Level Of Enzyme Production Research Articles

Abstract C079: Exploring acinar to ductal metaplasia in pancreatitis and precancerous contexts using FixNCut

Abstract Acinar to ductal metaplasia (ADM) is the process by which acinar cells transition to a duct-like state acquiring ductal markers and morphology. This process can be driven by pancreas inflammation or injury, such as pancreatitis, where it is thought to be an adaptive response to help mitigate damage and facilitate repair. ADM is also observed following acinar-specific oncogenic KRAS mutations and known to precede PDAC precancerous lesions termed pancreatic intraepithelial neoplasias (PanINs). While ADM is well characterized histologically, cellular and molecular details of ADM and PanIN formation remain less well understood, in part due to high levels of digestive enzyme production in acinar cells that hamper characterization. Leveraging our recently described DSP-fixation method, FixNCut, to improve capture and transcriptome quality of acinar and ADM populations in the pancreas, we characterize ADM arising in three contexts: acute pancreatitis, recurrent acute pancreatitis, and acute pancreatitis with an oncogenic KRAS mutation known to drive the formation of PanINs. FixNCut and single-cell RNA-sequencing enabled us to elucidate the transcriptomic landscape of ADM and define changes to the microenvironment in unprecedented detail. Our findings indicate that the cellular and transcriptional landscape of recurrent acute pancreatitis closely mirrors that of acute pancreatitis for up to 6 weeks (or 6 bouts of 2-day acute pancreatitis), which was the longest timepoint tested. In contrast, acute pancreatitis in the presence of an acinar-specific oncogenic KRAS mutation results in a distinct transcriptional profile for both ADM and the surrounding microenvironment compared to pancreatitis alone. Additionally, we use FixNCut to preserve the epigenomes of acinar cells, enabling us to sort acinar cells and profile their histone modifications in various contexts of ADM. We benchmark and validate our results against existing mouse and human datasets of precancer and cancer. Through this work, we aim to enhance our understanding of ADM in various contexts, providing insights into acinar cell plasticity and early PDAC precursor formation that may inform strategies for early detection and interception. Citation Format: Katherine Aney, Woo-Jeong Jeong, Pal Koak, Isabel Kim, Sahar Nissim. Exploring acinar to ductal metaplasia in pancreatitis and precancerous contexts using FixNCut [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C079.

Process optimization for the production of fibrinolytic enzyme by a novel marine fungus – Penicillium steckii KU1

The demand for microbial thrombolytic agents is alarmingly increasing, and the discovery of novel microbial species with significant level of fibrinolytic enzyme production are required to cope up with the current circumstances. Surprisingly, marine fungi remain largely unexplored in this regard, presenting an untapped potential for the production of fibrinolytic enzymes.A novel marine fungal strain (Penicillium steckii KU1) was isolated successfully from Kappil beach, Kerala. At the beginning Czapek-dox media was used for the production of fibrinolytic enzyme. Statistical optimization of the enzyme production was achieved initially by Plackett-Burman design (PB). Interactive effect of various process parameters was further scrutinized by Central Composite Design (CCD). Incubation time, inoculum size and concentration of casein were shown significant contribution towards production. Statistical optimization, resulted in a tenfold increment (454.49 ± 14.66 U/mL) [optimized conditions - 30 °C, pH 7, casein 2.7 % (w/v), inoculum 5.75 million spores/mL and 115 h incubation], against the predicted value 441.42 U/mL.

Use of Microbial L-asparaginase in Cancer Treatment -A Detail Review

Abstract The exceptional anticancer effects of l-asparaginases, which hydrolyze plasma l-asparagine and l-glutamine, have drew a lot of interest in recent years. This enzyme is effective against acute lymphoblastic leukemia (ALL) and lymphosarcoma, as well as ALL in children, neoplasia, and various other cancer .The l-asparaginase anticancer application technique has achieved amazing advances in modern oncology by depleting plasma l-asparagine to suppress cancer cell development, notably in youngsters. As bacterial alternative enzyme sources for anticancer therapy, high levels of l-asparaginase enzyme production by Escherichia coli, Erwinia species, Streptomyces, and Bacillus subtilis species are very desirable. Purification of L-asparaginase by Ammonium Sulphate precipitation and various chromatographic technique methods, and improvement of enzyme purity by dialysis confirmation of molecular weight with SDS page.

