Enzyme Production Research Articles

Production and Optimization of Fungal Silicase Using Rice Straw under Solid State Fermentation

Aim: The present study aimed to convert agricultural residue, specifically rice straw, into a valuable enzymatic product through solid-state fermentation (SSF), using three fungal strains—Penicillium limosum (WF1), Bipolaris sorokiniana (BF1), and Pleurotus ostreatus (P1). The objective was to optimize the production of the silica-degrading enzyme silicase, which catalyzes the transformation of silica into plant-available silicic acid. Study Area: Rice straw used in this study was sourced from agricultural fields in Haryana, India. The experimental work was carried out at the Department of Biotechnology, Kurukshetra University, Kurukshetra. Study Design and Methodology: A one-variable-at-a-time (OVAT) strategy was applied to optimize process variables such as substrate form (crude vs. powdered), incubation period, temperature, pH, rice straw-to-water ratio, and the use of nutrient additives. Silicase production was compared between submerged fermentation (SmF) and solid-state fermentation (SSF). Under SSF, the optimized medium consisted of crude rice straw with a 1:5 straw-to-water ratio, incubated at 30 °C and pH 7 for 8 days. Silicase activity was measured spectrophotometrically using a silicate assay kit. Results: Solid-state fermentation significantly outperformed submerged fermentation for enzyme production. Under optimal SSF conditions, silicase activity reached 0.98 U/mL/min for WF1, 0.96 U/mL/min for BF1, 1.00 U/mL/min for P1, and 0.99 U/mL/min for the fungal consortium (WF1+BF1+P1). Supplementation with jaggery and soy protein further enhanced enzyme yield. Crude rice straw supported higher enzyme activity than powdered forms, and incubation beyond 8 days or deviations in pH and temperature resulted in decreased activity. Conclusion: The study demonstrates that rice straw can be efficiently bioconverted into a high-value enzymatic product using solid-state fermentation with silicate-solubilizing fungi. The optimized SSF system, supplemented with cost-effective nutrient additives, provides an eco-friendly and scalable strategy for silicase production. These findings have potential applications in sustainable agriculture, silica bioremediation, and industrial processing of silica-rich biomass.

Safety evaluation of the food enzyme asparaginase from the non‐genetically modified Saccharomyces cerevisiae strain ARY‐1

The asparaginase (l‐asparagine amidohydrolase, EC 3.5.1.1) is produced by the non‐genetically modified Saccharomyces cerevisiae strain ARY‐1 by Renaissance BioScience Corporation. The food enzyme is not separated from the yeast cells during the enzyme production. The food enzyme is intended to be used to reduce acrylamide formation during food processing at high temperature and low moisture conditions by hydrolysing asparagine. Dietary exposure was estimated to be up to 32.646 mg TOS/kg body weight per day in European populations. Toxicity tests were considered unnecessary by the Panel because the production strain was considered safe and no issues of concern resulting from the food enzyme manufacturing process were identified. A search for the homology of the amino acid sequence of the asparaginase to known allergens was made and no match was found. The Panel considered that a risk of allergic reactions upon dietary exposure to the food enzyme cannot be excluded. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Recombinant Expression and Bioprocess Optimization of Priestia megaterium α-Amylase and Its Impact on Dough Fermentation Efficiency.

α-Amylase is a key hydrolytic enzyme in starch degradation, playing a crucial role in industrial bioprocesses such as dough fermentation. However, optimizing α-amylase production remains challenging due to variations in microbial sources, culture conditions, and induction strategies. In this study, Priestia megaterium α-amylase (PmAmy) production was optimized for the first time based on the biomass/IPTG ratio in a controlled bioprocess. The impact of enzyme supplementation with equal quantity and enzymatic activity on dough fermentation was also evaluated to ensure consistent performance and effective application. Under the bioprocess conditions of 1.0vvm airflow, 37°C, and 1000rpm, a biomass-to-IPTG ratio was optimized as 20gbiomassmmolIPTG -1 at pH 7.0. Fed-batch fermentation was conducted at a specific growth rate of µ=0.22h-1 for 22h, yielding an α-amylase activity of 67.7±4.0UmL-1 at a cell concentration of 22.3±5.3gL-1 Dough fermentation trials demonstrated 99% efficiency compared to commercial α-amylase, despite PmAmy being in its primary recovery form. These findings highlight its potential for industrial baking applications. This study offers a scalable and sustainable enzyme production strategy, contributing to improved fermentation efficiency, product quality, and economic feasibility in food biotechnology.

