A review on recent upgradation and strategies to enhance cyclodextrin glucanotransferase properties for its applications.

Cyclodextrin glycosyltransferase (CGTase) is a significant extracellular enzyme with diverse functions. CGTase is widely used in production of cyclic α-(1,4)-linked oligosaccharides (cyclodextrins) from starch via transglycosylation reaction. Recent discoveries of novel CGTases from different microorganisms have expanded its applications but natural CGTase have lower yield, leading to heterologous expression for increased production to meet various needs. Moreover, significant advancements in directed evolution approach have been explored to alter the molecular structure of CGTase to enhance its performance. This review comprehensively summarizes the strategies employed in heterologous expression to boost CGTase production and secretion in various host. It also outlines molecular engineering approaches aimed to improving CGTase properties, including product and substrate specificity, catalytic efficiency, and thermal stability. Additionally, a considerable stability against changes in temperature and organic solvents can be obtained by immobilization.

Understanding the Fermentation Potentiality For Gibberellic Acid (GA3) Production Using Fungi.

Gibberellins represent an important group of potent phytohormones, growth-promoting, closely related diterpenoid acids biologically derived from tetracyclic diterpenoid hydrocarbon. Among these, gibberellic acid (GA3) has received the greatest attention. GA3 is a highly valued plant growth regulator which has various applications in agriculture. It is extensively used for beneficial effects including stem elongation, elimination of dormancy, sex expression, seed germination, flowering, and fruit senescence. Along with plants, many microbes are also producing GA3 as their secondary metabolite, and among these, fungi are reported to produce a higher amount of GA3. Fermentation technology based on submerged fermentation and solid-state fermentation for the production of GA3 has been used with its merits and demerits using Fusarium moniliforme fungus in the industry. Several mathematical models and optimization tools were also designed for enhancing the fermentative yield by researchers. The detailed analysis is essential to understand all the fermentation aspects, various unit parameters, process operation approaches, reduction in cost, and assessment of the possible uses of these models in the production of GA3 for higher yield. Recently, exclusive research is executed to lower down the production cost of GA3 approaching various strategies.

Cyclodextrin glucanotransferase: fundamentals and biotechnological implications.

Cyclodextrin glucanotransferase (CGTase) is an extracellular enzyme of the GH13 α-amylase family that catalyzes a unique intramolecular reaction known as cyclization to transform α-1, 4-glucans and similar starches into cyclodextrins. They also catalyze intermolecular transglycosylation reactions namely coupling, disproportionation, and some hydrolyzing effects on starch. The monomeric structures of the CGTase exhibit five domains (A, B, C, D, and E domains) with different molecular weights and amino acid sequences depending on the source. Among bacteria, Bacillus genus covers approximately 90% of the CGTase producers, while other genera like Klebsiella, Paenibacillus, and Thermoanaerobacter also shown decent contributions in recent studies. CGTase production is highly supported by alkaliphilic bacteria under submerged fermentation rather than solid-state fermentation. The bacterial sources, biochemical properties, production conditions, and structure of CGTases are compiled in this review. Cyclodextrins have the unique property of making inclusion complexes with various compounds, hence widely used in the food, pharmaceutical, cosmetics, laundry, and chemical sectors. This review presents a comprehensive view of CGTase produced by Bacillus spp., and other bacterial genera like Klebsiella, Paenibacillus, and Microbacterium. It also gives insight of the properties and recent biotechnological applications of cyclodextrins. KEY POINTS: • Transglycosylation reactions catalyzed by CGTase and their structural properties. • Comparative data of CGTase production by various genera and Bacillus spp. • Structures, properties, and applications of different cyclodextrins.

Assessment of gut microbial ß-glucuronidase and ß-glucosidase activity in women with polycystic ovary syndrome.

