Search results for: enzyme reusability

Authors: Aleksandra Łochowicz, Daria Świętochowska, Loredano Pollegioni, Nazim Ocal, Franck Charmantray, Laurence Hecquet, Katarzyna Szymańska

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Multienzymatic cascade reactions are nowadays widely used in pharmaceutical, chemical and cosmetics industries to produce high valuable compounds. They can be carried out in two ways, step by step and one-pot. If two or more enzymes are in the same reaction vessel is necessary to work out the compromise to run the reaction in optimal conditions for each enzyme. So far most of the reports of multienzymatic cascades concern on usage of free enzymes. Unfortunately using free enzymes as catalysts of reactions accomplish high cost. What is more, free enzymes are soluble in solvents which makes reuse impossible. To overcome this obstacle enzymes can be immobilized what provides heterogeneity of biocatalyst that enables reuse and easy separation of the enzyme from solvents and reaction products. Usually, immobilization increase also the thermal and operational stability of enzyme. The advantages of using immobilized multienzymes are enhanced enzyme stability, improved cascade enzymatic activity via substrate channeling, and ease of recovery for reuse. The one-pot immobilized multienzymatic cascade can be carried out in mixed or coimmobilized type. When biocatalysts are coimmobilized on the same carrier the are in close contact to each other which increase the reaction rate and catalytic efficiency, and eliminate the lag time. However, in this type providing the optimal conditions both in the process of immobilization and cascade reaction for each enzyme is complicated. Herein, we examined immobilization of 3 enzymes: D-amino acid oxidase from Rhodotorula gracilis, commercially available catalase and transketolase from Geobacillus stearothermophilus. As a support we used silica monoliths with hierarchical structure of pores. Then we checked their stability and reusability in one-pot cascade of L-erythrulose and hydroxypuryvate acid synthesis.

Keywords: biocatalysts, enzyme immobilization, multienzymatic reaction, silica carriers

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Authors: Mirjana Petronijević, Sanja Panić, Aleksandra Cvetanović, Branko Kordić, Nenad Grba

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Enzyme peroxidases are biological catalysts and play a major role in phenolic wastewater treatments and other environmental applications. The most studied species from the peroxidases family is horseradish peroxidase (HRP). In environmental processes, HRP could be used in its free or immobilized form. Enzyme immobilization onto solid support is performed to improve the enzyme properties, prolong its lifespan and operational stability and allow its reuse in industrial applications. One of the enzyme supports of a newer generation is magnetic particles (MPs). Fe₃O₄ MPs are the most widely pursued immobilization of enzymes owing to their remarkable advantages of biocompatibility and non-toxicity. Also, MPs can be easily separated and recovered from the water by applying an external magnetic field. On the other hand, metals and metal oxides are not suitable for the covalent binding of enzymes, so it is necessary to perform their surface modification. Fe₃O₄ MPs functionalization could be performed during the process of their synthesis if it takes place in the presence of plant extracts. Extracts of plant material, such as wild plants, herbs, even waste materials of the food and agricultural industry (bark, shell, leaves, peel), are rich in various bioactive components such as polyphenols, flavonoids, sugars, etc. When the synthesis of magnetite is performed in the presence of plant extracts, bioactive components are incorporated into the surface of the magnetite, thereby affecting its functionalization. In this paper, the suitability of bio-magnetite as solid support for covalent immobilization of HRP across glutaraldehyde was examined. The activity of immobilized HRP at different pH values (4-9) and temperatures (20-80°C) and reusability were examined. Bio-MP was synthesized by co-precipitation method from Fe(II) and Fe(III) sulfate salts in the presence of water extract of the Allium cepa peel. The water extract showed 81% of antiradical potential (according to DPPH assay), which is connected with the high content of polyphenols. According to the FTIR analysis, the bio-magnetite contains oxygen functional groups (-OH, -COOH, C=O) suitable for binding to glutaraldehyde, after which the enzyme is covalently immobilized. The immobilized enzyme showed high activity at ambient temperature and pH 7 (30 U/g) and retained ≥ 80% of its activity at a wide range of pH (5-8) and temperature (20-50°C). The HRP immobilized onto bio-MPs showed remarkable stability towards temperature and pH variations compared to the free enzyme form. On the other hand, immobilized HRP showed low reusability after the first washing cycle enzyme retains 50% of its activity, while after the third washing cycle retains only 22%.

Keywords: bio-magnetite, enzyme immobilization, water extracts, environmental protection

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Authors: Charline Monnier, Rudolf Andrys, Irene Castellino, Lucie Zemanova

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Due to their inherent sustainability and compatibility with green chemistry principles, enzymes are attracting increasing attention for various applications like bioremediation or biocatalysis. These natural catalysts boast remarkable substrate specificity and operate under mild biological conditions. However, their intrinsic limitations, such as instability at high temperatures or in organic solvents, impede their wider applicability. Enzyme immobilization on supportive matrices emerges as a promising strategy to address these challenges. This approach not only facilitates enzyme reusability but also offers the potential to modulate their stability, activity, and selectivity. The present study investigates the immobilization and application of two distinct groups of hydrolases on supportive matrices: PETases, naturally capable of PolyEthylene Terephthalate (PET) degradation, and cholinesterases (ChEs), key enzymes in neurotransmitter regulation. All tested enzymes will be immobilized on porous and non-porous particles using both covalent and non-covalent methods. Additionally, the stability of PETases and cholinesterases will be explored, followed by exposure to denaturing conditions to assess their resilience under harsh conditions. Furthermore, due to the exceptional catalytic efficiency and selectivity, their biocatalytic efficiency will be tested using xenobiotic substrates, aiming to establish them as replacements for conventional chemical catalysts in environmentally friendly processes. By exploiting the power of enzyme immobilization, this research strives to unlock the full potential of these biocatalysts for sustainable and efficient technological advancements.