Beauveria Bassiana Amylase-Polygalacturonase Production Using Lignocellulosic Biomass and Application in Juice Processing.

The search for sources of industrial biocatalysts, which are non-pathogenic and can utilise cheap nutrient sources, has been a continuous endeavour in the ~ 7 billion USD enzyme industry. Beauveria bassiana, an endophytic fungal entomopathogen, is non-pathogenic and possesses the potential to secrete various bioproducts while utilising readily available lignocellulosic biomass. This study investigated the optimised production of two glycosyl hydrolases, amylase and polygalacturonase, by B. bassiana while utilising readily available agricultural residues. Subsequently, the industrial potential of the enzymes in the clarification of fruit juice was evaluated. Initially, seven agro residues were screened for the concomitant production of amylase and polygalacturonase by B. bassiana SAN01. Subsequently, statistical optimisation tools, Plackett Burman Design (PBD) and Central Composite Design (CCD), were employed for the optimisation of enzyme production. The enzyme mixture was partially purified and applied in the clarification of pineapple juice. The production of B. bassiana SAN01 amylase and polygalacturonase was found to be maximal while utilising wheat bran. Subsequent to PBD and CCD optimisation, the optimal conditions for enzyme production were identified to be at 30 °C, pH 6.0 and wheat bran concentration of ~40 g.L-1. Under these optimised conditions, heightened production levels of 34.82 and 51.05 U.mL-1 were recorded for amylase and polygalacturonase, respectively, which were 179% and 187% of the initial unoptimised levels. In addition, the most effective clarification of the juice (~90%) was observed at 35 °C after an incubation time of 120 min with no significant effect on the pH and total dissolved solids. B. bassiana, a well-known biocontrol agent, was shown to produce amylase and polygalacturonase using readily available agricultural residues for the first time. These enzyme production levels are the highest for these enzymes from any known endophytic fungal entomopathogen. This study further demonstrates the potential applicability of B. bassiana in other industrial processes besides its widespread use as a biopesticide.

A closed-loop strategy for on-site production of saccharolytic enzymes for lignocellulose biorefinery using internal lignocellulosic hydrolysates

The cost-effective production of lignocellulose-saccharolytic enzymes is crucial for the bioconversion of plant biomass into fuels and chemicals. The integrated mode for producing these enzymes using lignocellulosic feedstocks as natural inducers has a cost advantage; however, enzyme production level is limited because of the solid-state and complex composition of the raw materials. This study aimed to establish an innovative process for the integrated production of lignocellulolytic enzymes using lignocellulosic hydrolysate as the carbon source. The cellulase-producing workhorse Trichoderma reesei was reprogrammed by combinatorial engineering of transcription factors and the introduction of heterologous β-glucosidases. This enabled inducer-independent production of a complete lignocellulose-saccharolytic enzyme cocktail using lignocellulose-derived monosaccharides as sole carbon source. Via continuous feeding of the enzymatic hydrolysate of pretreated corn stover, crude enzymes with filter paper enzyme activity of 60.4 U/ml and β-glucosidase activity of 503 U/ml were produced. Crude fermentation broth can be used directly to prepare hydrolysates, enabling nex-round enzyme production by recycling 3.1% of the hydrolysate. These results demonstrate a closed-loop strategy for integrated enzyme production using internally produced lignocellulosic sugars from biorefinery plants.

The Immunological Facets Of Chondrocytes In Osteoarthritis: A Narrative Review.