Yeasts as Biofertilizers and Biocontrol Agents: Mechanisms and Applications.

Yeasts, a large and diverse group of microorganisms, are gaining increasing interest from both the scientific community and industry. Yeasts' natural association with plants has endowed them with a broad repertoire of mechanisms that facilitate a beneficial coexistence. Despite the ability of certain yeast species to enhance plant growth and demonstrate broad-spectrum antifungal activities, their commercialization remains limited. This mini-review focuses on recent insights into the mechanisms by which yeasts stimulate plant growth and protect plants from certain diseases. Yeast species support plant growth by increasing the supply or availability of essential nutrients or by producing phytohormones. The mode of action of yeasts as biological control agents includes competition for nutrients and space, mycoparasitism, formation of biofilms that inhibit pathogen growth, and production of killer toxins, hydrolytic enzymes, and volatile organic compounds. Additionally, yeasts can induce systemic resistance in host plants by enhancing defensive enzyme activity or upregulating pathogenesis-related gene expression. This mini-review explores the current and future applications of yeasts as biofertilizers and biocontrol agents, emphasizing the metabolic engineering of yeast strains and the engineering of plant microbiomes.

Biosynthesis in Streptomyces sp. NRRL S-1813 and regulation between oxazolomycin A and A2.

A novel actinomycete strain, Streptomyces sp. NRRL S-1813 was employed to study its secondary metabolites under different mediums to activate its cryptic gene clusters and produce antimicrobial secondary metabolites. During fermentation optimization, and purification, oxazolomycin A and oxazolomycin A2 were isolated from one strain simultaneously. Their structure was elucidated using a series of characterization techniques, including full wavelength scanning, mass spectrometry (MS), and nuclear magnetic resonance (NMR) spectroscopy. Oxazolomycin A2 was found not to be a typical enzymatic product of fermentation process. Instead, a spontaneous, non-enzymatic ring cleavage reaction was identified as mechanism for conversion of oxazolomycin A to oxazolomycin A2. Basing on these results, if the target product is oxazolomycin A2, the best fermentation condition of Streptomyces sp. NRRL S-1813 should be the Medium B under the alkalescence condition. For the biosynthesis of oxazolomycin A, the medium pH and reaction time were both important. A slightly acidic environment suppresses the side reactions such as hydrolysis of product, while reasonable reaction time minimizes accumulation of byproducts.

Identifying Extremophilic Actinomycetes Effective Against Multidrug-Resistant Bacterial Pathogens and Demonstrating High Enzyme Activities

Objective: This study focused on isolating actinomycetes from an unexplored arid region of Saudi Arabia. Methods: Actinobacteria were isolated on starch nitrate agar and identified using molecular techniques. All isolated screened for antibacterial and enzyme activities. Results: Twenty-two different isolates were obtained on starch nitrate agar. Identification via 16S rRNA sequencing showed that 86.4% belonged to the genus Streptomyces, with one isolate related to Lentzea albidocapillata. The actinomycetes were tested for enzyme production (amylase, protease, keratinase, gelatinase, chitinase, and lipase) and antibacterial activity against various pathogens, including Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), Enterococcus faecalis, Acinetobacter baumannii, Klebsiella oxytoca, and Escherichia coli. Eight isolates exhibited antibacterial activity, with AL3 being effective against all tested Gram-positive bacteria and showing the largest inhibition zones. The crude extract from AL3 demonstrated strong activity against E. faecalis. In conclusion, actinomycetes from extreme habitats can produce valuable antibiotics and enzymes, highlighting the potential for further bioprospecting in similar and unique ecological regions of Saudi Arabia.

