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Microbial Cell Factories May 2024Rhizomucor miehei (RM) lipase is a regioselective lipase widely used in food, pharmaceutical and biofuel industries. However, the high cost and low purity of the...
BACKGROUND
Rhizomucor miehei (RM) lipase is a regioselective lipase widely used in food, pharmaceutical and biofuel industries. However, the high cost and low purity of the commercial RM lipase limit its industrial applications. Therefore, it is necessary to develop cost-effective strategies for large-scale preparation of this lipase. The present study explored the high-level expression of RM lipase using superfolder green fluorescent protein (sfGFP)-mediated Escherichia coli secretion system.
RESULTS
The sfGFP mutant was fused to the C-terminus of RM lipase to mediate its secretion expression. The yield of the fusion protein reached approximately 5.1 g/L with high-density fermentation in 5-L fermentors. Unlike conventional secretion expression methods, only a small portion of the target protein was secreted into the cell culture while majority of the fusion protein was still remained in the cytoplasm. However, in contrast to intracellular expression, the target protein in the cytoplasm could be transported efficiently to the supernatant through a simple washing step with equal volume of phosphate saline (PBS), without causing cell disruption. Hence, the approach facilitated the downstream purification step of the recombinant RM lipase. Moreover, contamination or decline of the engineered strain and degradation or deactivation of the target enzyme can be detected efficiently because they exhibited bright green fluorescence. Next, the target protein was immobilized with anion-exchange and macropore resins. Diethylaminoethyl sepharose (DEAE), a weak-basic anion-exchange resin, exhibited the highest bind capacity but inhibited the activity of RM lipase dramatically. On the contrary, RM lipase fixed with macropore resin D101 demonstrated the highest specific activity. Although immobilization with D101 didn't improve the activity of the enzyme, the thermostability of the immobilized enzyme elevated significantly. The immobilized RM lipase retained approximately 90% of its activity after 3-h incubation at 80 °C. Therefore, D101 was chosen as the supporting material of the target protein.
CONCLUSION
The present study established a highly efficient strategy for large-scale preparation of RM lipase. This innovative technique not only provides high-purity RM lipase at a low cost but also has great potential as a platform for the preparation of lipases in the future.
Topics: Lipase; Rhizomucor; Escherichia coli; Enzymes, Immobilized; Green Fluorescent Proteins; Recombinant Fusion Proteins; Fermentation
PubMed: 38802857
DOI: 10.1186/s12934-024-02432-y -
Allergologie Select 2024None.
None.
PubMed: 38756207
DOI: 10.5414/ALX02444E -
Foods (Basel, Switzerland) Apr 2024Strong-flavor Daqu, as a fermentation agent, plays a significant role in shaping the quality of strong-flavor baijius, and fungal species in Daqu are important factors...
Strong-flavor Daqu, as a fermentation agent, plays a significant role in shaping the quality of strong-flavor baijius, and fungal species in Daqu are important factors affecting the quality of Daqu. Therefore, we selected strong-flavor Daqu from seven different origins to study the fungal composition and the effects of the fungal composition on the physicochemical properties and volatile organic compounds (VOCs). It was found that the fungal composition influences the physicochemical properties of Daqu. Specifically, there was a positive link between , , , and liquefying activity and a positive correlation between and fermenting activity. Furthermore, the relationships between esterifying activity and , , , , and were found to be positive. The VOCs in Daqu were affected by , , , and . was significantly positively correlated with benzeneethanol as well as and pentadecanoic acid ethyl ester, ethyl myristate. and were significantly negatively correlated with benzaldehyde and 2-furaldehyde. This study deepens our understanding of the relationship between VOCs, the physicochemical properties with microbial communities, and reference significance for the production of better-quality strong-flavor Daqu.
PubMed: 38672935
DOI: 10.3390/foods13081263 -
Marine Drugs Feb 2024Low levels of docosahexaenoic acid (DHA) in the brain have been related to neurological disorders, like Alzheimer's disease (AD). After ingestion, dietary DHA must cross...
