AbstractIn this paper, a Serratia marcescens fibrinolytic protease KD was covalently immobilized onto the electrospun prepared glutaraldehyde (GA)-functionalized chitosan/cellulose acetate membrane nanofibres. Enzyme immobilization has been optimized at some conditions such as different GA values, different crosslinking times, different enzyme and pH values, and different times of immobilization. Results exhibited that the optimized immobilization conditions were obtained in 5.0% GA, after 4 h of crosslinking time, after 8 h immobilization time, using 210 mg protein/g support at pH 9.0. Based on these optimal conditions, the best encapsulation yield (EY) and activity recovery (AR) were obtained about 85% and 121.3%, respectively. The immobilized protease showed a 52% enhancement in protease activity than the free protease in pH 10. Furthermore, results displayed that the Vmax values of free and immobilized enzymes towards casein were gained 0.491 and 0.79 µmol/min, respectively. Moreover, the activity of immobilized protease was retained about 75% after incubation at 60 °C for 180 min at pH 9.0, in which the free protease only preserved about 20% of its primary activity. Results exhibited that the protease-NFs kept nearly 73% of its initial activity after three weeks of storage, while the free protease retained about 20% of its initial activity at the same condition. Results showed that the free protease exhibited 31% clot lysis, whereas the immobilized enzyme exhibited 39% clot lysis. The highest hydrolysis value of both proteases was done 17 and 48% after 4 h at 40 °C, respectively. These results indicated that Chit/CA electrospun nanofibres are excellent membranes for protease immobilization with high application in the digestion of protein waste.

AbstractAn extracellular alkaline protease from Scytalidium thermophilum was produced in a glucose-containing medium supplemented with 5 mM NaCl for 3 days at pH 8.0 and 45 °C. The enzyme was 10-fold purified using ammonium sulfate precipitation followed by ion-exchange chromatography, and its molecular weight was calculated as 80 kDa from SDS-PAGE. The enzyme exhibited optimum activity at pH 8.0 and 60 °C. It was stable at pH and temperature range of 6.0–10.0 and 30–80 °C, respectively. Its half time was 30 h at pH 6.0, 7.0 and 8.0, while those were 22, 16, 8, and 3 h at 50 °C, 60 °C, 70 °C, and 80 °C, respectively. Kinetic parameters including Km (2 ± 0.02 mg/ml), Vmax (18.7 ± 1.5 µmole tyrosine ml−1 min−1), and kcat (2.5 x 103 min−1) were determined using casein. Ca2+ increased the enzyme activity, but it was slightly reduced by EDTA, Triton X-100, Tween 20, and Tween 80. It was active against reducing agents like β-mercaptoethanol but completely inhibited by phenyl methyl sulphonyl fluoride supporting the enzyme belonging to the serine protease family. Chloroform (143%), methanol (138%), and isopropanol (111%) increased the enzyme activity at 5% (v/v), while ethanol (71%) and acetone (81%) moderately reduced the proteolytic activity at the same concentration. Dimethyl sulfoxide (5%, v/v) did not significantly affect the enzyme. The enzyme was compatible with several detergents (1%, w/v), maintaining more than 90% of its original activity in almost all detergents tested. The stability of the enzyme presented against pH, temperature, organic solvents, and detergents indicates its potential use in various industrial applications, especially in peptide synthesis and the laundry industry.

AbstractChitin is an abundant, linear, insoluble β-1, 4-linked N-acetylglucosamine polymer (GlcNAc), found in the cuticles of insects, the cell walls of fungi and crustacean shells. Chitinases are hydrolytic enzymes which catalyze the breakdown of chitin and are produced by a diverse range of microorganisms, plants, mammals and insects. Microbial chitinases are primarily in charge of breaking down chitin and are essential for maintaining an ecosystem’s balance of carbon and nitrogen. They are gaining ground in the fields of medicine, agriculture, food, pharmaceuticals and environmental management due to their vast range of applications. In contrast to chemical pesticides, they have shown their ability to act as biocontrol agents against a variety of pathogenic fungus and insect pests. Considering the essential role of chitinases as biopesticides and in a variety of industrial and medicinal applications, this review focusses on the basic structure of chitin and chitinases, different classes of chitinases, their sources, purification, characterization and latest developments in implementation of chitinases in various fields such as biomedical, agricultural, environmental, etc.