Keywords: biocatalysis, bioremediation, enzyme efficiency, enzyme immobilization, green chemistry

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Authors: Bipasa Dey, Varsha Panwar, Tanmay Dutta

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Laccase, a multicopper oxidase (EC 1.10.3.2) have been at the forefront as a superior industrial biocatalyst. They are versatile in terms of bestowing sustainable and ecological catalytic reactions such as polymerisation, xenobiotic degradation and bioremediation of phenolic and non-phenolic compounds. Regardless of the wide biotechnological applications, the critical limiting factors viz. reusability, retrieval, and storage stability still prevail. This can cause an impediment in their applicability. Crosslinked enzyme aggregates (CLEAs) have emerged as a promising technique that rehabilitates these essential facets, albeit at the expense of their enzymatic activity. The carrier free crosslinking method prevails over the carrier-bound immobilisation in conferring high productivity, low production cost owing to the absence of additional carrier and circumvent any non-catalytic ballast which could dilute the volumetric activity. To the best of our knowledge, the ε-amino group of lysyl residue is speculated as the best choice for forming Schiff’s base with glutaraldehyde. Despite being most preferrable, excess glutaraldehyde can bring about disproportionate and undesirable crosslinking within the catalytic site and hence could deliver undesirable catalytic losses. Moreover, the surface distribution of lysine residues in Trametes versicolor laccase is significantly less. Thus, to mitigate the adverse effect of glutaraldehyde in conjunction with scaling down the degradation or catalytic loss of the enzyme, crosslinking with inert substances like gelatine, collagen, Bovine serum albumin (BSA) or excess lysine is practiced. Analogous to these molecules, sugars have been well known as a protein stabiliser. It helps to retain the structural integrity, specifically secondary structure of the protein during aggregation by changing the solvent properties. They are comprehended to avert protein denaturation or enzyme deactivation during precipitation. We prepared crosslinked enzyme aggregates (CLEAs) of laccase from T. versicolor with the aid of sugars. The sugar CLEAs were compared with the classic BSA and glutaraldehyde laccase CLEAs concerning physico-chemical properties. The activity recovery for the fructose CLEAs were found to be ~20% higher than the non-sugar CLEA. Moreover, the 𝐾𝑐𝑎𝑡𝐾𝑚⁄ values of the CLEAs were two and three-fold higher than BSA-CLEA and GACLEA, respectively. The half-life (t1/2) deciphered by sugar-CLEA was higher than the t1/2 of GA-CLEAs and free enzyme, portraying more thermal stability. Besides, it demonstrated extraordinarily high pH stability, which was analogous to BSA-CLEA. The promising attributes of increased storage stability and recyclability (>80%) gives more edge to the sugar-CLEAs over conventional CLEAs of their corresponding free enzyme. Thus, sugar-CLEA prevails in furnishing the rudimentary properties required for a biocatalyst and holds many prospects.

Keywords: cross-linked enzyme aggregates, laccase immobilization, enzyme reusability, enzyme stability

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Authors: Homa Torabizadeh, Mohaddeseh Mikani

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Inulinase from Aspergillus niger was covalently immobilized on magnetic nanoparticles (MNPs/Fe₃O₄) covered with soy protein isolate (SPI/Fe₃O₄) functionalized by bovine serum albumin (BSA) nanoparticles. MNPs are promising enzyme carriers because they separate easily under external magnetic fields and have enhanced immobilized enzyme reusability. As MNPs aggregate simply, surface coating strategy was employed. SPI functionalized by BSA was a suitable candidate for nanomagnetite coating due to its superior biocompatibility and hydrophilicity. Fe₃O₄@SPI-BSA nanoparticles were synthesized as a novel carrier with narrow particle size distribution. Step by step fabrication monitoring of Fe₃O₄@SPI-BSA nanoparticles was performed using field emission scanning electron microscopy and dynamic light scattering. The results illustrated that nanomagnetite with the spherical morphology was well monodispersed with the diameter of about 35 nm. The average size of the SPI-BSA nanoparticles was 80 to 90 nm, and their zeta potential was around −34 mV. Finally, the mean diameter of fabricated Fe₃O₄@SPI-BSA NPs was less than 120 nm. Inulinase enzyme from Aspergillus niger was covalently immobilized through gluteraldehyde on Fe₃O₄@SPI-BSA nanoparticles successfully. Fourier transform infrared spectra and field emission scanning electron microscopy images provided sufficient proof for the enzyme immobilization on the nanoparticles with 80% enzyme loading.

Keywords: high fructose syrup, inulinase immobilization, functionalized magnetic nanoparticles, soy protein isolate

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Authors: Talat Baran

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The Suzuki C(sp2)-C(sp2) coupling reaction is considered to be one of the best ways for the synthesis of biaryl compounds. There are many studies reporting the catalytic performance of palladium catalyst in Suzuki coupling reactions. Natural biopolymer (such as zeolite, carbon, silica, and chitosan) supporting catalysts have been lately attracted interest because of their low-cost, nontoxicity, and eco-friendliness. One of the most important natural biopolymer is cellulose, which is widely considered as an eco-friendly biopolymer due to its biodegradable, non-toxic and renewable nature. In this study, (1) cellulose supported Pd(II) catalyst was synthesized (2) its chemical structure was characterized by FT-IR, SEM/EDAX, XRD, TG-DTG, ICP-OES techniques (3) to investigate the performance of the catalyst in Suzuki coupling reactions by using microwave irradiation technique (4) reusability of the catalyst was done under optimum conditions. This cellulose supported Pd(II) catalyst exhibited high selectivity and efficiency in Suzuki coupling reactions under mild conditions (50°C). High TON and TOF values were recorded for the catalyst. Also, the reusability tests showed the catalysts could be used for several times in consequence of reusability tests.

Keywords: palladium, cellulose, Schiff base, reusability

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Authors: Sidra Pervez, Afsheen Aman, Shah Ali Ul Qader

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The importance of attaining an optimum performance of an enzyme is often a question of devising an effective method for its immobilization. Cross-linked enzyme aggregate (CLEAs) is a new approach for immobilization of enzymes using carrier free strategy. This method is exquisitely simple (involving precipitation of the enzyme from aqueous buffer followed by cross-linking of the resulting physical aggregates of enzyme molecules) and amenable to rapid optimization. Among many industrial enzymes, amyloglucosidase is an important amylolytic enzyme that hydrolyzes alpha (1→4) and alpha (1→6) glycosidic bonds in starch molecule and produce glucose as a sole end product. Glucose liberated by amyloglucosidase can be used for the production of ethanol and glucose syrups. Besides this amyloglucosidase can be widely used in various food and pharmaceuticals industries. For production of amyloglucosidase on commercial scale, filamentous fungi of genera Aspergillus are mostly used because they secrete large amount of enzymes extracellularly. The current investigation was based on isolation and identification of filamentous fungi from genus Aspergillus for the production of amyloglucosidase in submerged fermentation and optimization of cultivation parameters for starch saccharification. Natural isolates were identified as Aspergillus niger KIBGE-IB36, Aspergillus fumigatus KIBGE-IB33, Aspergillus flavus KIBGE-IB34 and Aspergillus terreus KIBGE-IB35 on taxonomical basis and 18S rDNA analysis and their sequence were submitted to GenBank. Among them, Aspergillus fumigatus KIBGE-IB33 was selected on the basis of maximum enzyme production. After optimization of fermentation conditions enzyme was immobilized on CLEA. Different parameters were optimized for maximum immobilization of amyloglucosidase. Data of enzyme stability (thermal and Storage) and reusability suggested the applicability of immobilized amyloglucosidase for continuous saccharification of starch in industrial processes.