Osteoarthritis (OA) is a disease in which the pathogenesis affects the joint and its surrounding tissues. Cartilage degeneration is the main hallmark of OA and chondrocytes within the cartilage regulate matrix production and degradation. In OA patients and animal models of OA, the pathology of the disease relates to disequilibrium between anabolic and catabolic states of the cartilage. Moreover, chondrocyte phenotype and function are also immunologically altered. Under inflammatory conditions, chondrocytes increase production levels of inflammatory cytokines and cartilage-degrading enzymes, which further drive cartilage destruction. Chondrocytes also have an innate immune function and respond to DAMPs and cartilage fragments via innate immune receptors. In addition, chondrocytes play a role in adaptive immune responses by acting as antigen presenting cells and presenting cartilaginous antigens to T cells. Indirectly, chondrocytes are stimulated by pathogen-associated molecular patterns (PAMPs) present in the joints, a result of the microbiota in the host. Chondrocytes have both direct and indirect relationships with immune cells and the immune compartment of OA patients. Therefore, chondrocytes serve as a target for immunotherapeutic approaches in OA. In this narrative review, we cover the aforementioned immune-related aspects of chondrocytes in OA.

SAT267 BMP4, GDF11 And TGFβ2 Repress Adrenal Cell Cortisol Production By Blocking CYP17A1 And CYP11B1 Expression

Abstract Disclosure: E.C. Fischbach: None. A. Lopez: None. M. Ullenbruch: None. J. Rege: None. W.E. Rainey: None. Introduction: The adrenal cortex is responsible for the biosynthesis of multiple steroid hormones that regulate electrolyte and carbohydrate balance. Cortisol, the principal glucocorticoid in humans, is primarily produced in the middle zone of the cortex known as the zona fasciculata (ZF). The enzymes responsible for cortisol biosynthesis include cytochrome P450c17 (17α-hydroxylase), encoded by CYP17A1 and cytochrome P450c11 (11β-hydroxylase), encoded by CYP11B1. Several paracrine/autocrine regulators, including members of the transforming growth factor beta (TGFβ) superfamily, have been implicated in controlling adrenocortical cell homeostasis and steroid hormone production. Previously, we reported a bone morphogenetic protein 4 (BMP4) expression gradient in the adrenal cortex with higher levels in the zona glomerulosa (ZG) and outer ZF compared to the zona reticularis (ZR). In this study, we sought to compare adrenal expression of members of the TGFβ family including BMP4, growth differentiation factor 11 (GDF11), and transforming growth factor beta 2 (TGFβ2), and determine their effect on adrenal cell cortisol biosynthesis. Methods: Growth factor mRNA levels were studied using microarray analysis of laser captured ZG, ZF and ZR from human adrenals. The adrenal cell line, HAC15 was used to determine the effects of the TGFβ family members alone and in combination. Output measures included cell cortisol production and mRNA levels of steroidogenic enzymes using ELISA and quantitative RT-PCR, respectively. Results: Microarray analysis indicated higher mRNA levels of BMP4, GDF11 and TGFβ2 in the ZG compared with the ZF and ZR regions of the adrenal. Treatment of HAC15 cells with either BMP4 (50 ng/mL), GDF11 (50 ng/mL) or TGFβ2 (10 ng/mL) significantly inhibited cortisol production, as well as CYP11B1 and CYP17A1 mRNA levels. The inhibition was observed under basal and forskolin-stimulated conditions. Furthermore, the inhibitory effects on cortisol production and enzyme expression were additive when factors were added together. Summary: Overall, our findings suggest that the human adrenal has a gradient of multiple members of the TGFβ family. The additive action of these growth factors on adrenal cell differentiation may explain, in part, the inability of the ZG to produce cortisol and its low expression of CYP11B1 and CYP17A1. Presentation: Saturday, June 17, 2023

The use of newly isolated fungal cultures for the selective delignification of bamboo culms.