AbstractRapamycin is a macrolide antibiotic with antifungal, immunosuppressive, antitumor, and lifespan-extension activities etc. In order to increase its structural diversity, microbial transformation of rapamycin was conducted. More than 3 converted compounds were shown in the microbial conversion culture of rapamycin by Bacillus subtilis CGMCC7764. By extensive NMR and MS spectroscopic analyses, the major compound was identified as 43-O-(β-D-glucoside)-rapamycin. This compound has an inhibitory effect on tumour growth, and its activity is similar to rapamycin.

AbstractBiocatalysts can accelerate the catalysis of a chemical reaction that is difficult to synthesize with typical chemical methods. The global enzyme market size is predicted to expand at a CAGR of 6.5% from 2021 to 2028. Enzymatic reactions are highly chemo, regio, and stereoselective and produce various fine chemicals such as drugs, agrochemicals, and fragrance molecules. Peroxidases (PO) (EC 1.11.1.x) are a large class of enzymes that play an important role in various biological processes. Chloroperoxidase (CPO, EC 1.1.1.10) is a versatile fungal haem-thiolate protein that is useful in the asymmetric synthesis of chiral building blocks and has an important role in a number of biological processes. CPO’s main biological role is chlorination, although it also catalyses haem PO, catalase (CAT), and reactions similar to cytochrome P450. However, CPO performs both oxidation and stereo-specific halogenation of chemical molecules. The haem and vanadium POs are produced by Caldariomyces fumago, and Curvularia inaequalis, respectively, and are capable of halogenating the flavanones, naringenin, and hesperetin, at C-6 and C-8 in the presence of either Cl− or Br−. In this review, we discussed the various applications of CPO including synthesis of epoxides, drugs, halogenation of thymol, nitriles, the Aza-Achmatowicz reaction, and biomedical applications such as cancer and biosensors. In light of these novel features, we have provided a detailed review of CPOs and their applications in various stereoselective chemical transformations of industrial relevance.

AbstractCarbohydrates and their analogues play a unique role in all living organisms and therefore have played a supremacy role in molecular recognition, energy supply, drug discovery, etc. During the last few decades, biocatalyst lipases have emerged as one of the greener and sustainable catalysts in comparison to traditional synthetic catalysts for the synthesis of modified carbohydrates and their analogues. Because lipase is a natural catalyst, it shows outstanding selectivity, reactivity, amazing tolerance, and assistance in carrying out eco-friendly greener methodology. The application of biocatalyst lipase as a chemo- and regio-selective catalyst is particularly relevant for organic chemists for the synthesis of modified carbohydrates, because carbohydrates contain several identical hydroxyl groups. Herein, we discussed the recent developments in the lipase Novozyme-435 mediated synthesis of modified carbohydrates and their biological significance.

AbstractSebacic esters have excellent lubricity, thermal stability, and biodegradability and therefore are widely used as aerospace lubricants, metal working oil, or engine oil. They are mainly produced by chemical synthesis which, however, may cause environmental pollution, its enzymatic synthesis represents a more environmentally friendly alternative. A few reports have described the synthesis of sebacic esters employing immobilised lipases, but these biocatalytic reactions were exclusively carried out in solvent-free systems and thus, could have been limited by slow reaction rates and high reaction temperatures due to poor enzyme dispersion, low substrate solubility, and high viscosity of the reaction mixture. The current study investigated the biosynthesis of dioctyl sebacate in toluene by Novozym 435, a commercial immobilised lipase. The reaction parameters were investigated using the single factor approach and an orthogonal array design. The optimal conditions obtained were as follows: 10 mL toluene, sebacic acid,1 mmol (202.25 mg); molar ratio of sebacic acid to 1-octanol, 1:3; Novozym 435, 0.03 g; 4 Å molecular sieves, 1.5 g; reaction temperature, 40 °C; reaction time, 30 h. A dioctyl sebacate conversion rate of 93% was achieved under these optimal conditions. In particular, the addition of molecular sieves to the reaction mixture markedly improved the product yield. The reaction temperature was low enough to make the operation easy and energy-efficient and therefore, well suited for large-scale production.