Keywords: aspergillus, immobilization, industrial processes, starch saccharification

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Authors: Devendra Sillu, Shekhar Agnihotri

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The 'nano-biocatalyst' utilizes an ordered assembling of enzyme on to nanomaterial carriers to catalyze desirable biochemical kinetics and substrate selectivity. The current study describes an inter-disciplinary approach for converting agriculture waste, sugarcane bagasse into D-glucose exploiting halloysite nanotubes (HNTs) decorated cellulase enzyme as nano-biocatalytic system. Cellulase was successfully immobilized on HNTs employing polydopamine as an eco-friendly crosslinker while iron oxide nanoparticles were attached to facilitate magnetic recovery of material. The characterization studies (UV-Vis, TEM, SEM, and XRD) displayed the characteristic features of both cellulase and magnetic HNTs in the resulting nanocomposite. Various factors (i.e., working pH, temp., crosslinker conc., enzyme conc.) which may influence the activity of biocatalytic system were investigated. The experimental design was performed using Response Surface Methodology (RSM) for process optimization. Analyses data demonstrated that the nanobiocatalysts retained 80.30% activity even at elevated temperature (55°C) and excellent storage stabilities after 10 days. The repeated usage of system revealed a remarkable consistent relative activity over several cycles. The immobilized cellulase was employed to decompose agro-waste and the maximum decomposition rate of 67.2 % was achieved. Conclusively, magnetic HNTs can serve as a potential support for enzyme immobilization with long term usage, good efficacy, reusability and easy recovery from solution.

Keywords: halloysite nanotubes, enzyme immobilization, cellulase, response surface methodology, magnetic recovery

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Authors: Gokhan Zengin, Abdurrahman Aktumsek

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The key enzyme inhibitory theory is one of the most accepted strategies in the treatment of global health problems including Alzheimer’s Disease and Diabetes mellitus. For this reason, the enzyme inhibitory potentials of different solvent extracts from Linaria genistifolia subsp. genistifolia were investigated against cholinesterase, and tyrosinase. The in vitro enzyme inhibitory potentials were measured with a microplate reader. The acetone and methanol extracts exhibited the strongest enzyme inhibitory effects on cholinesterase. However, the water extract was only active on tyrosinase. The results suggested that Linaria genistifolia subsp. genistifolia could be considered as a source of natural enzyme inhibitors for the treatment of major health problems.

Keywords: enzyme inhibitors, cholinesterase, tyrosinase, linaria, Turkey

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Authors: Furqan M. Kadhum, Asmaa A. Hussein, Maysaa Ch. Hatem

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Hyaluronidase enzyme was purified from the clinical isolate Staphyloccus aureus in three purification steps, first by precipitation with 90% saturated ammonium sulfate, ion exchange chromatography on DEAE-Cellulose, and gel filtration chromatography throughout Sephacryl S-300. Specific activity of the purified enzyme was reached 930 U/mg protein with 7.4 folds of purification and 46.5% recovery. The enzyme has an average molecular weight of about 69 kDa, with an optimum pH of enzyme activity and stability at pH 7, also the optimum temperature for activity was 37oC. The enzyme was stable with full activity at a temperature ranged between 30-40 oC. Metal ions showed variable inhibitory degree with the strongest effect for Fe+3, however, the chelating and reducing agents had no or little effects. Cytotoxic studies for purified and crude hyaluronidase against cancer cell Hep G2 type at different enzyme concentrations and exposure times showed that the inhibition effect of both crude and purified enzyme increased by increasing the enzyme concentration with no change was observed at 24hr, while at 48 and 72 hrs the same inhibition rate were observed for purified enzyme and differ for the crude filtrate.

Keywords: hyaluronidase, S. aureus, metal ions, cytotoxicity

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Authors: Ogbonnaya Nwokoro, Florence O. Anya

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Nutritional factors relating to the production of linamarase from Lactobacillus delbrueckii NRRL B–763 were investigated. The microorganism was cultivated in a medium containing 1% linamarin. Enzyme was produced using a variety of carbon substrates but the highest enzyme activity was detected in the presence of salicin (522 U/ml) after 48 h while the lowest yield was observed with CM cellulose (38 U/ml) after 72 h. Enzyme was not produced in the presence of cellobiose. Among a variety of nitrogen substrates tested, peptone supported maximum enzyme production (412 U/ml) after 48 h. Lowest enzyme production was observed with urea (40 U/ml). Organic nitrogen substrates generally supported higher enzyme productivity than inorganic nitrogen substrates. Enzyme activity was observed in the presence of Mn2+ (% relative activity = 216) while Hg2+ was inhibitory (% relative activity = 28). Locally-formulated media were comparable to MRS broth in supporting linamarase production by the bacterium. Higher enzyme activity was produced in media with surfactant than in media without surfactant. The enzyme may be useful in enhanced degradation of cassava cyanide.

Keywords: linamarase, locally formulated media, carbon substrates, nitrogen substrates, metal ions

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Authors: S. Lekhavat, T. Kajsongkram, S. Sang-han

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Dried ginger was extracted and investigated the effect of ethanol concentration and enzyme pre-treatment on its bioactive compounds in solvent extraction process. Sliced fresh gingers were dried by oven dryer at 70 °C for 24 hours and ground to powder using grinder which their size were controlled by passing through a 20-mesh sieve. In enzyme pre-treatment process, ginger powder was sprayed with 1 % (w/w) cellulase and then was incubated at 45 °C for 2 hours following by extraction process using ethanol at concentration of 0, 20, 40, 60 and 80 % (v/v), respectively. The ratio of ginger powder and ethanol are 1:9 and extracting conditions were controlled at 80 °C for 2 hours. Bioactive compounds extracted from ginger, either enzyme-treated or non enzyme-treated samples, such as total phenolic content (TPC), 6-Gingerol (6 G), 6-Shogaols (6 S) and antioxidant activity (IC50 using DPPH assay), were examined. Regardless of enzyme treatment, the results showed that 60 % ethanol provided the highest TPC (20.36 GAE mg /g. dried ginger), 6G (0.77%), 6S (0.036 %) and the lowest IC50 (625 μg/ml) compared to other ratios of ethanol. Considering the effect of enzyme on bioactive compounds and antioxidant activity, it was found that enzyme-treated sample has more 6G (0.17-0.77 %) and 6S (0.020-0.036 %) than non enzyme-treated samples (0.13-0.77 % 6G, 0.015-0.036 % 6S). However, the results showed that non enzyme-treated extracts provided higher TPC (6.76-20.36 GAE mg /g. dried ginger) and Lowest IC50 (625-1494 μg/ml ) than enzyme-treated extracts (TPC 5.36-17.50 GAE mg /g. dried ginger, IC50 793-2146 μg/ml).