The screening of ligninolytic enzyme-producing fungal species in samples led to the identification of Paracremonium sp. LCB1, Clonostachys compactiuscula LCD1 and C. compactiuscula LCN1. Both these strains produced high levels of hemicellulase and ligninolytic enzyme production over a relatively short fermentation period of 3-5days while exhibiting very low levels of cellulase activity. The results of the tests indicated that co-culturing LCB1 and LCN1 enhanced the ability to degrade lignin, and the ideal degrading circumstances and internal degrading mechanism of combined fungi were examined. The results showed that under conditions of temperature (30°C), pH (5), culture time (40 d), solid-liquid ratio (1:2.5), the pretreatment of bamboo culms with a co-culture of LCB1 and LCN1 resulted in a pronounced 76.37% drop in lignin weight and a high lignin/cellulose loss ratio (>10). Fourier transform infrared spectroscopy, X-ray diffractometry, and scanning electron microscopy were used to characterize the physicochemical properties of these bio-pretreated bamboo culms, further confirming that LCB1 and LCN1 co-culture represents an effective approach to bamboo delignification.

Unleashing the Potential of Bacterial Isolates from Apple Tree Rhizosphere for Biocontrol of Monilinia laxa: A Promising Approach for Combatting Brown Rot Disease.

Monilinia laxa, a notorious fungal pathogen responsible for the devastating brown rot disease afflicting apples, wreaks havoc in both orchards and storage facilities, precipitating substantial economic losses. Currently, chemical methods represent the primary means of controlling this pathogen in warehouses. However, this study sought to explore an alternative approach by harnessing the biocontrol potential of bacterial isolates against brown rot in apple trees. A total of 72 bacterial isolates were successfully obtained from the apple tree rhizosphere and subjected to initial screening via co-cultivation with the pathogen. Notably, eight bacterial isolates demonstrated remarkable efficacy, reducing the mycelial growth of the pathogen from 68.75 to 9.25%. These isolates were subsequently characterized based on phenotypic traits, biochemical properties, and 16S rRNA gene amplification. Furthermore, we investigated these isolates' production capacity with respect to two enzymes, namely, protease and chitinase, and evaluated their efficacy in disease control. Through phenotypic, biochemical, and 16S rRNA gene-sequencing analyses, the bacterial isolates were identified as Serratia marcescens, Bacillus cereus, Bacillus sp., Staphylococcus succinus, and Pseudomonas baetica. In dual culture assays incorporating M. laxa, S. marcescens and S. succinus exhibited the most potent degree of mycelial growth inhibition, achieving 68.75 and 9.25% reductions, respectively. All the bacterial isolates displayed significant chitinase and protease activities. Quantitative assessment of chitinase activity revealed the highest levels in strains AP5 and AP13, with values of 1.47 and 1.36 U/mL, respectively. Similarly, AP13 and AP6 exhibited the highest protease activity, with maximal enzyme production levels reaching 1.3 and 1.2 U/mL, respectively. In apple disease control assays, S. marcescens and S. succinus strains exhibited disease severity values of 12.34% and 61.66% (DS), respectively, highlighting their contrasting efficacy in mitigating disease infecting apple fruits. These findings underscore the immense potential of the selected bacterial strains with regard to serving as biocontrol agents for combatting brown rot disease in apple trees, thus paving the way for sustainable and eco-friendly alternatives to chemical interventions.

Screening of tomato (Solanum lycopersicon L.) genotypes by inducing systemic resistance against early blight disease caused by Alternaria solani

Early blight of tomato (Solanum lycopersicon L.) incited by Alternaria solani is highly destructive disease in the world. Environmental factors significantly impact early blight epidemics, leading to the loss of up to 78 per cent of tomato production. Twenty tomato genotypes were used in this study to identify the early blight resistant and susceptible genotypes selected to represent a range of reactions when screened under field conditions. The tomato plants were evaluated for early blight disease by using Per cent Disease Index (PDI). Pusa Uphar (20.18%) and Sankaranti (20.18%) showed resistance to early blight disease among the twenty genotypes. Anaka Kerala (61.25%), Arka Vikas (61.76%), Pusa Rohini (53.65%), Ashoka (50.60%) and Paiyur1 (56.08%) genotypes were found highly susceptible based on early blight disease intensity. Spore inoculation of A. solani was sprayed into tomato plants, it was discovered that the number of defense-inducing compounds viz., total phenols, peroxidase (PO), polyphenol oxidase (PPO) and phenylalanine ammonia lyase (PAL) has increased. Among the genotypes, Pusa Uphar (T2) and Sankaranti (T1) genotypes showed a high level of defense enzyme production. After tomato plants were exposed to pathogens through artificial inoculation, the activity of these defensive enzymes and compounds was highly induced in the resistant and sensitive germplasm than in the control.