AbstractMicrobial biotransformation is an efficient process for making high-value natural products in a controlled and environment friendly manner. Bioconversion of low value monoterpenes to high value products using fungi offers an effective strategy for producing desirable products. Efficient conversion of D-limonene, a monoterpene, into carveol is reported in this study by using an endophytic fungus, Aspergillus flavus IIIMF4012, isolated from the root of Rosa damascena. By optimizing different parameters, isolated strain resulted in the hydroxylation of D-limonene to carveol effectively and the process was carried out at 1 L scale which resulted in the formation of a mixture of carveol isomers (cis- carveol 23.82%, trans- carveol 17.21%). Further, the biotransformation was scaled up in 5 L fermenter and the product was characterized by GC-MS and NMR (1H & 13C). As a result, an efficient procedure was established for biotransforming a low-value molecule i.e., D-limonene into a high-value product i.e.,carveol.

AbstractChalcones and flavanones are both present as secondary metabolites in edible and medicinal plants as isomers. Mostly of them are isolated in low yields from natural sources, along with the difficulties faced by the total synthesis, make them attractive for biotechnological studies in order to obtain new derivatives. Prompted by the enormous pharmacological interest in chalcones and flavanones compounds, their alimentary and chemical applications, this review covers their biotransformation as an alternative way for achieving chemical analogues with high selectivity, using mild and eco-friendly conditions. Stereoselective bioreductions of chalcones were the most reported biotransformation reactions, and they could be performed by bacteria, cyanobacteria, non-conventional yeasts (NCYs), and fungi strains with good yields. Flavanones have also been derivatized under stereochemical control by several microorganisms. All reported biotransformations contribute to increasing the availability of new and valuable leads for the market and industrial fields.

AbstractHerein, we report the use of whole cells of Brazilian marine-derived fungi in the biotransformation and biodegradation of organic compounds, particularly, Knoevenagel adducts. A preliminary screening with five marine-derived fungi revealed that these microorganisms promoted the bioreduction and biodegradation of four Knoevenagel adducts. Additionally, a biotransformation pathway for these compounds was proposed, which suggested the presence of ene-reductase and alcohol dehydrogenase in the marine-derived fungi, which catalysed the microbial transformation of the substrates. In summary, the fast biodegradation of organic molecules has an ecological relevance since several of these compounds and their biotransformed products have aquatic environment impact.

AbstractAlong with the cellulase enzyme, xylanase plays an efficient role in the production of biofuel from agricultural wastes by degrading the xylan sugar present in the hemicellulose of cell wall. This study aims to improve the sugar production from biomass by the use of different enzymes with surfactant. The objective of this study is to compare sugar production and bioethanol production from sodium hydroxide along polyethylene glycol pre-treated paddy straw (Oryza sativa L.) with different combination of xylanase and cellulose enzymes along with lignin-degrading laccase enzyme. In results, 10.87 g/l of ethanol with saccharification of 64.51% ± 0.90 was obtained when xylanase and laccase were used, while 18.40 ± 0.56 g/l of ethanol with saccharification of 84.01%±1.09 was obtained when cellulase and laccase enzymes were used. Maximum bioethanol production was found to be 19.20 ± 0.26g/l, which was obtained by combination of xylanase, cellulase and laccase enzymes together at 37 °C after 36 h.

Abstract5-Hydroxymethylfurfural oxidase (HMFO) has demonstrated to be a useful biocatalyst for the selective oxidation of alcohols employing oxygen as mild oxidant with no requirement of expensive organic cofactors. This wild-type HMFO biocatalyst and an engineered thermostable variant have been tested in the kinetic resolution of different benzofused alcohols. The use of natural deep eutectic solvents was also explored in HMFO-catalysed oxidation of alcohols. The oxidation of racemic 1-indanol showed a higher conversion and selectivity in presence of 60% v/v of different NADES, especially for those containing carbohydrates. By choosing properly the biocatalyst and the NADES, good enantioselectivity values can be obtained, demonstrating the advantages of employing these neoteric solvents in biocatalysed processes.

AbstractThe area of combining an enzyme and transition metals to catalyse the sequence of reactions in one-pot which improve total conversion and operational stability has seen tremendous growth in recent years. Herein, we describe the synthesis and substrate controlled modification of β-aminocarbonyl using α-amylase enzyme and Pd-based catalyst in the one-pot. Moreover, the chemo-enzymatic approach provides substituted indole derivatives unprecedentedly when isocyanide was used in the reaction. In the second phase, isocyanide was replaced by phenyl boronic acid which in turn provides the substituted amino biaryls in good yield. Next, the feasibility of one-pot chemo-enzymatic approach was proved by employing substituted 2-bromo aniline along with different isocyanide/phenyl boronic acid and obtained the corresponding products in 56–84% isolated yield.

