2 Strain

Isolation of wound healing compounds from Heliotropium indicumDodehe Yeo, Barthélémy Attioua, Christine Lehalle, Metowogo Kossi, Jean David N’guessan, Allico Joseph Djaman, Annelise Lobstein, Nelly Frossard

Fungal biotransformation of synthetic levodopa to stable dopamine in l-ascorbate-mediated aerobic-thermophilic biochemical process.

In the present study, fungal biotransformation of synthetic levodopa to stable dopamine in an l-ascorbate-mediated thermophilic-aerobic biochemical reaction was investigated. A mutant strain of Aspergillus oryzae EMS-6 was used for the preparation of mycelial biomass. The mutant was previously developed through EMS-induced mutagenesis and repressed against l-cysteine HCl. Growth parameters such as rate of cultivation (48h), initial pH (6) and incubation temperature (30°C) supported 18.84g/l biomass with 23g/l glucose consumption. Thermophilic behaviour of culture was observed at 25-40°C. Kinetic variables notably µ = 0.385 /h and Qs, exhibited consistent growth pattern. Biochemical reactions were performed aerobically using mycelial biomass as the source of enzyme 'tyrosinase' in a digital hotplate equipped with magnetic stirrers. The reaction conditions included 5mg/ml biomass and 2.5mg/ml levodopa as basal substrate in a thermophilic reaction of 25min duration acidified with l-ascorbic acid. TLC and HPLC analysis of reaction mixture confirmed the presence of levodopa and dopamine using a CN-9dth (R) column. Activation enthalpy and entropy of dopa decarboxylase (DDC) and its thermal inactivation showed an improved biotransformation of levodopa to dopamine at the optimal temperature (30°C) as compared to other temperatures being employed. Overall, 3.68mg/ml dopamine (4.55mg/ml proteins) synthesis from 2.38mg/ml levodopa was accomplished. The enhancement in metabolic activity of the mutant strain is ~ 2.75-fold improved when compared to the unoptimized reaction conditions, which is highly significant (HS) indicating an eco-commercially viable (LSD ~ 0.412) bioprocess.

Use of chick neural tube for optimizing the PSM and epithelial somites electroporation parameters: A detailed protocol.

Somite myogenesis is one of the crucial early embryonic events that lead to the formation of muscular tissue. A complex of dynamic gene regulatory networks masters this event. To understand and analyze these networks, there remains a genuine need for the use of a reproducible and highly efficient gene transfer technique. In vivo electroporation has proven to be amongst the best approaches in achieving a high level of gene transfer. However, unoptimized electroporation conditions can directly cause varying degrees of cellular damage which may induce abnormal embryonic development as well as changes in the endogenous gene expression. Presegmented mesoderm and epithelial somites are not easy to electroporate. Chick neural tube has served in many functional studies as an ideal experimental model organ which is both robust and easily manipulated. In the current detailed protocol, the neural tube was used as a tool to optimize the electroporation conditions which were subsequently applied in the electroporation of the presegmented mesoderm and epithelial somites. The protocol highlights important notes and hints that enable reproducible results and could be applied in the in vivo electroporation of other chick embryo tissues.

Screening of Yeasts Isolated from Brazilian Environments for the 2-Phenylethanol (2-PE) Production

Phenylethanol alcohol, or 2-phenylethanol (2-PE) production by yeasts has been considered a promising alternative to its chemical synthesis. In order to evaluate the potential of yeast strains isolated from different Brazilian environments, we evaluated the 2-PE production of 267 strains. Among them, the Kluyveromyces marxianus CCT 7735 yeast stood out as being the best 2-PE producer. The K. marxianus CCT 7735 growth was impaired by 2-PE; nevertheless, this effect is less pronounced than the inhibition reported for certain Saccharomyces cerevisiae strains. The maximum 2-PE titer obtained under optimized conditions was 3.44 g/L, 28% higher than the titer achieved under unoptimized conditions. The optimized conditions were: 30oC, and glucose and L-phe concentrations of 3.0 and 4.0 g/L, respectively. Moreover, the specific production rate of 2-PE increased twofold compared to the unoptimized conditions.