Low levels of docosahexaenoic acid (DHA) in the brain have been related to neurological disorders, like Alzheimer's disease (AD). After ingestion, dietary DHA must cross the blood-brain barrier, where it is absorbed as lysophosphatidylcholine (LPC), due to its role as a preferential DHA carrier in the brain. This work aimed at the production of LPC-DHA extracts to be used in supplementation/food fortification intended neural enrichment in DHA. As it is rich in DHA, especially its phospholipids (PL), Atlantic mackerel (, caught in Spring/2022) was used as a raw material. The polar lipids fraction was separated and hydrolysed with lipase, to enzymatically convert phosphatidylcholine (PC) into LPC. The fish (muscle and by-products) lipids fraction was used for total lipids (TL) content, lipid classes (LC) and fatty acid (FA) profile evaluation, whilst polar lipids extracts were studied for LC production and FA analysis. Muscle TL ranged between 1.45 and 4.64 g/100 g (WW), while by-products accounted for 7.56-8.96 g/100 g, with the highest contents being found in March. However, PL were more abundant in muscle (22.46-32.20% of TL). For polar lipids extracts, PL represented 50.79% of TL, among which PC corresponded to 57.76% and phosphatidylethanolamine to 42.24%. After hydrolysis, nearly half of this PC was converted into LPC. When compared to the initial PC, DHA relative content (33.6% of total FA) was significantly higher after hydrolysis: 55.6% in PC and 73.6% in LPC. Such extract, obtained from this undervalued species, may represent a promising strategy to increase DHA uptake into brain cells while allowing this species to upgrade.
Topics: Animals; Phospholipids; Docosahexaenoic Acids; Brain; Blood-Brain Barrier; Phosphatidylcholines; Fatty Acids; Lysophosphatidylcholines
PubMed: 38535457
DOI: 10.3390/md22030116 -
EFSA Journal. European Food Safety... Mar 2024The food enzyme mucorpepsin (EC 3.4.23.23) is produced with the non-genetically modified strain LP-N836 by Meito Sangyo Co., Ltd. The native enzyme can be chemically...
The food enzyme mucorpepsin (EC 3.4.23.23) is produced with the non-genetically modified strain LP-N836 by Meito Sangyo Co., Ltd. The native enzyme can be chemically modified to produce a more thermolabile form. The food enzyme is free from viable cells of the production organism. It is intended to be used in the processing of dairy products for the production of cheese and fermented dairy products. Dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.108 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 95 mg TOS/kg bw per day, the mid-dose tested, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 880. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and four matches with respiratory allergens and one with a food allergen (mustard) were found. The Panel considered that the risk of allergic reactions upon dietary exposure to this food enzyme, particularly in individuals sensitised to mustard proteins, cannot be excluded. Based on the data provided, the Panel concluded that both the native and thermolabile forms of this food enzyme do not give rise to safety concerns under the intended conditions of use.
PubMed: 38450083
DOI: 10.2903/j.efsa.2024.8631 -
EFSA Journal. European Food Safety... Feb 2024The food enzyme mucorpepsin (EC 3.4.23.23) is produced with the non-genetically modified strain M19-21 by Meito Sangyo Co., Ltd. The enzyme is chemically modified to...
The food enzyme mucorpepsin (EC 3.4.23.23) is produced with the non-genetically modified strain M19-21 by Meito Sangyo Co., Ltd. The enzyme is chemically modified to produce a thermolabile form. The food enzyme was considered free from viable cells of the production organism. It is intended to be used in the processing of dairy products for the production of cheese and fermented dairy products. Based on the maximum use levels, dietary exposure was estimated to be up to 0.108 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 226 mg TOS/kg bw per day, the highest dose tested, which, when compared with the estimated dietary exposure, results in a margin of exposure of at least 2093. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and four matches to respiratory allergens and one match to a food allergen (mustard) were found. The Panel considered that the risk of allergic reactions upon dietary exposure to this food enzyme, particularly in individuals sensitised to mustard proteins, 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.
PubMed: 38410150
DOI: 10.2903/j.efsa.2024.8633 -
Frontiers in Microbiology 2024This study investigated the microbial community in three-color sauce-flavor (black, yellow, and white) throughout their maturation processes, together with their...