New Yarrowia lipolytica chassis strains for industrial enzyme production

BackgroundYarrowia lipolytica has emerged as a well-established platform for producing a wide range of biomolecules, including recombinant proteins (rProteins). Its robust metabolism and resistance to various environmental stressors make it particularly well-suited as a microbial cell factory. However, additional physiological modifications are still required to fully meet industrial demands. Over years of strain development, Y. lipolytica has been engineered to carry auxotrophic markers, streamline the secretory pathway via deletion of native secretory proteins, prevent filamentation, and enable inducible gene expression systems.ResultsIn this study, we continued the fine-tuning of Y. lipolytica as a platform for rProtein synthesis, building on previous work. Specifically, we: (i) introduced a third auxotrophy to facilitate more complex genetic engineering strategies, (ii) removed bacterial vector elements (including antibiotic resistance genes) from previous constructs, and (iii) carried out extensive deletions of extracellular proteases and a peroxidase gene. The newly constructed chassis strains, JMY9438 and JMY9451/9452, both bear triple auxotrophies. The latter strain additionally lacks proteolytic activity due to the deletion of five protease genes. We evaluated the rProtein production efficiency of these strains harboring one, two or three integrated copies of the target gene. rProtein expression levels increased with copy number up to two; however, no further improvement was observed with three copies. Notably, the strain with protease deletions and a single gene copy showed the highest rProtein production per cell, while the strain retaining proteases but harboring two copies yielded the highest absolute rProtein levels.ConclusionsWe present a new generation of Y. lipolytica chassis strains specifically optimized for recombinant protein production. Our results demonstrate that extensive protease deletions can provide a high-performance genetic background, enabling high-level rProtein production without relying on multi-copy expression strategies.

Fungal diversity in Antarctic lignocellulosic substrates and their production of enzymes and lipids with potential industrial applications

Antarctica is characterized by extreme conditions including low temperatures, strong winds, desiccation, high UV radiation, high salinity, freeze-thaw cycles and pH variations. As a result, the resident diversity is dominated by extremophilic microorganisms with adaptations that enable their survival and attract significant biotechnological interest. The present study aimed to recover culturable fungi from different lignocellulosic substrates obtained on Deception Island, maritime Antarctica, and evaluate their ability to produce enzymes and lipids of interest. A total of 47 fungal isolates were recovered from different substrates, representing 16 genera and 23 taxa of the phyla Ascomycota and Basidiomycota. The most abundant genus was Coniochaeta, followed by Cadophora, Pseudogymnoascus, Mrakia and Leucosporidium. The fungal community detected in this study displayed high diversity, richness, and dominance indices. The highest number of fungi produced amylase degradation halos, followed by inulinase and cellulase. However, inulinase was produced the highest number of good-producing fungi. The two strains isolated of the yeast, Solicoccozyma terricola, were able to produce intracellular lipid at low temperatures. Our data indicates the presence of a high diversity and dominance of decomposer taxa. Some of these fungi may have been introduced in wood originally imported for the construction of whaling station or research station buildings, or arrived on Deception Island in different ways. The wood substrates may also have served as bait for the resident mycobiota. The spectrum of enzymatic activity of the cultured fungi corroborates previous studies, confirming the importance of these enzymes for microorganism survival in Antarctica’s habitats. The enzymes produced may have biotechnological potential as more sustainable alternatives in industrial processes involving enzymes active at low temperatures. The oleaginous yeast, S. terricola, demonstrated growth across a wide temperature range, which may favor its presence in Antarctica’s cold but also variable temperature soils. This species also displays biotechnological potential as a potential lipid source, for instance for use as biofuel feedstock.