PCOS is a prevalent endocrine disorder among women of reproductive age, characterized by hormonal imbalances and metabolic disturbances. This study explores the correlation between gut microbial ß-glucuronidase and ß-glucosidase and PCOS, focusing on their association with hormone levels and other clinical parameters. In this case-control study, fecal samples were collected from women of reproductive age, both with and without PCOS. The analysis of gut ß-glucuronidase and ß-glucosidase enzymes was conducted with the other clinical parameters, including body mass index, hormone levels, and hirsutism. These factors were then subjected to correlation analysis. PCOS women showed significantly higher levels of ß-glucuronidase activity with a statistically significant P-value (0.05 ± 0.1 vs. 0.04 ± 0.1; P = 0.006) as well as ß-glucosidase activity (0.13 ± 0.08 vs. 0.09 ± 0.05; P = 0.06) compared to the controls. This study also revealed intriguing connections between the selected enzymes and hormone levels, particularly testosterone and estradiol. Gut microbial enzymes ß-glucuronidase and ß-glucosidase may be used as biomarkers for early detection and monitoring of PCOS in women with metabolic challenges. It could lead to improved diagnostic tools and treatment options.

Plant growth promoting activities and effect of fermented panchagavya isolate Klebsiella sp. PG-64 on Vigna radiata.

A natural bacterial isolate from fermented panchagavya named as PG-64, exhibits multiple plant growth-promoting traits. This Gram-negative bacteria was identified as Klebsiella sp. PG-64 by 16S rRNA gene sequencing. The Klebsiella sp. PG-64 has shown production of indole acetic acid (106.0 µg/ml), gibberellic acid (20.0 µg/ml), ammonia (7.12 µmol/ml), exopolysaccharide (2.04% w/v) and phosphate solubilization (106.0 µg/ml). It produced 437 µg/ml IAA with 0.75% (w/v) L-tryptophan supplementation and was increased to 575 µg/ml in a laboratory-scale fermenter. The PG-64 has shown tolerance to abiotic stress conditions like pH (5.0-12.0), temperature (28-46 °C), salt (0.5-10.0% w/v NaCl) and osmotic resistance (1-10% w/v PEG-6000). The PG-64 also produced 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase (0.3 ng α-ketobutyrate/mg protein/h) indicating its potential for drought tolerance. Owing to its diverse properties, the effect of Klebsiella sp. PG-64 on Vigna radiata (Mung bean) was examined. The seeds treated with PG-64 culture showed 92% germination with a good seedling vigour index (202). In the pot study, Vigna radiata growth showed 2.23, 1.55, 2.00, 1.65, 1.73, 1.88, 5.00, 5.00, 1.57 times increase in primary root length, dry root weight, root hair numbers, leaf width, leaf numbers, leaf area, fruits number, flower number and chlorophyll content, respectively after 75 days. The application of Klebsiella sp. PG-64 culture resulted in substantial growth enhancement of Vigna radiata. The Klebsiella sp. PG-64 has multiple plant growth-promoting properties along with capabilities to tolerate abiotic stresses, making it a promising liquid biofertilizer contender for various crops.

Exopolysaccharides from marine microbes with prowess for environment cleanup.

A variety of both small and large biologically intriguing compounds can be found abundantly in the marine environment. Researchers are particularly interested in marine bacteria because they can produce classes of bioactive secondary metabolites that are structurally diverse. The main secondary metabolites produced by marine bacteria are regarded as steroids, alkaloids, peptides, terpenoids, biopolymers, and polyketides. The global urbanization leads to the increased use of organic pollutants that are both persistent and toxic for humans, other life forms and tend to biomagnified in environment. The issue can be addressed, by using marine microbial biopolymers with ability for increased bioremediation. Amongst biopolymers, the exopolysaccharides (EPS) are the most prominent under adverse environmental stress conditions. The present review emphasizes the use of EPS as a bio-flocculent for wastewater treatment, as an adsorbent for the removal of textile dye and heavy metals from industrial effluents. The biofilm-forming ability of EPS helps with soil reclamation and reduces soil erosion. EPS are an obvious choice being environmentally friendly and cost-effective in processes for developing sustainable technology. However, a better understanding of EPS biosynthetic pathways and further developing novel sustainable technologies is desirable and certainly will pave the way for efficient usage of EPS for environment cleanup.