This study investigated the microbial community in three-color sauce-flavor (black, yellow, and white) throughout their maturation processes, together with their physicochemical factors, culturable microbes, flavor components, and fermenting vitalities. Results from high-throughput sequencing revealed distinct microbial diversity, with more pronounced variations in bacterial community than in fungal community. Firmicutes and Ascomycota emerged as the most dominant bacterial and fungal phyla, respectively, during maturation. Genus-level analysis identified , , and as dominant bacteria in black , yellow , and white , severally, while was shared as the core dominant fungi for these . Physicochemical factors, particularly acidity, were found to exert a significant impact on microbial community. was the key bacteria influencing the color formation of these . Furthermore, correlations between dominant microbes and flavor compounds highlighted their role in quality. Molds (, , and ), excepting , played a crucial role in the formation of pyrazine compounds. Consequently, this study offers innovative insights into the microbial perspectives on color and pyrazine formation, establishing a groundwork for future mechanized production and quality control of sauce-flavor baijiu.
PubMed: 38328433
DOI: 10.3389/fmicb.2024.1345772 -
Frontiers in Microbiology 2024Proso millet, a high-quality fermentation material used for Chinese yellow wine production, can produce special flavored substances; however, its role in improving the...
INTRODUCTION
Proso millet, a high-quality fermentation material used for Chinese yellow wine production, can produce special flavored substances; however, its role in improving the flavor and altering microbial communities of light-flavored Baijiu during fermentation remain unknown. Thus, we aimed to investigate the effect of proso millet on improving the flavor of light-flavored Baijiu and altering microbial communities during different fermentation stages.
METHODS
The dynamic changes in the microbial communities and flavor of proso millet (50%) + sorghum (50%) mixed fermentation samples were analyzed through intermittent sampling on days 7, 14, 21, and 28 of the fermentation process. Microbial high-throughput sequencing and the analysis of flavor characteristics were conducted through 16S DNA/ ITS amplicon sequencing and gas chromatography (multi-capillary column)-ion mobility spectrometry, respectively.
RESULTS
Proso millet significantly changed the core flavor compound composition of traditional light-flavored Baijiu from ethyl acetate, ethyl hexanoate, ethyl hexanoate dimer, ethyl butanoate, ethyl lactate, and butyl acetate to oct-2-ene, 2-butanol, propyl propanoate, 2-pentenal, and 4-methylpentanal. The amplicon sequencing analysis revealed that the alpha diversity parameters of bacterial and fungal communities, including the Chao1, Pielou_e, Shannon, and Simpson indices, for proso millet-sorghum mixed fermentation samples were significantly higher than those for sorghum fermentation samples ( < 0.05). Of the 40 most significant microbial genera in two treatments, proso millet significantly increased the abundance of 12 bacterial and 18 fungal genera. Among the 40 most significant bacterial and fungal species, 23 bacterial species belonged to the genus, whereas the 30 primary fungal species belonged to 28 different genera. The analysis of the relationship between microbial changes and the main flavor compounds of light-flavored Baijiu showed that bacteria from the , , , , , , , , , , , genera and fungi from the , , , , , , and genera significantly inhibited the synthesis of ethyl hexanoate, ethyl butanoate, ethyl lactate ethyl lactate, and butyl acetate but increased the synthesis of ethyl acetate ( < 0.05). Moreover, these microbes exhibited a significantly greater abundance in proso millet-sorghum mixed fermentation samples than in sorghum samples. The synthesis of special flavored compounds in proso millet Baijiu was significantly positively correlated with the presence of fungi from the , , , , and genera but negative correlated with the presence of bacteria from the , , , , , and genera. Regarding ethanol content, the low alcohol content of Fenjiu may be due to the significantly high abundance of fungi from the genus and bacteria from the , , , and genera during fermentation. In summary, proso millet significantly altered the flavor of light-flavored Baijiu by inducing the formation of a special microbial community; however, it did not increase alcohol concentration.
DISCUSSION
This study lays the foundation for future research on Baijiu fermentation. Additionally, the study findings may help improve the production efficiency and elevate the quality and flavor of the final product.