AbstractEnantiomerically pure secondary alcohols are useful in the synthesis of several natural products and as active pharmaceutical intermediates (API). Due to the high demand for these chiral compounds, much progress has been made in the areas of asymmetric synthesis and catalysis. In this context, biocatalysis together with continuous flow technology can be a valuable tool for more versatile and sustainable methods, with lower cost, greater stereoselectivity and less environmental impact. This work aims to obtain an enantiomerically pure alcohol of industrial interest, (4 Fluorophenyl) (furan-2-yl) methanol (3), by performing a kinetic resolution using immobilized Candida antarctica lipase B (Novozyme 435, N435) under continuous-flow conditions. Initial study was carried out to optimize batch reaction conditions. The best results were obtained using isooctane as solvent, 37.7 mg of N435 and three equivalents of isopropenyl acetate as acyl donor at 60 °C for 24 h. Under these conditions, a conversion of 49% and 91 of enantiomeric ratio was obtained. Optimized batch conditions were translated to the continuous flow reactor leading to the desired product in 30 min of residence time, 47% conversion and an enantiomeric ratio of 61.

AbstractTo augment the keratinolytic ability of Bacillus halotolerans L2EN1 isolated from the poultry farm (Nahan, District Sirmour) of Himachal Pradesh, different cultural conditions were optimised using One Variable at a Time (OVAT) approach accompanied by Response Surface Methodology (RSM). Optimisation (OVAT) results revealed that after 3rd day of incubation, maximum enzyme activity was attained at 45 °C, pH 11.0 with 12.5% of inoculum size in the presence of Mn2 + and EDTA in the production medium. Sucrose (1.5%) and yeast extract (2.0%) were observed to be best carbon and nitrogen sources, respectively. A significant increase of 73.19 per cent in the keratinase activity was observed using Central Composite Design (CCD) of RSM. The SDS-PAGE results revealed that crude keratinase is a heterotetramer made up of four polypeptide chains with molecular weights of 17, 37, 40 and 60 kDa. Partial purification by 90 − 100 percent ammonium sulphate gave maximum keratinase production of 22.66 U/mL with purification of 1.68 and yield of 11.47 per cent. The enzyme showed compatibility with different commercial detergents and retained its activity in the order: Reshma (97.77%) > Speed (93.44%) > Tide (79.93%) >Ariel (70.18%) > Surf excel (67.98%) at 50 °C after 1 h of incubation. Wash performance analysis demonstrated that washing with tap water at 18, 28, 35 and 45 °C for 30, 45 and 60 min removed some amount of blood stains from the cotton cloth pieces. However, replacement of detergent’s enzyme (Reshma) with crude keratinase achieved complete blood stain removal under same conditions, suggesting its suitability as a potential cleaning additive in detergents for the removal of blood (proteinaceous) stains for long washing cycles (1 h).

AbstractNatural deep eutectic solvents (NADESs) were investigated for the activity and stability of the laccase enzyme. Laccase was found to be active only in a low amount of choline chloride containing NADESs while it maintained its activity both in high and low amounts of betaine containing NADESs. Rutin, as a flavonoid monomer shows high antioxidant, antibacterial, antiviral and anti-inflammatory properties and these properties are enhanced in the polymerised form. In this study, the organic co-solvent that is conventionally used in the oligomerization of rutin was replaced with two different green solvents, choline chloride-ethylene glycol (1:2 molar ratio) and betaine-mannose (5:2 molar ratio). LC-MS results revealed spontaneous derivatization of rutin as well as the oligomerization of the derivatives besides rutin. The final products were found to have enhanced superoxide radical activity. In this study, enzymatic oligomerization of rutin was investigated for the first time in the presence of eco-friendly green solvents presenting an alternative pathway without any toxic components.