Optimization of the fermentation parameters to maximize the production of cellulases and xylanases using DDGS as the main feedstock in stirred tank bioreactors

Lignocellulolytic enzymes such as cellulase and xylanases are needed on the industrial scales for low-cost production of biofuels and the other value-added products from lignocellulosic biomass such as distillers' dried grains with solubles (DDGS). Optimization of fermentation variables such as agitation, aeration, and inoculum size for fungal enzyme production by submerged fermentation can enhance the enzyme production levels. Therefore, this research focused on the statistical optimization of fungal inoculum size (1–10%), aeration (0.5-2vvm) and agitation rates (100–500rpm) through response surface methodology (RSM) to obtain highest amounts of cellulases and xylanases. Aspergillus niger (NRRL 330) was grown in dilute acid treated DDGS supplemented with peptone, yeast extract, and ammonium sulfate in benchtop bioreactors to produce these enzymes. The optimal fermentation results were determined to be 6.5% inoculum size, 310 rpm agitation rate and 1.4 vvm aeration, which revealed increases of cellulase activity from 0.6 to 0.82 IU/ml and xylanase activity from 3.99 to 52.76 IU/ml after optimization. The results prove the need for optimization of culture conditions such as aeration, agitation and inoculum size for higher production of hydrolytic enzymes by A. niger using DDGS as the main feedstock.

Spartina argentinensis valorization and process optimization for enhanced production of hydrolytic enzymes by filamentous fungus

Hemicellulose is a major component of plant cell walls and xylan is the most predominant polysaccharide. Xylan degrading enzymes integrate the xylanolytic system. Xylanolytic enzymes were produced by fermentation by Aspergillus niger and Thermomyces lanuginosus grown on grass considered unsuitable for farming called Spartina argentinensis . Significant parameters: type of fermentation used (SmF or SSF), type of leaves of S. argentinensis (green or senescent) and conidia final concentration for xylanase production were screened and optimized. The main results showed that the highest levels of xylanolytic enzyme production were obtained by 1 × 10 5 conidia/mL of A. niger in SmF at 96 h, 30ºC, with a mixture of 20.20% of senescent and 79.80% of green leaves. The xylanase specific activity obtained was 62 U/mg, higher than the activity obtained (23 U/mg) in previous work ( Taddia et al., 2019 ), and the concentration of xylanolytic production over that of glucanase activity was maximized five times. The optimized enzymatic extract obtained was characterized by LC-MS and HPLC of carbohydrates. Six enzymes were identified as constituents of the xylanolytic complex and seven carbohydrates. Moreover, the xylanolytic enzyme extract was stable for 30 days at 20 °C. Thus, S. argentinensis can be used within the framework of a circular economy, rendering a synergistic combination of the xylanolytic enzymes with industrial applications. • Lignocellulosic biomass; Spartina argentinensis was utilized in the fermentation. • Enzyme statistical design production was optimized. • Maximum xylanase specific activity of 61 U/mL was obtained. • The enzymatic extract maintained 100% its activity after 30 days at 20°C.

Improved β-galactosidase Secretion in Fresh Water Fungi through Cultural Optimization