Keywords: antioxidant activity, enzyme, extraction, ginger

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Authors: Irina E. Sukovataya, Oleg S. Sutormin, Valentina A. Kratasyuk

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Effect of viscosity of media on kinetic parameters of the coupled enzyme system NADH:FMN-oxidoreductase–luciferase was investigated with addition of organic solvents (glycerol and sucrose), because bioluminescent enzyme systems based on bacterial luciferases offer a unique and general tool for analysis of the many analytes and enzymes in the environment, research, and clinical laboratories and other fields. The possibility of stabilization and increase of activity of the coupled enzyme system NADH:FMN-oxidoreductase–luciferase activity in vicious aqueous-organic mixtures have been shown.

Keywords: coupled enzyme system of bioluminescence bacteria NAD(P)H:FMN-oxidoreductase–luciferase, glycerol, stabilization of enzymes, sucrose

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Authors: Y. Moldes-Diz, M. Gamallo, G. Eibes, C. Vazquez-Vazquez, G. Feijoo, J. M. Lema, M. T. Moreira

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Laccases (benzenediol oxygen oxidoreductases, EC 1.10.3.2) are excellent biocatalysts for biotechnological and environmental applications because of their high activity, selectivity, and specificity. Specifically, these characteristics allow them to perform the oxidation of recalcitrant compounds with simple requirements for the catalysis (presence of molecular oxygen). Nevertheless, the low stability under unfavorable conditions (pH, inactivating agents or temperature) and high production costs still limits their use for practical applications. Immobilization of enzymes has proven particularly valuable to avoid some of the aforementioned drawbacks. Magnetic nanoparticles (MNPs) have received increasing attention as carriers for enzyme immobilization since they can potentially provide an easy recovery of the biocatalyst from the reaction medium under an external magnetic field. In the present work, silica-coated magnetic nanoparticles (Fe3O4@SiO2) were prepared, characterized and used for laccase immobilization by covalent binding. The synthesis of Fe3O4@SiO2 was performed in a two-step procedure: co-precipitation and reverse microemulsion. The influence of immobilization conditions: concentrations of the functionalization agent (3-aminopropyl-triethoxy-silane) and the cross-linker (glutaraldehyde) as well as the influence of pH, T or inactivating agents were evaluated. In general, immobilized laccase showed superior stability compared to that of free enzyme. The reusability of the biocatalyst was demonstrated in successive batch reactions, where enzyme activity was maintained above 65% after 8 cycles of oxidation of the substrate 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate).

Keywords: silica-coated magnetic nanoparticles, laccase, immobilization, regeneration

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Authors: Shanna Swart, Caryn Fenner, Athanasios Kotsiopoulos, Susan Harrison

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Linear alkanes are produced as by-products from the increasing use of gas-to-liquid fuel technologies for synthetic fuel production and offer great potential for value addition. Their current use as low-value fuels and solvents do not maximize this potential. Therefore, attention has been drawn towards direct activation of these aliphatic alkanes to more useful products such as alcohols, aldehydes, carboxylic acids and derivatives. Cytochrome P450 monooxygenases (P450s) can be used for activation of these aliphatic alkanes using whole-cells or cell-free systems. Some limitations of whole-cell systems include reduced mass transfer, stability and possible side reactions. Since the P450 systems are little studied as cell-free systems, they form the focus of this study. Challenges of a cell-free system include co-factor regeneration, substrate availability and enzyme stability. Enzyme immobilization offers a positive outlook on this dilemma, as it may enhance stability of the enzyme. In the present study, 2 different P450s (CYP153A6 and CYP102A1) as well as the relevant accessory enzymes required for electron transfer (ferredoxin and ferredoxin reductase) and co-factor regeneration (glucose dehydrogenase) have been expressed in E. coli and purified by metal affinity chromatography. Glucose dehydrogenase (GDH), was used as a model enzyme to assess the potential of various enzyme immobilization strategies including; surface attachment on MagReSyn® microspheres with various functionalities and on electrospun nanofibers, using self-assembly based methods forming Cross Linked Enzymes (CLE), Cross Linked Enzyme Aggregates (CLEAs) and spherezymes as well as in a sol gel. The nanofibers were synthesized by electrospinning, which required the building of an electrospinning machine. The nanofiber morphology has been analyzed by SEM and binding will be further verified by FT-IR. Covalent attachment based methods showed limitations where only ferredoxin reductase and GDH retained activity after immobilization which were largely attributed to insufficient electron transfer and inactivation caused by the crosslinkers (60% and 90% relative activity loss for the free enzyme when using 0.5% glutaraldehyde and glutaraldehyde/ethylenediamine (1:1 v/v), respectively). So far, initial experiments with GDH have shown the most potential when immobilized via their His-tag onto the surface of MagReSyn® microspheres functionalized with Ni-NTA. It was found that Crude GDH could be simultaneously purified and immobilized with sufficient activity retention. Immobilized pure and crude GDH could be recycled 9 and 10 times, respectively, with approximately 10% activity remaining. The immobilized GDH was also more stable than the free enzyme after storage for 14 days at 4˚C. This immobilization strategy will also be applied to the P450s and optimized with regards to enzyme loading and immobilization time, as well as characterized and compared with the free enzymes. It is anticipated that the proposed immobilization set-up will offer enhanced enzyme stability (as well as reusability and easy recovery), minimal mass transfer limitation, with continuous co-factor regeneration and minimal enzyme leaching. All of which provide a positive outlook on this robust multi-enzyme system for efficient activation of linear alkanes as well as the potential for immobilization of various multiple enzymes, including multimeric enzymes for different bio-catalytic applications beyond alkane activation.