AbstractFast depleting fossil fuel and its associated negative impacts on environment and climate change raising concern over clean fuel and sustainability. Thus, in line scientific community are thinking to have alternative fuels like hydrogen, natural gas, syngas, and biofuels. Furthermore, hydrogen gas is superior among alternative fuels due to its renewable nature, zero emissions, and generate large content of energy during combustion. Researcher cites on various biological methods like bio photolysis of water, dark fermentation, photo-fermentation, and microbial electrolysis cell. However, biohydrogen production from wastewater as substrate is becoming popular due to less energy intensive and sustainable way to fulfil the future energy demands. With rapid increase in industrialization and urbanization will further increase wastewater generation globally. Hence, wastewaters could have huge potential for the green hydrogen production which ultimately provides clean energy, and the low-cost wastewater treatment. Literature reveals that wastewater from various industries like citric acid, cheese whey, paper mill, rice mill, beverage, cassava starch processing, palm oil, starch processing, pharmaceutical, food processing, distillery, and sugar industry have been utilized for biohydrogen production. Hence, the present review is focused on various wastewaters based green hydrogen production which have overall positive impacts on social, economic, and environment for the future generation.

AbstractA substantial reduction in the cost of biodiesel production necessitates the identification of less expensive lipid-bearing substrates and a cost-effective process. The present study demonstrates the use of biomass suspension of Aspergillus sps. as a whole-cell catalyst for the generation of various alkyl esters from acid oil and cottonseed oil with different alcohols (methanol to decanol) as acyl acceptors. The yield of alkyl esters increased from methanol (79%) to pentanol (87%), followed by a decrease from hexanol (80%) to decanol (55%) in the case of acid oil. The extent of transesterification was significantly higher [P < 0.05] in case of acid oil, with most of the acyl acceptors as compared to cottonseed oil. The study reveals the potential use of biomass suspension of fungus as a catalyst and acid oil as an alternative, low-cost bearing, and quality feedstock for the generation of biodiesel for a diverse variety of industrial/commercial use.

AbstractMelanins are a complex natural pigment that darken the skin, a phenomenon regarded as an esthetic problem in tropical countries such as Indonesia. Melanin can be degraded by various oxidative enzymes such as laccase (Lac) and manganese peroxidase (MnP) which are produced by white rot fungi (WRF). In the current study, we successfully obtained a partial purified Lac-MnP from a tropical WRF called Trametes hirsuta OK271075. Co-catalysis of Lac and MnP in the melanin degradation existed as indicated by the enhancement of degradation upon the addition of mediators for the respective enzymes. The highest degradation was up to 42% in the presence of TEMPO (0.2 mM) and MnSO4-H2O2 (0.75 mM, 0.2 mM) as mediators. The increment was up to 20% compared to treatment without mediators. A higher degradation rate achieved when specific mediators of the enzymes were utilized indicated a synergistic effect of both enzymes that could be used simultaneously for melanin degradation. FTIR and FESEM analysis revealed significant changes in melanin structure between enzyme-treated and untreated melanin. Melanin granules were cracked and exfoliated due to an enzyme attack. Based on Pyrolysis-GCMS analysis, several compounds such as phenols, epoxides, ketones, and carboxylic acids were found in higher amount in the enzyme-treated than untreated melanin. This finding revealed the potency of a new isolated T. hirsuta for new enzyme applications in biotechnology fields, especially to degrade melanin for application in natural whitening cosmetics.

AbstractThis study aimed to establish a novel method for catalysing salidroside synthesis by immobilized β-D-glucosidase with deep eutectic solvents (DESs) as a new green non-aqueous medium. To this end, the catalytic properties of β-D-glucosidase (i.e. activity, stability, and enzymatic properties) were analysed within sixteen different DESs systems. Although most DESs significantly improved enzyme stability, they also reduced enzymatic activity. This was verified at the molecular level by employing fluorescence spectroscopy to evaluate conformational changes in β-D-glucosidase molecules. Subsequently, chitosan microspheres were used to immobilize β-D-glucosidase; the structures of the microspheres and immobilized enzyme were observed by scanning electron microscopy (SEM). Through single factor experiments and application of a response surface design, the optimal conditions for production of salidroside by the reaction of tyrosol with immobilized β-D-glucosidase were established. These conditions comprised 80 vol% ChCl/G (1:2), 50 °C reaction temperature, 5.8 pH, 100 h reaction time, 45 U/mL enzyme dosage, and a tyrosol/D-glucose molar ratio of 10. Under these conditions, the average conversion rate of the tyrosol substrate was 31.6%. Moreover, a salidroside crude product was obtained with a purity >70% (g/g) and a substrate conversion rate of immobilized β-D-glucosidase >50% of its initial value after five repeated uses. Collectively, this study establishes a novel green reaction system for the efficient preparation of salidroside and other glycoside compounds, while also providing a theoretical foundation for the analysis of β-D-glucosidase catalytic reactions in DESs systems.