The wide range of applications of β-galactosidase has made it a candidate enzyme of study for improved yield. Most studies focused on expressing this enzyme in expressible vector(s) while sources of this enzyme are rarely investigated. Aquatic habitats harbor arrays of microorganisms that can participate in demineralization, nutrient restoration, circulation and hence confer significant properties on the habitats and significantly influence microbial interactions and metabolism. In view of this, aquatic fungal isolates were qualitatively and quantitatively screened for intracellular and extracellular β-galactosidase production. Four fungal isolates tagged SB01 and SC02 from stream identified as Aspergillus oryzae and Aspergillus niger and SC01 and SA01 from pond identified as A. oryzae and A. niger were studied. β-galactosidase gene was successfully amplified from the four isolates and their enzyme production levels were further studied. All the isolates showed considerably higher extracellular secretion than the intracellular synthesis on glucose-based medium. The production level was probed by xylose supplementation but surprisingly, the intracellular synthesis was higher than the extracellular secretion for all the isolates. In an attempt to boost the enzyme production, temperature was varied and 25 °C showed improved β-galactosidase synthesis and secretion than the 30 °C. This study revealed these filamentous fungi as candidates for β-galactosidase production and broaden the understanding of harnessing microorganisms from an aquatic reservoir for bio-production, and more importantly, revealed the intracellular accumulation of the enzyme as a bottleneck for industrial production.
 HIGHLIGHTS
 
 The wide range of applications of β-galactosidase has made it a candidate enzyme of study for improved yield
 Most studies focused on expressing this enzyme in expressible vector(s) while sources of this enzyme are rarely investigated
 Aquatic fungal isolates were qualitatively and quantitatively screened for intracellular and extracellular β-galactosidase production
 All the isolates showed considerable higher extracellular secretion than the intracellular synthesis on glucose-based medium, and vice-versa on xylose-supplemented medium, with the highest β-galactosidase synthesis and secretion efficiency occurring at 25 °C in both instances
 This study revealed these filamentous fungi as candidates for β-galactosidase production and broaden the understanding of harnessing microorganisms from an aquatic reservoir for bio-production
 
 GRAPHICAL ABSTRACT

Optimization of protease production by Bacillus cereus HMRSC30 for simultaneous extraction of chitin from shrimp shell with value-added recovered products.

Chitin extraction from shrimp shell powder (SSP) using protease-producing microbes is an attractive approach for valorizing shrimp shell waste because it is simple and environmentally friendly. In this study, the protease production and chitin extraction from SSP by Bacillus cereus HMRSC30 were simultaneously optimized using statistical approaches. As a result, fermentation in medium composed of 30g/L SSP, 0.2g/L MgSO4 · 7H2O, 3g/L (NH4)2SO4, 0.5g/L K2HPO4, and 1.5g/L KH2PO4 (pH 6.5) for 7days maximized protease production (197.75 ± 0.33 U/mL) to approximately 1.64-fold compared to unoptimized condition (126.8 ± 0.047 U/mL). This level of enzyme production was enough to achieve 97.42 ± 0.28% deproteinization (DP) but low demineralization (DM) of 53.76 ± 0.21%. The high DM of 90% could be easily accomplished with the post-treatment using 0.4M HCl and acetic acid. In addition, the study evaluated the possible roadmap to maximize the value of generated products and obtain additional profits from this microbial process. The observation showed the possibility of serving crude chitin as a bio-adsorbent with the highest removal capacity against Coomassie brilliant blue (97.99%), followed by methylene blue (74.42%). The recovered protease exhibited the function to remove egg yolk stain, indicating its potential for use as a detergent in de-staining. The results corroborated the benefits of microbial fermentation by B. cereus HMRSC30 as green process for comprehensive utilization of shrimp shell waste as well as minimizing waste generation along the established process.

Bacterial Endophyte Community Dynamics in Apple (Malus domestica Borkh.) Germplasm and Their Evaluation for Scab Management Strategies.