Efficient secretion of a plastic degrading enzyme from the green algae Chlamydomonas reinhardtii

Plastic pollution has become a global crisis, with microplastics contaminating every environment on the planet, including our food, water, and even our bodies. In response, there is a growing interest in developing plastics that biodegrade naturally, thus avoiding the creation of persistent microplastics. As a mechanism to increase the rate of polyester plastic degradation, we examined the potential of using the green microalga Chlamydomonas reinhardtii for the expression and secretion of PHL7, an enzyme that breaks down post-consumer polyethylene terephthalate (PET) plastics. We engineered C. reinhardtii to secrete active PHL7 enzyme and selected strains showing robust expression, by using agar plates containing a polyester polyurethane (PU) dispersion as an efficient screening tool. This method demonstrated the enzyme’s efficacy in degrading ester bond-containing plastics, such as PET and bio-based polyurethanes, and highlights the potential for microalgae to be implemented in environmental biotechnology. The effectiveness of algal-expressed PHL7 in degrading plastics was shown by incubating PET with the supernatant from engineered strains, resulting in substantial plastic degradation, confirmed by mass spectrometry analysis of terephthalic acid formation from PET. Our findings demonstrate the feasibility of polyester plastic recycling using microalgae to produce plastic-degrading enzymes. This eco-friendly approach can support global efforts toward eliminating plastic in our environment, and aligns with the pursuit of low-carbon materials, as these engineered algae can also produce plastic monomer precursors. Finally, this data demonstrates C. reinhardtii capabilities for recombinant enzyme production and secretion, offering a “green” alternative to traditional industrial enzyme production methods.

Sono-Destructive Polymeric Nanocapsules Enable Spatiotemporal Orchestration of DC-T Cell Crosstalk in Combined In Situ Vaccination and Cytokine Therapy.

Antigen-directed T cell cancer immunotherapy is increasingly shifting toward combination regimens with complementary mechanisms to enhance efficacy without exacerbated toxicity. However, rationally designing efficient combination strategies in this realm remains a challenge. Herein, we engineered a sono-destructive and clinically approved β-amino ester random copolymer (PBAE) nanoplatform that integrates sonosensitizers and interleukin-12 (IL-12) to spatially and temporally regulate antitumor immunity. The PBAE polymer features a thioacetal backbone that enables ultrasound (US)-triggered complete fragmentation, allowing for precise and rapid spread of tumor antigens, immunogenic molecules, and a single low dose of IL-12 (one-tenth of the high doses typically used for therapeutic benefit). In a preclinical human papillomavirus (HPV) 16 oncoproteins E6/E7-expressing tumor model, this combination therapy demonstrates superior antitumor efficacy alongside a robust safety profile. Mechanistically, US-mediated in situ vaccination activates multiple subsets of dendritic cells (DCs), including conventional type I DC (cDC1), cDC2, monocyte-derived DCs, and plasmacytoid DCs, enhancing the presentation of released E7 antigens to prime T cells. IL-12 further amplifies the cytotoxic activity of E7-specific CD8+ T cells and drives the differentiation of T helper 1 cells, complementing therapeutic effect through increased production of cytotoxic enzymes, and cytokines. This work presents an advanced combination regimen for streamlined cancer immunotherapy.

Sustainable recycling of agri‐food waste for industrially important enzyme production: An exclusive overview

Abstract Increasing population around the world is leading to higher food demand and enhanced food and crop production. However, a considerable amount of waste is also being generated worldwide, which is difficult to manage. China is the largest agri‐food waste producer in the world, with 91.65 million metric tonnes per year, followed by India with 68.76 million metric tonnes. Landfilling and burning of these waste products are aiding environmental pollution with the release of GHGs. Hence, a sustainable management method is required to tackle the agri‐food waste. The organic content present in such waste provides an opportunity for utilization for industrially important enzyme production. The production of enzymes requires a specific substrate to help in the growth of the microorganism, along with high enzyme production. Furthermore, the nutrient requirement for enzyme production may differ from bacterial growth. Hence, the complex consortia of agri‐food waste may provide a suitable substrate for the production of various enzymes. However, the utilization of such waste for enzyme production can be challenging in terms of the complexity and unpredictability of the composition. Other factors, such as microplastics and heavy metal contamination, may produce positive or negative results during fermentation. This review discusses global agri‐food waste production and the harmful effects of traditional disposal methods. Furthermore, the main discussion follows the composition of agri‐food waste and its utilization for the production of various industrially important enzymes. Additionally, the review links the idea of agri‐food waste utilization for enzyme production with green chemistry and UN sustainability goals.