Calcium Alginate Bead-mediated Enhancement of the Selective Recovery of a Lead Novel Antifungal Bacillomycin Variant

In the pursuit of new antifungal compounds, five coproduced lipopeptide variants (AF1 to AF5) from wild-type Bacillus subtilis RLID 12.1 were identified in our previous study. Out of five, AF4 was identified as a novel lead molecule belonging to the bacillomycin family showing less cytotoxicity at its respective minimum inhibitory concentrations (MIC) evaluated against 81 strains of Candida and Cryptococcus species (including clinical isolates); besides this, AF4 purified in the present study exhibited encouraging MIC values against 10 clinical mycelial fungi. Aiming for a selective production augmentation of AF4 lipopeptide variant, a new fermentation media comprising malt extract (1.01%), dextrose (0.55%), peptone (1.79%), MnSO4 (2mM), and NaCl (0.5%) was formulated. Maximum production of 954.8 ± 10.8mg/L was achieved with 44% selectivity at 30°C compared to unoptimized conditions (186.4 ± 6.1mg/L). Use of calcium alginate beads in the formulated media during the onset of lipopeptide production resulted in an augmentation in the selectivity of the most efficacious AF4 variant to about 72% presumably due to attenuation of other coproduced lipopeptide variants AF1 and AF2. Difference in yield of lipopeptides varied with bead size, bead preparation ratios, and sodium alginate concentrations. Use of Ca-alginate beads in the upstream production process of the lead AF4 variant may be considered as a novel strategy to address the potential challenge that may arise during the scale-up and downstream processing steps. Another significant finding derived from the study is that the proportion of bacillomycin variants of B. subtilis RLID 12.1 could be controlled by temperature and metal ions under static and shaking conditions.

Functional analysis and heterologous expression of bifunctional glutathione synthetase from Lactobacillus

Bifunctional glutathione synthetase (GshF) has recently been reported to simultaneously catalyze the 2-step ATP-dependent biosynthesis of reduced glutathione (GSH). In this work, 19 putative gshF were mined from the complete sequenced genome of 20 representative Lactobacillus species. To functionally analyze these putative GshF, GshF from Lactobacillus plantarum and Lactobacillus casei were selected and successfully expressed in Escherichia coli. Compared with the control without expressing GshF, GSH titers were enhanced significantly in E. coli with overexpression of GshF, demonstrating that putative GshF from Lactobacillus have functional activities on GSH biosynthesis. Moreover, with the expression of GshF from L. plantarum in E. coli as a paradigm, GSH yield (286.5 μM) was strongly improved by 177.9% with optimized induced conditions and precursor concentration compared with the control under unoptimized conditions. Transcriptional analysis showed that key genes of endogenous GSH metabolism and precursor biosynthesis were remarkably suppressed by GshF expression, indicating that the increase of GSH titer was attributed to heterologous expression of GshF. Overall, our results suggested that gshF is enriched in Lactobacillus and that heterologous expression of GshF is an efficient strategy for improving GSH biosynthesis.

Enhanced saturated fatty acids accumulation in cultures of newly-isolated strains of Schizochytrium sp. and Thraustochytriidae sp. for large-scale biodiesel production