Microbial surfactants: A journey from fundamentals to recent advances.

Microbial surfactants are amphiphilic surface-active substances aid to reduce surface and interfacial tensions by accumulating between two fluid phases. They can be generically classified as low or high molecular weight biosurfactants based on their molecular weight, whilst overall chemical makeup determines whether they are neutral or anionic molecules. They demonstrate a variety of fundamental characteristics, including the lowering of surface tension, emulsification, adsorption, micelle formation, etc. Microbial genera like Bacillus spp., Pseudomonas spp., Candida spp., and Pseudozyma spp. are studied extensively for their production. The type of biosurfactant produced is reliant on the substrate utilized and the pathway pursued by the generating microorganisms. Some advantages of biosurfactants over synthetic surfactants comprise biodegradability, low toxicity, bioavailability, specificity of action, structural diversity, and effectiveness in harsh environments. Biosurfactants are physiologically crucial molecules for producing microorganisms which help the cells to grasp substrates in adverse conditions and also have antimicrobial, anti-adhesive, and antioxidant properties. Biosurfactants are in high demand as a potential product in industries like petroleum, cosmetics, detergents, agriculture, medicine, and food due to their beneficial properties. Biosurfactants are the significant natural biodegradable substances employed to replace the chemical surfactants on a global scale in order to make a cleaner and more sustainable environment.

Valorization of agro-food wastes: Ease of concomitant-enzymes production with application in food and biofuel industries.

The novel and greener approach toward the co-production of hydrolytic enzymes in a single-cultivation medium with inexpensive substrates can bring down the production costs. Likewise, the natural and industrial organic biomass/solid are all nutritionally rich substrates waiting for free use in industries such as food, biofuel, etc. Valorization must broaden its applications in industries and households with a step towards a sustainable environment. The biofuel approach can be projected as one of the most promising deputations to meet future energy demands, in reduction of the environmental pollution due to excessive fossil fuel consumption. The present review highlights the multifaceted stature of microbial enzymes in this direction and possible implications mainly in the food industry and biofuel with the global impact of similar bio-based industries. In this review, design scale-up, fermentation cost, energy needs,and agro-food waste management have been meticulously delineated.

Plant chitinases and their role in plant defense: A comprehensive review.

Chitinases are present in diverse form of organisms from bacteria, fungi, insects, plants. Plant chitinases are part of pathogenesis-related proteins. When plant (host) cells are under pathogen stress, plant chitinases are strongly expressed and hence plant chitinases play a critical part against fungal pathogens. Chitinases are also found to be involved in various abiotic stress responses like wounding, osmotic pressure, cold, heavy metal stress, salt in plants. Understanding of the plant chitinases will provide an insight for improving the pathogenic activity of various potential biocontrol strains and to develop novel pathogen resistant strategies for exploring their roles with regards to plant defense. The present review covers the detailed account of potential and relevance of plant chitinases for controlling pathogens infection in plant and prospecting to improve plant defense, growth and yield.

Statistical optimization of medium components for biosurfactant production by Pseudomonas guguanensis D30.

Biosurfactant production by Pseudomonas guguanensis D30 was reported using mineral oil in submerged condition. Twelve medium components were tested at two levels by Plackett-Burman design, among them, mineral oil, yeast extract, peptone, MgSO4, and CaCl2 found significant on the basis of emulsification index. These five significant components were further optimized through central composite design (CCD). The experimental design was successfully used for regression analysis and the significant model suggested the solution of 10% (v/v) mineral oil, 3.0 g/L (w/v) yeast extract and 0.2 g/L (w/v) peptone for 13.14 g/L predicted biosurfactant production. We kept the suggested concentrations of medium components and got 13.34 ± 0.08 g/L biosurfactant production, which is almost double the conventional one-factor-at-a-time production (7.126 ± 0.12 g/L). It reduced the surface tension of the medium up to 28 ± 1.2 mN/m. We found ethyl acetate a suitable solvent for biosurfactant extraction amongst methanol, chloroform, and methanol:chloroform. The partially purified biosurfactant was chemically characterized as lipopeptide by Fourier transform infrared spectroscopy (FT-IR).