Hyperproduction of nattokinase from Bacillus subtilis VIT MS2 using random mutagenesis and statistical optimization through central composite design

BackgroundAn increase in worldwide death rates attributed to ischemic stroke and myocardial infarction explains the demand to search for new thrombolytic drugs. The current study investigates the therapeutic effect of Nattokinase, a fibrinolytic protein from the mutant strain Bacillus subtilis VITMS 2 isolated from fermented milk of Vigna unguiculata.ResultsThe enzyme production was improved using random mutagenesis combined with statistical optimization through composite central design (CCD) with agro-residual substrates. Among all the different combinations employed, 10% (v/v) cane molasses, 12.5 g/L soybean waste, 12.5 g/L eggshell powder, and 27.5 g/L brewer’s spent grain demonstrated a significant influence on fibrinolytic enzyme yield was 4639.43 ± 10.65 FU/mL (Fibrinolytic units/milliliter). This represents a ~ 37.75-fold increase when compared to the unoptimized wild-type strain. The CCD model demonstrated high significance (p < 0.0001) with a strong correlation (R2 = 0.9963), indicating a reliable model fit. The identity and purity of the enzyme was confirmed via MALDI-TOF.ConclusionThe combination of strain improvement through mutagenesis and media optimization enhanced Nattokinase production, offering a promising approach to develop an enzyme therapy for ischemic stroke and myocardial infarction.

Development of an efficient heterologous protein expression platform in Aspergillus niger through genetic modification of a glucoamylase hyperproducing industrial strain

BackgroundAspergillus niger is widely used in industrial enzyme production due to its strong secretion capacity and the status of generally recognized as safe (GRAS). However, heterologous protein expression in A. niger is frequently constrained by high levels of background endogenous protein secretion, limited access to native high transcription loci, and limitations in the efficiency of the secretory machinery. To address these limitations, this study genetically engineered a chassis strain based on an industrial glucoamylase-producing A. niger strain AnN1 for constructing the improved heterologous protein expression.ResultsIn this study, by using CRISPR/Cas9-assisted marker recycling, we deleted 13 of the 20 copies of the heterologous glucoamylase TeGlaA gene and disrupted the major extracellular protease gene PepA, resulting in the low-background strain AnN2. Compared to the parental strain AnN1, AnN2 exhibited 61% less extracellular protein and significantly reduced glucoamylase activity, while retaining multiple transcriptionally active integration loci. Four diverse proteins were integrated into the high-expression loci originally occupied by the TeGlaA gene in the chassis AnN2. These recombinant protein included a homologous glucose oxidase (AnGoxM), a thermostable pectate lyase A (MtPlyA), a bacterial triose phosphate isomerase (TPI), and a medical protein Lingzhi-8 (LZ8). All target proteins were successfully expressed and secreted within 48–72 h, with yields ranging from 110.8 to 416.8 mg/L in 50 mL shake-flasks cultivation. The enzyme activities of AnGoxM, MtPlyA and TPI reached ~ 1276 − 1328 U/mL, ~ 1627. 43 − 2105.69 U/mL, and ~ 1751.02 to 1906.81 U/mg after 48 h, respectively. Additionally, Overexpression of Cvc2, a COPI vesicle trafficking component, further enhanced MtPlyA production by 18%, highlighting the benefit of combining transcriptional and secretory pathway engineering.ConclusionsOur results demonstrated that the chassis AnN2 served as a robust, modular, and time-efficient platform for heterologous protein expression in A. niger. Through site-specific integration of target genes into native high-expression loci and strategic modulation of the secretory pathway, we successfully enabled the rapid production of functional enzymes and bioactive proteins from diverse origins. This dual-level optimization strategy, which integrates rational genomic engineering with targeted enhancement of the secretory pathway, enabled high-yield expression while minimizing background interference. Together, these findings offer a practical framework for constructing versatile fungal expression systems and highlight the potential of combining genetic and cellular engineering to improve recombinant protein production in filamentous fungi.