Keywords: alkane activation, cytochrome P450 monooxygenase, enzyme catalysis, enzyme immobilization

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Authors: Afsheen Aman, Zainab Bibi, Shah Ali Ul Qader

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Introduction: Xylan is a heteropolysaccharide composed of xylose monomers linked together through 1,4 linkages within a complex xylan network. Owing to wide applications of xylan hydrolytic products (xylose, xylobiose and xylooligosaccharide) the researchers are focusing towards the development of various strategies for efficient xylan degradation. One of the most important strategies focused is the use of heat tolerant biocatalysts which acts as strong and specific cleaving agents. Therefore, the exploration of microbial pool from extremely diversified ecosystem is considerably vital. Microbial populations from extreme habitats are keenly explored for the isolation of thermophilic entities. These thermozymes usually demonstrate fast hydrolytic rate, can produce high yields of product and are less prone to microbial contamination. Another possibility of degrading xylan continuously is the use of immobilization technique. The current work is an effort to merge both the positive aspects of thermozyme and immobilization technique. Methodology: Geobacillus stearothermophilus was isolated from soil sample collected near the blast furnace site. This thermophile is capable of producing thermostable endo-β-1,4-xylanase which cleaves xylan effectively. In the current study, this thermozyme was immobilized within a synthetic and a non-synthetic matrice for continuous production of metabolites using entrapment technique. The kinetic parameters of the free and immobilized enzyme were studied. For this purpose calcium alginate and polyacrylamide beads were prepared. Results: For the synthesis of immobilized beads, sodium alginate (40.0 gL-1) and calcium chloride (0.4 M) was used amalgamated. The temperature (50°C) and pH (7.0) optima of immobilized enzyme remained same for xylan hydrolysis however, the enzyme-substrate catalytic reaction time raised from 5.0 to 30.0 minutes as compared to free counterpart. Diffusion limit of high molecular weight xylan (corncob) caused a decline in Vmax of immobilized enzyme from 4773 to 203.7 U min-1 whereas, Km value increased from 0.5074 to 0.5722 mg ml-1 with reference to free enzyme. Immobilized endo-β-1,4-xylanase showed its stability at high temperatures as compared to free enzyme. It retained 18% and 9% residual activity at 70°C and 80°C, respectively whereas; free enzyme completely lost its activity at both temperatures. The Immobilized thermozyme displayed sufficient recycling efficiency and can be reused up to five reaction cycles, indicating that this enzyme can be a plausible candidate in paper processing industry. Conclusion: This thermozyme showed better immobilization yield and operational stability with the purpose of hydrolyzing the high molecular weight xylan. However, the enzyme immobilization properties can be improved further by immobilizing it on different supports for industrial purpose.

Keywords: immobilization, reusability, thermozymes, xylanase

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Authors: Jiraporn Burakorn, Pran Pinthong, Supida Hutabaedya

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Commercial traditional pork sausages (Moo Yaw) were produced by added more than 30% of pork fat for appetite customer. The pork sausages texture were softness, firmness, juiciness and smooth. If the pork sausages contained less fat, their textures were hardness, dryness and incoherence. This research investigated production of low fat traditional pork sausage containing transglutaminase for improved its sensory properties and nutritive values. The enzyme pork sausage composed of transglutaminase, soybean cake, rice bran oil and other ingredients. Consumer acceptance test was done by comparing the enzyme pork sausage with the 3 commercial pork sausage with 95 consumer. The enzyme pork sausage was accepted 92.6% and was preferred in all attributes over the 3 commercial pork sausages such as appearance, color, flavor, taste, firmness and overall liking. The enzyme pork sausage was high protein but low total calories, calories from fat, total fat, saturated fat, cholesterol and carbohydrate. The enzyme pork sausage was lower calorie (90 kcal) than the commercial reference pork sausage (150 kcal) 64%. The morphological texture of the enzyme pork sausage was smooth and consistency when analyzed by SEM.

Keywords: low fat, Moo Yaw, pork sausage, transglutaminase

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Authors: Mailin Misson, Bo Jin, Sheng Dai, Hu Zhang

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Advances in biotechnology have witnessed a growing interest in enzyme applications for the development of green and sustainable bio processes. While known as powerful bio catalysts, enzymes are no longer of economic value when extended to large commercialization. Alternatively, immobilization technology allows enzyme recovery and continuous reuse which subsequently compensates high operating costs. Employment of enzymes on nano structured materials has been recognized as a promising approach to enhance enzyme catalytic performances. High porosity, inter connectivity and self-assembling behaviors endow nano fibers as exciting candidate for enzyme carrier in bio reactor systems. In this study, nano fibers were successfully fabricated via electro spinning system by optimizing the polymer concentration (10-30 %, w/v), applied voltage (10-30 kV) and discharge distance (11-26 cm). Microscopic images have confirmed the quality as homogeneous and good fiber alignment. The nano fibers surface was modified using strong oxidizing agent to facilitate bio molecule binding. Bovine serum albumin and β-galactosidase enzyme were employed as model bio catalysts and immobilized onto the oxidized surfaces through covalent binding. Maximum enzyme adsorption capacity of the modified nano fibers was 3000 mg/g, 3-fold higher than the unmodified counterpart (1000 mg/g). The highest immobilization yield was 80% and reached the saturation point at 2 mg/ml of enzyme concentration. The results indicate a significant increase of activity retention by the enzyme-bound modified nano fibers (80%) as compared to the nascent one (60%), signifying excellent enzyme-nano carrier bio compatibility. The immobilized enzyme was further used for the bio conversion of dairy wastes into value-added products. This study demonstrates great potential of acid-modified electrospun polystyrene nano fibers as enzyme carriers.