The large genetic evolution due to the sexual reproduction-mediated gene assortments and propensities has made Venturia inaequalis (causing apple scab) unique with respect to its management strategies. The resistance in apple germplasm against the scab, being controlled for by more than fifteen genes, has limited gene alteration-based investigations. Therefore, a biological approach of bacterial endophyte community dynamics was envisioned across the apple germplasm in context to the fungistatic behavior against V. inaequalis. A total of 155 colonies of bacterial endophytes were isolated from various plant parts of the apple, comprising 19 varieties, and after screening for antifungal behavior followed by morphological, ARDRA, and sequence analysis, a total of 71 isolates were selected for this study. The alpha diversity indices were seen to fluctuate greatly among the isolation samples in context to microflora with antifungal behavior. As all the isolates were screened for the presence of various metabolites and some relevant genes that directly or indirectly influence the fungistatic behavior of the isolated microflora, a huge variation among the isolated microflora was observed. The outstanding isolates showing highest percentage growth inhibition of V. inaequalis were exploited to raise a bio-formulation, which was tested against the scab prevalence in eight apple varieties under controlled growth conditions. The formulation at all the concentrations caused considerable reductions in both the disease severity and disease incidence in all the tested apple varieties. Red Delicious being most important cultivar of the northwestern Himalayas was further investigated for its biochemical behavior in formulation and the investigation revealed different levels of enzyme production, chlorophyll, and sugars against the non-inoculated control.

Biotechnological potential of psychrophilic microorganisms as the source of cold-active enzymes in food processing applications.

Microorganisms striving in extreme environments and exhibiting optimal growth and reproduction at low temperatures, otherwise known as psychrophilic microorganisms, are potential sources of cold-active enzymes. Owing to higher stability and cold activity, these enzymes are gaining enormous attention in numerous industrial bioprocesses. Applications of several cold-active enzymes have been established in the food industry, e.g., β-galactosidase, pectinase, proteases, amylases, xylanases, pullulanases, lipases, and β-mannanases. The enzyme engineering approaches and the accumulating knowledge of protein structure and function have made it possible to improve the catalytic properties of interest and express the candidate enzyme in a heterologous host for a higher level of enzyme production. This review compiles the relevant and recent information on the potential uses of different cold-active enzymes in the food industry.

Wastewater from the Edible Oil Industry as a Potential Source of Lipase- and Surfactant-Producing Actinobacteria.

Wastewaters generated from various stages of edible oil production in a canola processing facility were collected with the aim of determining the presence of lipase-producing actinobacteria of potential industrial significance. The high chemical oxygen demand (COD) readings (up to 86,700 mg L−1 in some samples) indicated that the wastewater exhibited the nutritional potential to support bacterial growth. A novel approach was developed for the isolation of metagenomic DNA from the oil-rich wastewater samples. Microbiota analysis of the buffer tank and refinery condensate tank wastewater samples showed a dominance of Cutibacterium acnes subsp. defendens, followed by a limited number of other actinobacterial genera, indicating the presence of a highly specialized actinobacterial population. Cultured isolates with typical actinobacterial morphology were analyzed for their ability to produce lipases and biosurfactants. Two strains, designated as BT3 and BT4, exhibited the highest lipase production levels when grown in the presence of tributyrin and olive oil (1.39 U mg−1 crude protein and 0.8 U mg−1 crude protein, respectively) and were subsequently definitively identified by genome sequencing to be related to Streptomyces albidoflavus. Cultivation of the strains in media containing different types of oils did not markedly increase the level of enzyme production, with the exception of strain BT4 (1.0 U mg−1 crude protein in the presence of peanut oil). Genome sequencing of the two strains, BT3 and BT4, revealed the presence of a range of lipase and esterase genes that may be involved in the production of the enzymes detected in this study. The presence of gene clusters involved in the production of biosurfactants were also detected, notably moreso in strain BT3 than BT4.

Lignocellulolytic activity of Pleurotus ostreatus under solid state fermentation using silage, stover, and cobs of maize

Lignocellulolytic white-rot fungi allow the bioconversion of agricultural wastes into value-added products that are used in a myriad of applications. The aim of this work was to use corn residues (Zea mays L.) to produce valuable products under solid-state fermentation (SSF) with Pleurotus ostreatus. White-rot fungus P. ostreatus was isolated from maize silage (MS) and thereafter it was inoculated on MS as substrate and compared with maize stover (MSt) and maize cobs (MC) to determine the best lignocellulosic substrate for the production of lignocellulolytic enzymes and extracellular protein. The MS gave the highest productivity of CMCase (368.2 U/mL), FPase (170.5 U/mL), laccase (11.4 U/mL), and MnPase (6.6 U/mL). This is compared to productivity on MSt of 222 U/mL, 50.2 U/mL, 4.55 U/mL, and 2.57 U/mL, respectively; and productivity on MC at the same incubation period as 150.5 U/mL, 48.2 U/mL, 3.58 U/mL, and 2.5 U/mL, respectively. The levels of enzyme production declined with increasing incubation period after 15 and 20 days using MS and MC, respectively, as substrates. Maximum liberated extracellular protein content (754 to 878 µg/mL) was recorded using MS, while a low amount (343 to 408 µg/mL) was liberated with using MSt and MC.