Carrier-Based Application of Phyto-Benefic and Salt-Tolerant Bacillus wiedmannii and Bacillus paramobilis for Sustainable Wheat Production Under Salinity Stress

Plant growth-promoting rhizobacteria (PGPR) are beneficial soil microorganisms that enhance plant growth and stress tolerance through various mechanisms, including phytohormone production, EPS production, phosphate solubilization, and extracellular enzyme production. These bacteria establish endosymbiotic relationships with plants, improving nutrient availability and overall crop productivity. Despite extensive research on PGPR isolation, their practical application in agricultural fields has faced challenges due to environmental stresses and limited survival during storage. To address these limitations, the present study aimed to isolate salt-tolerant bacterial strains and formulate them with organic carriers to enhance their stability and effectiveness under saline conditions. The isolated bacterial strains exhibited high salt tolerance, surviving NaCl concentrations of up to 850 millimolar. These strains demonstrated basic key plant growth-promoting traits, including phosphate solubilization, auxin production, and nitrogen fixation. The application of carrier-based formulations with both strains, Bacillus wiedmannii (RR2) and Bacillus paramobilis (RR3), improved physiological and biochemical parameters in wheat plants subjected to salinity stress. The treated plants, when subjected to salinity stress, showed notable increases in chlorophyll a (73.3% by Peat + RR3), chlorophyll b (41.1% by Compost + RR3), carotenoids (51.1% by Peat + RR3), relative water content (77.7% by Compost + RR2), proline (75.8% by compost + RR3), and total sugar content (12.4% by peat + RR2), as compared to the stressed control. Plant yield parameters such as stem length (35.1% by Peat + RR3), spike length (22.5% by Peat + RR2), number of spikes (67.6% by Peat + RR3), and grain weight (39.8% by Peat + RR3) were also enhanced and compared to the stressed control. These results demonstrate the potential of the selected salt-tolerant PGPR strains (ST-strains) to mitigate salinity stress and improve wheat yield under natural field conditions. The study highlights the significance of carrier-based PGPR applications as an effective and sustainable approach for enhancing crop productivity in saline-affected soils.

Exogenous putrescine enhances lime stress tolerance in grapevine rootstock-scion combinations

BackgroundClimate change and drought are increasing soil lime content globally, a major threat to crop productivity and quality, especially for viticulture. High lime concentrations disrupt nutrient uptake, leading to chlorosis and reduced growth in plants. While exogenous putrescine applications have shown potential in enhancing plant defense responses against various environmental stresses, their efficacy in modulating lime stress has not been previously explored. In this regard, we studied the effects of putrescine on grapevine saplings under varying lime conditions. The experiment used the Karaerik grape variety, an economically important indigenous cultivar from Eastern Black Sea Region of Turkey, grafted onto three American rootstocks with different lime tolerances: Fercal (high), 5 BB (moderate), and 3309 C (low). These were tested under four lime levels (0%, 20%, 40%, and 60% CaO) and treated with different putrescine concentrations (0, 0.05, 0.1, and 0.2 mM) to examine their morphological, biochemical, and physiological responses under lime stress conditions.ResultsThe results indicated that lime stress adversely affected the growth and development of grapevine saplings by reducing growth parameters, SPAD index, stomatal conductance, and relative water content (RWC), while increasing membrane damage and malondialdehyde (MDA) content. However, different rootstock/cultivar combinations exhibited distinct strategies to cope with lime stress. The highly tolerant Fercal/KE combination maintained higher chlorophyll content, better RWC, and stomatal conductance, supported by increased production of protective enzymes, along with higher proline and protein accumulation, ultimately leading to improved growth and development under lime stress. Exogenous putrescine applications effectively improved plant performance across all rootstock/cultivar combinations, with different concentrations showing optimal results for each combination. The 0.2 mM putrescine treatment significantly enhanced growth parameters, chlorophyll content, stomatal conductance, RWC, and protective enzyme levels while reducing membrane damage and lipid peroxidation in 3309 C/KE combination, which typically shows low lime tolerance. For the moderately tolerant 5 BB/KE, 0.1 mM putrescine was most effective, while the already lime-tolerant Fercal/KE responded best to 0.05 mM putrescine applications. These optimized concentrations demonstrated that putrescine can effectively enhance lime stress tolerance, particularly in more sensitive rootstock/cultivar combinations.ConclusionThis study demonstrated that exogenous putrescine applications effectively mitigated lime stress effects in grapevine saplings by improving physiological parameters and reducing oxidative damage, with optimal concentrations varying according to rootstock tolerance levels (0.2 mM for 3309 C/KE, 0.1 mM for 5 BB/KE, and 0.05 mM for Fercal/KE). The findings revealed that putrescine’s protective effects were most pronounced in lime-sensitive rootstock combinations, suggesting its potential as a protective compound for improving lime stress tolerance in grapevine cultivation.