PubMed: 38318340
DOI: 10.3389/fmicb.2024.1333466 -
Open Forum Infectious Diseases Jan 2024Mucormycosis is a potentially lethal mycosis. We reviewed peer-reviewed publications on mucormycosis to assess therapeutic outcomes. (Review)
Review
BACKGROUND
Mucormycosis is a potentially lethal mycosis. We reviewed peer-reviewed publications on mucormycosis to assess therapeutic outcomes.
METHODS
A systematic literature search using the Ovid MEDLINE and EMBASE databases identified manuscripts describing human mucormycosis diagnosed according to European Organization for Research and Treatment of Cancer and the Mycoses Study Group criteria with therapeutic outcomes published from 2000 to 2022.
RESULTS
In 126 articles, 10 335 patients were described, most from Asia (n = 6632, 66%). Diabetes was the most frequent underlying disease (n = 6188, 60%); 222 (2.1%) patients had no underlying diseases. The dominant clinical form was rhino-orbitocerebral (n = 7159, 69.3%), followed by pulmonary (n = 1062, 10.3%). Of 5364 patients with outcome data, amphotericin B monotherapy (n = 3749, mortality 31.5%) was most frequent, followed by amphotericin B + azole (n = 843, mortality 6.6%; < .0001), amphotericin B followed by azole (n = 357, mortality 13.7%; < .0001), posaconazole only (n = 250, mortality 17.2%; < .0001), and isavuconazole only (n = 65, mortality 24.6%; = .24). Duration and dose of antifungals varied widely. Documented outcomes from surgical resections in 149 patients found that 47 of 125 died (37.6%), compared with 16 of 24 (66.7%) patients who did not undergo surgery ( = .008).
CONCLUSIONS
Mucormycosis is more frequently reported in Asia than in Europe and is often linked to diabetes. Antifungal therapy, usually with surgery, is frequently effective for mucormycosis.
PubMed: 38288347
DOI: 10.1093/ofid/ofad704 -
Glycobiology Apr 2024The marine environment, contains plentiful renewable resources, e.g. macroalgae with unique polysaccharides, motivating search for enzymes from marine microorganisms to...
The marine environment, contains plentiful renewable resources, e.g. macroalgae with unique polysaccharides, motivating search for enzymes from marine microorganisms to explore conversion possibilities of the polysaccharides. In this study, the first GH17 glucanosyltransglycosylase, MlGH17B, from a marine bacterium (Muricauda lutaonensis), was characterized. The enzyme was moderately thermostable with Tm at 64.4 °C and 73.2 °C, but an activity optimum at 20 °C, indicating temperature sensitive active site interactions. MlGH17B uses β-1,3 laminari-oligosaccharides with a degree of polymerization (DP) of 4 or higher as donors. Two glucose moieties (bound in the aglycone +1 and +2 subsites) are cleaved off from the reducing end of the donor while the remaining part (bound in the glycone subsites) is transferred to an incoming β-1,3 glucan acceptor, making a β-1,6-linkage, thereby synthesizing branched or kinked oligosaccharides. Synthesized oligosaccharides up to DP26 were detected by mass spectrometry analysis, showing that repeated transfer reactions occurred, resulting in several β-1,6-linked branches. The modeled structure revealed an active site comprising five subsites: three glycone (-3, -2 and -1) and two aglycone (+1 and +2) subsites, with significant conservation of substrate interactions compared to the only crystallized 1,3-β-glucanosyltransferase from GH17 (RmBgt17A from the compost thriving fungus Rhizomucor miehei), suggesting a common catalytic mechanism, despite different phylogenetic origin, growth environment, and natural substrate. Both enzymes lacked the subdomain extending the aglycone subsites, found in GH17 endo-β-glucanases from plants, but this extension was also missing in bacterial endoglucanases (modeled here), showing that this feature does not distinguish transglycosylation from hydrolysis, but may rather relate to phylogeny.
Topics: Phylogeny; Oligosaccharides; Flavobacteriaceae; Polysaccharides; Substrate Specificity
PubMed: 38271624
DOI: 10.1093/glycob/cwae007