Heterotrophic marine protists (Thraustochytrids) have received increasingly global attention as a renewable, sustainable and alternative source of biodiesel because of their high ability of saturated fatty acids (SFAs) accumulation. Yet, the influence of extrinsic factors (nutrients and environmental conditions) on thraustochytrid culture and optimal conditions for high SFAs production are poorly described. In the present study, two different thraustochytrid strains, Schizochytrium sp. PKU#Mn4 and Thraustochytriidae sp. PKU#Mn16 were studied for their growth and SFAs production profiles under various conditions (carbon, nitrogen, temperature, pH, KH2PO4, salinity, and agitation speed). Of the culture conditions, substrates (C and N) source and conc., temperature, and agitation speed significantly influenced the cell growth and SFAs production of both strains. Although both the strains were capable of growth and SFAs production in the broad range of culture conditions, their physiological responses to KH2PO4, pH, and salinity were dissimilar. Under their optimal batch culture conditions, peak SFAs productions of 3.3g/L and 2.2g/L with 62% and 49% SFAs contents (relative to total fatty acids) were achieved, respectively. The results of 5-L fed-batch fermentation under optimal conditions showed a nearly 4.5-fold increase in SFAs production (i.e., 7.5g/L) by both strains compared to unoptimized conditions. Of the two strains, the quality of biodiesel produced from the fatty acids of PKU#Mn4 met the biodiesel standard defined by ASTM6751. This study, to the knowledge of the authors, is the first comprehensive report of optimal fermentation conditions demonstrating enhanced SFAs production by strains belonging to two different thraustochytrid genera and provides the basis for large-scale biodiesel production.

Optimizing secretory expression of recombinant human interferon gamma from Kluyveromyces lactis

ABSTRACTBiosimilar/biotherapeutic production is becoming a major area of focus for a big chunk of biotechnology industry. Easy licensing and already approved status for clinical use have given it a boost. In the present study, recombinant human interferon gamma (IFNG) was expressed for the first time in Kluyveromyces lactis expression system and its expression was optimized by varying growth parameters and carbon source concentration with the aim of increasing recombinant protein production level. Human IFNG gene was cloned in the genomic DNA of K. lactis by homologous recombination and under unoptimized conditions in shake flask, IFNγ protein was secreted in the fermentation medium at a level of 175 µg/L quantified by ELISA assay. After the optimization of expression conditions using one-variable-at-a-time technique, expression level was enhanced by 2.2-folds. Substrate inhibition studies revealed that up to 80 g/L of lactose is well tolerated by K. lactis cells for its growth but more than 80 g/L of lactose causes remarkable reduction in biomass production.

Statistical Optimization of the production of protease from Bacillus sp. MSK-01 in submerged fermentation

Protease has been in increasing demand in industries due to its hydrolytic nature. In industries, high yield of enzyme is required to meet the industrial need at a relatively cheaper cost. In the present study, the protease from Bacillus sp. MSK-01 was produced in large quantity by submerged fermentation. Statistical techniques including Plackett-Burman and Response surface methodology are useful tools for optimizing many parameters at a time and are used for increasing the protease production from Bacillus sp. MSK-01. 19 different parameters were chosen, out of which 15 factors had positive effect on protease yield. Four maximum influencing factors were peptone, magnesium sulphate, skim milk powder and casein were chosen to further increase the protease yield. 397.3 IU ml-1 of enzyme yield was obtained under optimized conditions which lead to 198 fold increase in the yield of protease from unoptimized condition.

Optimization of fermentation condition for co-production of ethanol and 2,3-butanediol (2,3-BD) from hemicellolosic hydrolysates by Klebsiella oxytoca XF7

ABSTRACTMicrobial production of ethanol and 2,3-butanediol (2,3-BD) from agro-residues has been attracting interest because of their applications in various industries, including generation of biofuel molecules. In the present investigation, the hemicellulosic fraction of corncob was hydrolyzed by indigenous holocellulase from novel psychrotolerant Aspergillus niger SH3 resulting in high xylose release (34.61 g L−1), followed by the bioconversion of xylose to ethanol and 2,3-BD. Taguchi design was adopted to optimize the process which resulted in 5.25- and 3.31-fold increase in 2,3-BD (12.18 ± 0.53 g L−1) and ethanol (4.08 ± 0.03 g L−1), as compared with un-optimized condition. For the first time, co-production of ethanol and 2,3-BD from the corncob hemicellulosic hydrolysate was performed using a newly isolated Klebsiella oxytoca XF7 strain, under the optimized fermentation conditions. These results suggest that K. oxytoca XF7 is a promising candidate for co-production of ethanol and 2,3-BD, with high xylose conversion efficiency (96.65%), facilitating the economical production of biofuel molecules.