The fate of Candida tropicalis in the black soldier fly larvae and its nutritional effect suggest indirect interactions

Bacteria are known to colonize the insect gut and determine a positive effect on their host’s fitness, for example, by providing essential nutrients or improving digestion efficiency. However, information on the colonization of the insect gut by fungi and their nutritional contribution is still scarce and fragmentary. In this study, the presence of Candida tropicalis, a fungus abundant in the black soldier fly (Hermetia illucens, BSF) larvae’s gut and environment, was determined in the different gut regions. In addition, metabolites present in larvae fed with a fungus-containing diet were determined by untargeted metabolomics and compared to the C. tropicalis metabolic composition and metabolic changes in the feeding substrate supplemented with the microorganism. Our results indicate that C. tropicalis ceased to be present in the BSF gut after its supplementation in the feeding substrate was stopped, indicating that C. tropicalis does not colonize the gut. Larvae that were reared on diet supplemented with C. tropicalis displayed an increase in the fatty acid biosynthesis pathway, due to an increase in the palmitic and myristic acids that are abundant in C. tropicalis. The presence of C. tropicalis in the substrate caused an increase in threonine, leucine, and isoleucine biosynthesis pathways in the larvae and suggests indirect feeding from the fungal excretions in the substrate. In addition, the lysozyme activity in the larval gut was reduced by the presence of C. tropicalis, suggesting the fungal involvement in the digestive process for increasing fungal survival. This study suggests indirect symbiotic interactions, in which C. tropicalis thrives in the BSF larvae’s environment and manipulates BSF digestive enzyme production to survive in this environment, but on the other hand, BSF larvae benefit metabolically from the C. tropicalis presence in its surrounding environment.

Advancements in Strain Development and Genetic Engineering for Cellulase Production: A Systematic Review

Strains, defined as subtypes or genetic variants of microorganisms, are selectively developed at an industrial scale to enhance the production of cellulases and other metabolites. These strains, bacterial or fungal, are genetically engineered to improve traits such as rapid growth, genetic stability, productivity, and ease of cultivation. Genetic modifications are typically aimed at increasing or altering gene expression to maximize output. Cellulases are enzymes that catalyze the breakdown of cellulose into simpler sugars, enabling its conversion into biofuels and other value-added products. In particular, bacterial cellulose produced by Acetobacteraceae is recognized for its purity, biocompatibility, and natural origin, making it highly suitable for applications in material science. To further enhance cellulase yields, researchers identify and utilize strains capable of high productivity under low-nitrogen conditions. Recombinant DNA technology is employed to sequence genomes, introduce genetic constructs, and express target proteins in host cells. In fungal systems, advanced approaches such as the Reconstruction of Expression Regulatory Network (REXRN) have been applied to fine-tune gene expression and enhance the production of cellulase enzymes involved in cellulose degradation. Together, these strategies support the development of high-performance microbial strains for industrial biotechnology applications.