Keywords: immobilization, enzyme, nanocarrier, nanofibers

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Authors: Chetna Tyagi. G. B. V. S. Lakshmi, Ambuj Tripathi, D. K. Avasthi

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Graphene oxide provides a good host matrix for preparing nanocomposites due to the different functional groups attached to its edges and planes. Being biocompatible, it is used in therapeutic applications. As enzyme-based biosensor requires complicated enzyme purification procedure, high fabrication cost and special storage conditions, we need enzyme-less biosensors for use even in a harsh environment like high temperature, varying pH, etc. In this work, we have prepared both enzyme-based and enzyme-less graphene oxide-based biosensors for glucose detection using glucose-oxidase as enzyme and gold nanoparticles, respectively. These samples were characterized using X-ray diffraction, UV-visible spectroscopy, scanning electron microscopy, and transmission electron microscopy to confirm the successful synthesis of the working electrodes. Electrochemical measurements were performed for both the working electrodes using a 3-electrode electrochemical cell. Cyclic voltammetry curves showed the homogeneous transfer of electron on the electrodes in the scan range between -0.2V to 0.6V. The sensing measurements were performed using differential pulse voltammetry for the glucose concentration varying from 0.01 mM to 20 mM, and sensing was improved towards glucose in the presence of gold nanoparticles. Gold nanoparticles in graphene oxide nanocomposite played an important role in sensing glucose in the absence of enzyme, glucose oxidase, as evident from these measurements. The selectivity was tested by measuring the current response of the working electrode towards glucose in the presence of the other common interfering agents like cholesterol, ascorbic acid, citric acid, and urea. The enzyme-less working electrode also showed storage stability for up to 15 weeks, making it a suitable glucose biosensor.

Keywords: electrochemical, enzyme-less, glucose, gold nanoparticles, graphene oxide, nanocomposite

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Authors: Joseph A. Bentil, Anders Thygesen, Lene Langea, Moses Mensah, Anne Meyer

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The study investigated the saccharification potential of in-house enzymes produced from a white-rot basidiomycete strain, Polyporus ciliatus CBS 366.74. The in-house enzymes were produced by growing the fungus on mono and composite substrates of cocoa pod husk (CPH) and green seaweed (GS) (Ulva lactuca sp.) with and without the addition of 25mM ammonium nitrate at 4%w/v substrate concentration in submerged condition for 144 hours. The crude enzyme extracts preparations (CEE 1-5 and CEE 1-5+AN) obtained from the fungal cultivation process were sterile-filtered and used as enzyme sources for enzymatic hydrolysis of hydrothermally pretreated corn stover using a commercial cocktail enzyme, Cellic Ctec3, as benchmark. The hydrolysis was conducted at 50ᵒC with 50mM sodium acetate buffer, pH 5 based on enzyme dosages of 5 and 10 CMCase Units/g biomass at 1%w/v dry weight substrate concentration at time points of 6, 24, and 72 hours. The enzyme activity profile of the in-house enzymes varied among the growth substrates with the composite substrates (50-75% GS and AN inclusion), yielding better enzyme activities, especially endoglucanases (0.4-0.5U/mL), β-glucosidases (0.1-0.2 U/mL), and xylanases (3-10 U/mL). However, nitrogen supplementation had no significant effect on enzyme activities of crude extracts from 100% GS substituted substrates. From the enzymatic hydrolysis, it was observed that the in-house enzymes were capable of hydrolysing the pretreated corn stover at varying degrees; however, the saccharification yield was less than 10%. Consequently, theoretical glucose yield was ten times lower than Cellic Ctec3 at both dosage levels. There was no linear correlation between glucose yield and enzyme dosage for the in-house enzymes, unlike the benchmark enzyme. It is therefore recommended that the in-house enzymes are used to complement the dosage of commercial enzymes to reduce the cost of biomass saccharification.

Keywords: enzyme production, hydrolysis yield, feedstock, enzyme blend, Polyporus ciliatus

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Authors: R. Majidzadeh Heravi, M. Sankian, H. Kermanshahi, M. R. Nassiri, A. Heravi Moussavi, S. A. Lari, A. R. Varasteh

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The use of probiotics engineered to express specific enzymes has been the subject of considerable attention in poultry industry because of increased nutrient availability and reduced cost of enzyme supplementation. Phytase enzyme is commonly added to poultry feed to improve digestibility and availability of phosphorus from plant sources. To construct a probiotic with potential of phytate degradation, phytase gene (appA) from E. coli was cloned and transformed into two probiotic bacteria Lactobacillus salivarius and Lactococcus lactis. L. salivarous showed plasmid instability, unable to express the gene. The expression of appA gene in L. lactis was analyzed by detecting specific RNA and zymography assay. Phytase enzyme was isolated from cellular extracts of recombinant L. lactis, showing a 46 kDa band upon the SDS-PAGE analysis. Zymogram also confirmed the phytase activity of the 46 kDa band corresponding to the enzyme. An enzyme activity of 4.9U/ml was obtained in cell extracts of L. lactis. The growth of native and recombinant L. lactis was similar in the presence of two concentrations of ox bile.

Keywords: Lactobacillus salivarus, Lactococcuslactis, recombinant, phytase, poultry

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Authors: Ashish Shukla, K. P. Mishra, Pushplata Tripathi

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This paper investigates the production and optimization of β-galactosidase enzyme using synthetic medium by isolated wild strains (S1, S2) mutated strains (M1, M2) through SSF and SmF. Among the different cell disintegration methods used, the highest specific activity was obtained when the cells were permeabilized using isoamyl alcohol. Wet lab experiments were performed to investigate the effects of carbon and nitrogen substrates present in Vogel’s medium on β-galactosidase enzyme activity using S1, S2, and M1, M2 strains through SSF. SmF experiments were performed for effects of carbon and nitrogen sources in YLK2Mg medium on β-galactosidase enzyme activity using S1, S2 and M1, M2 strains. Effect of pH on β-galactosidase enzyme production was also done using S1, S2, and M1, M2 strains. Results were found to be very appreciable in all the cases.

Keywords: β-galactosidase, cell disintegration, permeabilized, SSF, SmF

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Authors: Nuha Mansour Alhazmi

Abstract:

Replacement of petro-based plastics by a biodegradable plastic are vastly growing process. Poly-β-hydroxybutyrate (PHB) is a biodegradable biopolymer, synthesized by some bacterial genera. The objective of the current study is to explore the ability of some fungi to biodegrade PHB. The degradation of (PHB) was detected in Petri dish by the formation of a clear zone around the fungal colonies due to the production of depolymerase enzyme which has an interesting role in the PHB degradation process. Among 10 tested fungi, the most active PHB biodegraded fungi were identified as Trichoderma asperellum using morphological and molecular characters. The highest PHB degradation was at 25°C, pH 7.5 after 7 days of incubation for the tested fungi. Finally, the depolymerase enzyme was isolated, purified using column chromatography and characterized. In conclusion, PHB can be biodegraded in solid and liquid medium using depolymerase enzyme from T. asperellum.