Recovery of polyhydroxyalkanoates from municipal secondary wastewater sludge

In the current study, the feasibility of utilizing municipal secondary wastewater sludge for Polyhydroxyalkanoate (PHA) extraction was improved by optimization of various parameters (temperature, duration and concentration of sludge solids). Optimized process parameters resulted in PHA recovery of 0.605 g, significantly higher than un-optimized conditions. The characterization of PHA was carried out by GC–MS, FT-IR and NMR (1H and 13C) spectroscopy. The PHA profile was found to be dominated by mcl PHA (58%) along with other diverse PHA. The results of the present study show rich diversity of PHA extracted from a raw material which is readily available at minimal cost. In conclusion, exploring the potential of wastes for production of bioplastics not only reduces the cost of bioplastic production, but also provides a sustainable means for waste management.

Augmented cellulase production by Bacillus subtilis strain MU S1 using different statistical experimental designs.

The production of cellulase by Bacillus subtilis MU S1, a strain isolated from Eravikulam National Park, was optimized using one-factor-at-a-time (OFAT) and statistical methods. Physical parameters like incubation temperature and agitation speed were optimized using OFAT and found to be 40 °C and 150 rpm, respectively, whereas, medium was optimized by statistical tools. Plackett-Burman design (PBD) was employed to screen the significant variables that highly influence cellulase production. The design showed carboxymethyl cellulose (CMC), yeast extract, NaCl, pH, MgSO4 and NaNO3 as the most significant components that affect cellulase production. Among these CMC, yeast extract, NaCl and pH showed positive effect whereas MgSO4 and NaNO3 were found to be significant at their lower levels. The optimum levels of the components that positively affect enzyme production were determined using response surface methodology (RSM) based on central composite design (CCD). Three factors namely CMC, yeast extract and NaCl were studied at five levels whilst pH of the medium was kept constant at 7. The optimal levels of the components were CMC (13.46 g/l), yeast extract (8.38 g/l) and NaCl (6.31 g/l) at pH 7. The maximum cellulase activity in optimized medium was 566.66 U/ml which was close to the predicted activity of 541.05 U/ml. Optimization of physical parameters and medium components showed an overall 3.2-fold increase in activity compared to unoptimized condition (179.06 U/ml).

Isolation, characterization, statistical optimization, and application of a novel broad-spectrum capsular depolymerase against Klebsiella pneumoniae from Bacillus siamensis SCVJ30

Background: Bacterial resistance is one of the most challenging and emerging public healthcare crisis in the modern era. Along with antibiotic degrading strategies, bacteria also evolved to produce extracellular polymers such as capsular polysaccharide (CPS) which not only provides immune protection but also act as a permeability barrier to antibiotics. The use of therapeutic enzymes alone and in combination with antibiotics has opened a new window for clinicians and researchers. Methods: In the present study, isolation of broad-spectrum capsular depolymerase bacterium was attempted from a number of environmental samples followed by 16SrRNA characterization. Optimization of capsular depolymerase production was performed by the one variable at time (OVAT) method and response surface methodology (RSM). Capsular depolymerase was partially purified using ammonium sulfate saturation method. Capsule stripping effects of depolymerase were analyzed using microscopic visualization of the capsule and antibiotic susceptibility test. Results: Thirty-two capsular depolymerase producing bacteria were isolated in this study and broad-spectrum depolymerase producing Isolate-30 was characterized as Bacillus siamensis SCVJ30 according to the 16srRNA sequencing. Depolymerase production was optimized using OVAT method and RSM. Relatively high yields (1.92 IU/ml) of capsular depolymerase were obtained in a medium containing 1 mg magnesium sulfate, 7 mg peptone and at 9 pH. A 115% increase in capsular depolymerase production was observed under optimal conditions than unoptimized conditions. Microscopic visualization of the capsule and antibiotic susceptibility testing postulates the positive effect of depolymerase on antibiotic effi cacy against Klebsiella pneumoniae. Conclusion: Further characterization of the enzyme will help in developing broad-spectrum depolymerase as a potent therapeutic agent against drug-resistant strains.