Enhancing cheese yield and milk clotting efficiency with pineapple bromelain enzyme through response surface methodology

The high demand for cheese products has impacted rennet enzyme production as the availability of ruminant stomachs becomes less and less limited. Therefore, the plantbased enzyme was the potential solution as an alternative to an animal-based enzyme. Bromelain enzyme is chosen due to its ability to coagulate milk as a proteolytic enzyme. This study aimed to obtain the optimum milk clotting activity (MCA) and yield of soft cheese by bromelain using response surface methodology (RSM) and to evaluate the proteolysis properties of soft cheese. Experimental design was generated using RSM by MINITAB software version 15 with three selected variables including bromelain concentration (0.2-1%), incubation time (1-5 hrs) and incubation temperature (45-55℃). The optimum condition for MCA and yield of soft cheese was achieved at a bromelain concentration of 0.53%, incubation time of 2.33 hrs and incubation temperature of 54.2℃, with predicted MCA and yield of 60.81 SU/mL and 29.88%, respectively. The optimum condition was verified with the MCA and the yield obtained was 60.16 SU/mL and 32.06%, respectively. Since there was no significant difference (p&gt;0.05) between the predicted and verified MCA and yield values, thus it indicates that the predicted optimum condition by RSM can be accepted. The proteolysis of soft cheese produced using bromelain enzyme at optimum conditions was also evaluated and compared with commercial soft cheese. The result of proteolysis showed that proteolysis products such as alpha (α), beta (β) and kappa (κ) casein were visible at molecular bands of 30 kDa for all cheese samples. In conclusion, the bromelain enzyme from Josapine pineapple peel has a potential for rennet alternative with a significant value of MCA and yield and expected product of proteolysis.

Variations in virulence factors, antifungal susceptibility and extracellular polymeric substance compositions of cryptic and uncommon Candida species from oral candidiasis

BackgroundInfections caused by uncommon Candida spp. (species other than C. albicans, C. parapsilosis, C. glabrata, C. tropicalis, and C. krusei) have been continuously reported worldwide. However, virulence factors and antifungal susceptibility profiles of uncommon Candida species remain limited. This study aimed to investigate the extracellular hydrolytic enzyme activities, antifungal susceptibility in planktonic and biofilm forms of cryptic and uncommon Candida species as well as their biofilm structures and extracellular polymeric substance (EPS) compositions.MethodsCandida species was identified by sequence analysis of the internal transcribed spacer (ITS) regions of ribosomal RNA. The phospholipase and proteinase activities were evaluated by agar plate method. Biofilm-forming activity was analyzed using XTT-metabolic assay. Antifungal susceptibility tests for fluconazole, voriconazole, 5-flucytosine, and caspofungin were performed with broth microdilution following the CLSI guideline. Candida biofilms were stained with fluorescent dyes for fungal cells (SYTO9), extracellular proteins (SYPRO Ruby), exopolysaccharides (Alexa Fluor 635-conjugated Concanavalin A) and analyzed using confocal laser scanning microscopy (CLSM).ResultsA total of 25 uncommon Candida isolates including C. metapsilosis, C. orthopsilosis, C. guilliermondii (new nomenclature: Meyerozyma guilliermondii), C. fermentati (Meyerozyma caribbica), C. lusitaniae (Clavispora lusitaniae), C. rugosa (Diutina rugosa), C. pararugosa and C. nivariensis (Nakaseomyces nivariensis) were identified, with 8% and 40% of the oral Candida isolates producing phospholipase and proteinase, respectively. All Candida isolates in this study were susceptible to fluconazole, voriconazole, and 5-flucytosine whereas 2 C. rugosa isolates (8% of all tested isolates) reduce susceptibility to caspofungin. The antifungal concentrations required to inhibit biofilm (sMIC) of C. rugosa and C. guilliermondii complex were higher than those of other species. In addition, the C. rugosa and C. guilliermondii biofilms had relatively high quantities of proteins, exopolysaccharides, and extracellular DNA (eDNA) compared to other species. Significant positive correlations between eDNA and exopolysaccharides (rs = 0.77, p < 0.01) and proteins (rs = 0.60, p < 0.01) in Candida biofilms were observed.ConclusionOur results indicated variation in the expression of hydrolytic enzyme production and biofilm forming activities among uncommon Candida spp. All Candida isolates exhibited susceptibility to azole and 5-flucytosine. This study also presented the visualization of biofilm structures and distribution of EPS components in Candida biofilms.

Production of two novel antifungal peroxidase isoenzymes from Tabernaemontana catharinensis using a bubble-column bioreactor.

Production of two novel antifungal peroxidase isoenzymes from Tabernaemontana catharinensis using a bubble-column bioreactor.