Understanding the Basis of Occurrence, Biosynthesis, and Implications of Thermostable Alkaline Proteases.

The group of hydrolytic enzymes synonymously known as proteases is predominantly most favored for the class of industrial enzymes. The present work focuses on the thermostable nature of these proteolytic enzymes that occur naturally among mesophilic and thermophilic microbes. The broad thermo-active feature (40-80 °C), ease of cultivation, maintenance, and bulk production are the key features associated with these enzymes. Detailing of contemporary production technologies, and controllable operational parameters including the purification strategies, are the key features that justify their industrial dominance as biocatalysts. In addition, the rigorous research inputs by protein engineering and enzyme immobilization studies add up to the thermo-catalytic features and application capabilities of these enzymes. The work summarizes key features of microbial proteases that make them numero-uno for laundry, biomaterials, waste management, food and feed, tannery, and medical as well as pharmaceutical industries. The quest for novel and/or designed and engineered thermostable protease from unexplored sources is highly stimulating and will address the ever-increasing industrial demands.

Bio-prospecting the future in perspective of amidohydrolase L-glutaminase from marine habitats.

In the current scenario, considerable attention is being given to the enzyme L-glutaminase (EC 3.5.1.2). It belongs to the amidohydrolase class adherent to the family of serine-reliant ß-lactamases and the penicillin-binding proteins due to its higher affinity to polymerize and modify peptidoglycan synthesis. However, based on the catalytic proficiency, L-glutaminase is characterized as a proteolytic endopeptidase that cleaves peptide linkage and emancipates various byproducts, viz. ammonia along with glutamate. L-glutamine is considered the key amino acid reportedly involved in multiple metabolic pathways such as nitrogen metabolism. The present review is focused on the recent development and aspects concomitant to the biotechnological applicability of L-glutaminase predominantly from the marine habitat. Additionally, a majority of L-glutaminases finds application in cancer therapy as therapeutic agents, especially for acute lymphocytic leukaemia. The in vitro studies have been effective against various human cancer cell lines. L-glutaminase enhances the growth of probiotic bacteria. Apart from all these applications, it is suitably applicable in fermented foods as a flavour enhancer especially the umami flavour and content. Marine habitats have largely been exploited for their bio-catalytic potential but very scarcely for therapeutic enzymes. Some of the reports of such marine bacterial isolates from Bacillus sp., Pseudomonas sp. and Vibrio sp. are in the domain, but none highlights the therapeutic applications predominantly as anticancer and anti-proliferative agents. KEY POINTS: The exploration of marine habitats along the Gujarat coasts mainly for bacteria secreting L-glutaminase is scarcely reported, and even more scarce are the amidohydrolases from these marine niches as compared to their terrestrial counterparts. Microbial sourced amidohydrolase has wide bio-applicability that includes food, cosmetics and therapeutics especially as anticancer/anti-proliferative agent making it of immense biotechnological significance.

ß-Cyclodextrin Production by Cyclodextrin Glucanotransferase from an Alkaliphile Microbacterium terrae KNR 9 Using Different Starch Substrates.

Cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) is an important member of α-amylase family which can degrade the starch and produce cyclodextrins (CDs) as a result of intramolecular transglycosylation (cyclization). ß-Cyclodextrin production was carried out using the purified CGTase enzyme from an alkaliphile Microbacterium terrae KNR 9 with different starches in raw as well as gelatinized form. Cyclodextrin production was confirmed using thin layer chromatography. Six different starch substrates, namely, soluble starch, potato starch, sago starch, corn starch, corn flour, and rice flour, were tested for CD production. Raw potato starch granules were found to be the best substrate giving 13.46 gm/L of cyclodextrins after 1 h of incubation at 60°C. Raw sago starch gave 12.96 gm/L of cyclodextrins as the second best substrate. To achieve the maximum cyclodextrin production, statistical optimization using Central Composite Design (CCD) was carried out with three parameters, namely, potato starch concentration, CGTase enzyme concentration, and incubation temperature. Cyclodextrin production of 28.22 (gm/L) was achieved with the optimized parameters suggested by the model which are CGTase 4.8 U/L, starch 150 gm/L, and temperature 55.6°C. The suggested optimized conditions showed about 15% increase in ß-cyclodextrin production (28.22 gm/L) at 55.6°C as compared to 24.48 gm/L at 60°C. The degradation of raw potato starch granules by purified CGTase was also confirmed by microscopic observations.

Screening and Selection of Medium Components for Cyclodextrin Glucanotransferase Production by New Alkaliphile Microbacterium terrae KNR 9 Using Plackett-Burman Design.

Cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) production using new alkaliphile Microbacterium terrae KNR 9 was investigated by submerged fermentation. Statistical screening for components belonging to different categories, namely, soluble and raw starches as carbon sources, complex organic and inorganic nitrogen sources, minerals, a buffering agent, and a surfactant, has been carried out for CGTase production using Plackett-Burman factorial design. To screen out k (19), number of variables, k + 1 (20), number of experiments, were performed. Among the fourteen components screened, four components, namely, soluble starch, corn flour, yeast extract, and K2HPO4, were identified as significant with reference to their concentration effect and corresponding p value. Although soluble starch showed highest significance, comparable significance was also observed with corn flour and hence it was selected as a sole carbon source along with yeast extract and K2HPO4 for further media optimization studies. Using screened components, CGTase production was increased to 45% and 87% at shake flask level and laboratory scale fermenter, respectively, as compared to basal media.

A novel cyclodextrin glucanotransferase from an alkaliphile Microbacterium terrae KNR 9: purification and properties.

Cyclodextrin glucanotransferase (CGTase, EC. 2.1.1.19) produced using new alkaliphile Microbacterium terrae KNR 9 has been purified to homogeneity in a single step by the starch adsorption method. The specific activity of the purified CGTase was 45 U/mg compared to crude 0.9 U/mg. This resulted in a 50-fold purification of the enzyme with 33 % yield. The molecular weight of the purified enzyme was found to be 27.72 kDa as determined by SDS-PAGE. Non-denaturing gel electrophoresis and activity staining confirmed the presence of CGTase in crude and the ammonium sulfate precipitate fraction. The purified CGTase has a pI value of 4.2. The optimum pH of 6.0 and 60 °C temperature were found to be the best for CGTase activity. Purified CGTase showed 5.18 kcal/mol activation energy (Ea). The CGTase activity was increased in the presence of metal ions (5 mM): Ca+2 (130 %), Mg+2 (123 %), Mn+2 (119 %) and Co+2 (116 %). The enzyme activity was strongly inhibited in the presence of Hg+2 (0.0 %), Cu+2 (0.0 %) and Fe+2 (3.8 %). Inhibitor N-bromosuccinimide (5 mM) showed the highest 96 % inhibition of CGTase activity. SDS and triton X-100 among different detergents and surfactants (1.0 %, w/v) tested showed 92 % inhibition. Among the organic solvents checked for their effect on enzyme activity, 5 % (v/v) toluene resulted in 48 % increased activity. Polyethylene glycol-6000 showed a 26 % increase in the CGTase activity. The kinetic parameters K m and V max were 10 mg/ml and 146 µmol/mg min, respectively, for purified CGTase.