Keywords: degradation, depolymerase enzyme, PHB, Trichoderma asperellum

Procedia PDF Downloads 142

Authors: Jamal Abd Awadallak, Thiago Olinek Reinehr, Eduardo Raizer, Deise Molinari, Edson Antonio, Camila da Silva da Silva

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The hydrolysis of soy oil catalyzed by 1,3-specific enzyme (Lecitase Ultra) in a well-stirred bioreactor was studied. Two forms of applications of the ultrasound were evaluated aiming to increase reaction rates, wherein the use of probe ultrasound associated with the use of surfactant to pre-emulsify the substrate showed the best results. Two different reaction periods were found: the first where the ultrasound has great influence on reaction rates, and the second where ultrasound influence is minimal. Studies on the time of pre-emulsification, surfactant concentration and enzyme concentration showed that the initial rate of hydrolysis depends on the interfacial area between the oil phase and the aqueous phase containing the enzyme.

Keywords: specific enzyme, free fatty acids, Hydrolysis, lecitase ultra, ultrasound

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Authors: Misha Ali, Qayyum Husain, Nida Alam, Masood Ahmad

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Improving the activity and stability of the enzyme is an important aspect in bioremediation processes. Immobilization of enzyme is an efficient approach to amend the properties of biocatalyst required during wastewater treatment. The present study was done to immobilize partially purified ginger peroxidase on amino functionalized silica coated titanium dioxide nanocomposite. Interestingly there was an enhancement in enzyme activity after immobilization on nanosupport which was evident from effectiveness factor (η) value of 1.76. Immobilized enzyme was characterized by transmission electron microscopy, scanning electron microscopy and Fourier transform infrared spectroscopy. Immobilized peroxidase exhibited higher activity in a broad range of pH and temperature as compared to free enzyme. Also, the thermostability of peroxidase was strikingly improved upon immobilization. After six repeated uses, the immobilized peroxidase retained around 62% of its dye decolorization activity. There was a 4 fold increase in Vmax of immobilized peroxidase as compared to free enzyme. Circular dichroism spectroscopy demonstrated conformational changes in the secondary structure of enzyme, a possible reason for the enhanced enzyme activity after immobilization. Immobilized peroxidase was highly efficient in the removal of acid yellow 42 dye in a stirred batch process. Our study shows that this bio-remediating system has remarkable potential for treatment of aromatic pollutants present in wastewater.

Keywords: acid yellow 42, decolorization, ginger peroxidase, immobilization

Procedia PDF Downloads 216

Authors: Suphalucksana, Wichai, Sangsoponjit Settasit, Soytong Kasem

Abstract:

A study on the efficiency for enzyme production of fungi isolated from stomach of buffalo was conducted. The fungi were collected from 4 parts of stomach as rumen, reticulum, omasum and abomasums. The objective to study the efficiency of fungi from stomach of buffalo had effected to produced enzyme and to selected fungi for their ability to produced enzyme cellulase, hemicellulase and ligninase. Results shown that the fungi isolated from rumen were: Eupenicillium sp. (B-RU-01-1), Eupenicillium sp. (B-RU-02-3G), Rhyzopus stolonifer (B-RU-01-4) and Trichoderma sp. (B-RU-01-2). From the reticulum, Aspergillus glaucus (B-RET-02-3), Aspergillus orchraceus (B-RET-02-2) and Penicillium sp. (B-RET-02-4) were found. In the omasum Aspergillus fumigatus (B-OMA-01-1G), Eurotium sp. (B-OMA-01-4) and Rhizopus stolonifer (B-OMA-02-3) were isolated and in the abomasums Aspergillus flavas (B-ABO-02-3), Aspergillus fumigatus (B-ABO-02-1), Aspergillus niger (B-ABO-01-3G), Aspergillius terreus (B-ABO-02-4) and Mucor sp. (B-ABO-02-4G). Results of enzyme analysis revealed that cellulase was produced by isolated: Eupenicillium sp. (B-RU-02-3G), Eupenicillium sp. (B-RU-01-1), Penicillium sp. (B-RET-02-4), Aspergillius glaucus (B-RET-02-3), Aspergillus ochraceus (B-RET-02-2), Aspergillius fumigatus (B-OMA-01-1G), Eurotium sp. (B-OMA-01-4), Aspergillius flavus (B-ABO-02-3), Aspergillius fumigatus (B-ABO-02-1), Aspergillius niger (B-ABO-01-3G), Aspergillius terreus (B-ABO-02-4). Hemicellulase was produced Eupenicillium sp. (B-RU-02-3G), Eupenicillium sp. (B-RU-01-1), Rhizopus stolonifer (B-RU-01-4), Trichoderma sp. (B-RU-01-2), Aspergillius glaucus (B-RET-02-3), Aspergillus ochraceus (B-RET-02-2), Penicillium sp. (B-RET-02-4), Aspergillius fumigatus (B-OMA-01-1G), Eurotium sp. (B-OMA -01-4), Aspergillius flavus (B-ABO-02-3), Aspergillius fumigatus (B-ABO-02-1) Aspergillius niger (B-ABO-01-3G), Aspergillius terreus (B-ABO-02-4), Mucor sp. (B-ABO-02-4G). For the enzyme ligninase, two isolates were found to produced this enzyme namely : Trichoderma sp. (B-RU-01-2) and Mucor sp. (B-ABO-02-4G).

Keywords: enzyme production from fungi, enzyme production, fungi, agricultural technology

Procedia PDF Downloads 349

Authors: Kashif Ahmed

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Recent years researchers have been motivated towards extensive exploring of living organism, which could be utilized effectively in intense industrial conditions. The present study shows enhanced production, purification and characterization of industrial enzyme, invertase (Beta-D-fructofuranosidase) from Penicillium lilacinum. Various agricultural based by-products (cotton stalk, sunflower waste, rice husk, molasses and date syrup) were used as energy source. The highest amount of enzyme (13.05 Units/mL) was produced when the strain was cultured on growth medium containing date syrup as energy source. Yeast extract was used as nitrogen source after 96 h of incubation at incubation temperature of 40º C. Initial pH of medium was 8.0, inoculum size 6x10⁶ conidia and 200 rev/min agitation rate. The enzyme was also purified (7 folds than crude) and characterized. Molecular mass of purified enzyme (65 kDa) was determined by 10 % SDS-PAGE. Lineweaver-Burk Plot was used to determine Kinetic constants (Vmax 178.6 U/mL/min and Km 2.76 mM). Temperature and pH optima were 55º C and 5.5 respectively. MnCl₂ (52.9 %), MgSO₄ (48.9 %), BaCl₂ (24.6 %), MgCl₂ (9.6 %), CoCl₂ (5.7 %) and NaCl (4.2 %) enhanced the relative activity of enzyme and HgCl₂ (-92.8 %), CuSO₄ (-80.2 %) and CuCl₂ (-76.6 %) were proved inhibitors. The strain was showing enzyme activity even at extreme conditions of temperature (up to 60º C) and pH (up to 9), so it can be used in industries.