Optimizing Protein-Glutaminase Expression in Bacillus subtilis.

Protein-glutaminase (PG) is a promising protein deaminase. It only hydrolyzes the side chain amido groups of protein-bound to generate ammonia and protein-L-glutamic acid and does not catalyze any other undesirable changes in protein structures. Deamidation of proteins via PG can influence the solubility, emulsification, foaming, and gelation properties of proteins, which are important properties for some food proteins. Therefore, there is great potential for the application of PG in the food industry. PG is derived from Chryseobacterium proteolyticum (C. proteolyticum); however, wild strains are difficult to industrialize because of their low levels of enzyme production. In this article, we studied different strategies for PG expression in B. subtilis. Results showed that PG produced from C. proteolyticum could be successfully secreted in B. subtilis WB800N, and actively secreted in B. subtilis 168(BS168) or DB403 containing a pro-peptide (pro-PG). The secreted PG from B. subtilis WB800N was inactive unless digested by exogenous proteases, such as trypsin, alkaline protease, and neutral protease. However, active PG was secreted by the self-processing of BS168 and DB403. The specific activity of purified PG reached 20.9 U/mg. PG showed maximum activity at pH 5.5, 55°C and more than 80% of PG activity was retained within a range of pH 3.5-6.5. When Cbz-Gln-Gly was used as the substrate, PG activity was 31.1 ± 0.9μMmin-1mg-1. Mg2+, Ca2+, and Zn2+ stabilized and even activated PG activity. These strategies concerning PG expression in B. subtilis and the enzymatic properties of PG provide efficient alternatives for PG research and contribute to the industrial-scale production of PG.

Low-level lead exposure promotes hepatic gluconeogenesis and contributes to the elevation of fasting glucose level

BackgroundLead (Pb) is considered an endocrine-disrupting chemical. However, few studies have investigated the effects of low-level Pb exposure on plasma glucose levels. Herein, we aimed to investigate whether low-level Pb exposure causes elevated plasma glucose levels and the possible mechanisms involved. MethodsWe conducted a cross-sectional study of 5747 participants from 16 sites in China. The participants underwent measurements of anthropometric factors, blood lead level (BLL) and fasting plasma glucose (FPG). Wistar rats were exposed to 0.05% Pb through drinking water or fed with a high-fat diet (HFD) for 28 weeks. The relevant parameters of glucose homeostasis, hepatic glucose production (HGP) and gene expression levels of hepatic gluconeogenesis enzymes, including phosphoenolpyruvate carboxy kinase (PEPCK), glucose-6-phosphatase (G6PC) and fructose-1,6-bisphosphatase (FBP1), were measured. In addition, gene expression levels of gluconeogenesis enzymes were also measured in HepG2 cells administered with different concentrations of lead acetate for 24 h. ResultsIn humans, after adjusting for confounders, the odds of having High_FPG (≥5.6 mmol/L) were significantly increased by 25% in the participants in the fourth BLL quartile (OR 1.25, 95% CI 1.05, 1.49). In the animals exposed to 0.05% Pb, FPG, HGP and hepatic gene expression levels of PEPCK, G6PC and FBP1 were increased. In addition, the mRNA expression levels of PEPCK, G6PC and FBP1 in HepG2 cells were also increased in response to Pb exposure. ConclusionsThese findings support the possibility that low-level Pb exposure may increase HGP by affecting key enzymes of hepatic gluconeogenesis, eventually resulting in impaired FPG and hyperglycemia.