Keywords: invertase, Penicillium lilacinum, submerged fermentation, industrial enzyme

Procedia PDF Downloads 111

Authors: Sibashish Baksi, Ujjaini Sarkar, Sudeshna Saha

Abstract:

In the present study, hydrolysis of Pinus roxburghi wood powder was carried out with Viscozyme, and kinetics of the hydrolysis has been investigated. Finely ground sawdust is submerged into 2% aqueous peroxide solution (pH=11.5) and pretreated through autoclaving, probe sonication, and alkaline peroxide pretreatment. Afterward, the pretreated material is subjected to hydrolysis. A chain of experiments was executed with delignified biomass (50 g/l) and varying enzyme concentrations (24.2–60.5 g/l). In the present study, 14.32 g/l of glucose, along with 7.35 g/l of xylose, have been recovered with a viscozyme concentration of 48.8 g/l and the same condition was treated as optimum condition. Additionally, thermal deactivation of viscozyme has been investigated and found to be gradually decreasing with escalated enzyme loading from 48.4 g/l (dissociation constant= 0.05 h⁻¹) to 60.5 g/l (dissociation constant= 0.02 h⁻¹). The hydrolysis reaction is a pseudo first-order reaction, and therefore, the rate of the hydrolysis can be expressed as a fractal-like kinetic equation that communicates between the product concentration and hydrolytic time t. It is seen that the value of rate constant (K) increases from 0.008 to 0.017 with augmented enzyme concentration from 24.2 g/l to 60.5 g/l. Greater value of K is associated with stronger enzyme binding capacity of the substrate mass. However, escalated concentration of supplied enzyme ensures improved interaction with more substrate molecules resulting in an enhanced de-polymerization of the polymeric sugar chains per unit time which eventually modifies the physiochemical structure of biomass. All fractal dimensions are in between 0 and 1. Lower the value of fractal dimension, more easily the biomass get hydrolyzed. It can be seen that with increased enzyme concentration from 24.2 g/l to 48.4 g/l, the values of fractal dimension go down from 0.1 to 0.044. This indicates that the presence of more enzyme molecules can more easily hydrolyze the substrate. However, an increased value has been observed with a further increment of enzyme concentration to 60.5g/l because of diffusional limitation. It is evident that the hydrolysis reaction system is a heterogeneous organization, and the product formation rate depends strongly on the enzyme diffusion resistances caused by the rate-limiting structures of the substrate-enzyme complex. Value of the rate constant increases from 1.061 to 2.610 with escalated enzyme concentration from 24.2 to 48.4 g/l. As the rate constant is proportional to Fick’s diffusion coefficient, it can be assumed that with a higher concentration of enzyme, a larger amount of enzyme mass dM diffuses into the substrate through the surface dF per unit time dt. Therefore, a higher rate constant value is associated with a faster diffusion of enzyme into the substrate. Regression analysis of time curves with various enzyme concentrations shows that diffusion resistant constant increases from 0.3 to 0.51 for the first two enzyme concentrations and again decreases with enzyme concentration of 60.5 g/l. During diffusion in a differential scale, the enzyme also experiences a greater resistance during diffusion of larger dM through dF in dt.

Keywords: viscozyme, glucose, fractal kinetics, thermal deactivation

Procedia PDF Downloads 85

Authors: Shweta Sinha, Mukesh Doble, Manju S. L.

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Pyrazole scaffold is an important group of compound in heterocyclic chemistry and is found to possess numerous uses in chemistry. Pyrazole derivatives are also known to possess important biological activities including antitumor, antimicrobial, antiviral, antifungal, anticancer and anti-inflammatory. Inflammation is associated with pain, allergy and asthma. Leukotrienes are mediators of various inflammatory and allergic disorders. 5-Lipoxygenase (5-LOX) is an important enzyme involved in the biosynthesis of leukotrienes and metabolism of arachidonic acid (AA) and thus targeted for anti-inflammation. In vitro inhibitory activity of pyrazol-3-yl thiazole 4-carboxylic acid derivatives is tested against enzyme 5-LOX. Most of these compounds exhibit good inhibitory activity against this enzyme. Binding mode study of these compounds is determined by computational tool. Further experiments are being done to understand the mechanism of action of these compounds in inhibiting this enzyme. To conclude, these compounds appear to be a promising target in drug design against 5-LOX.

Keywords: inflammation, inhibition, 5-lipoxygenase, pyrazole

Procedia PDF Downloads 211

Authors: Parmjit S. Panesar, Rupinder Kaur, Ram S. Singh

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Enzymes are the biocatalysts which catalyze the biochemical processes and thus have a wide variety of applications in the industrial sector. β-Galactosidase (E.C. 3.2.1.23) also known as lactase, is one of the prime enzymes, which has significant potential in the dairy and food processing industries. It has the capability to catalyze both the hydrolytic reaction for the production of lactose hydrolyzed milk and transgalactosylation reaction for the synthesis of prebiotics such as lactulose and galactooligosaccharides. These prebiotics have various nutritional and technological benefits. Although, the enzyme is naturally present in almonds, peaches, apricots and other variety of fruits and animals, the extraction of enzyme from these sources increases the cost of enzyme. Therefore, focus has been shifted towards the production of low cost enzyme from the microorganisms such as bacteria, yeast and fungi. As compared to yeast and bacteria, fungal β-galactosidase is generally preferred as being extracellular and thermostable in nature. Keeping the above in view, the present study was carried out for the isolation of the β-galactosidase producing fungal strain from the food as well as the agricultural wastes. A total of more than 100 fungal cultures were examined for their potential in enzyme production. All the fungal strains were screened using X-gal and IPTG as inducers in the modified Czapek Dox Agar medium. Among the various isolated fungal strains, the strain exhibiting the highest enzyme activity was chosen for further phenotypic and genotypic characterization. The strain was identified as Rhizomucor pusillus on the basis of 5.8s RNA gene sequencing data.

Keywords: beta-galactosidase, enzyme, fungal, isolation

Procedia PDF Downloads 217