Improving thermostability of Bacillus amyloliquefaciens alpha-amylase by multipoint mutations.

The medium-temperature alpha-amylase of Bacillus amyloliquefaciens is widely used in the food and washing process. Enhancing the thermostability of alpha-amylases and investigating the mechanism of stability are important for enzyme industry development. The optimal temperature and pH of the wild-type BAA and mutant MuBAA (D28E/V118A/S187D/K370 N) were all 60 °C and 6.0, respectively. The mutant MuBAA showed better thermostability at 50 °C and 60 °C, with a specific activity of 206.61 U/mg, which was 99.1% greater than that of the wild-type. By analyzing predicted structures, the improving thermostability of the mutant MuBAA was mainly related to enhanced stabilization of a loop region in domain B via more calcium-binding sites and intramolecular interactions around Asp187. Furthermore, additional intramolecular interactions around sites 28 and 370 in domain A were also beneficial for improving thermostability. Additionally, the decrease of steric hindrance at the active cavity increased the specific activity of the mutant MuBAA. Improving the thermostability of BAA has theoretical reference values for the modification of alpha-amylases.

Considerations regarding affinity determinants for aflatoxin B1 in binding cavity of fungal laccase based on in silico mutational and in vitro verification studies.

Laccase, a multicopper oxidase, is well known for its industrial potentials to remove environmental pollutants due to its low substrate specificity to oxidize phenols and thus catalytic versatility. Many efforts focused on the metabolic mechanism, yet to decipher the structural determinants responsible for the differentiation between substrates. Aflatoxin B1 (AFB1), a new substrate for laccase, is a mycotoxin with a formidable environmental threat to public health and food safety. In the present study, we combined biochemical, in silico mutational and molecular-docking data to gain an insight to the function of key residues in the active cavity close to the T1 copper site in a characterized recombinant laccase from Cerrena unicolor (rCuL). Kinetic data for computer-assisted virtual mutants established the binding affinity of hydrogen bonds and residues (Asn336, Asp207, Val391, and Thr165) in rCuL to AFB1. The augmented binding affinity to AFB1 may be related to the conformational rearrangements of the laccase and its ability to hydrogen-bond with the substrate. Furthermore, the optimal pH and temperature for rCuL and variants mediated AFB1 degradation may depend on their pH stability and thermostability. Our findings reinforce the importance of the structure-function relationship of fungal laccases in degrading AFB1, providing mechanistic guidance for future biocatalyst and bioengineering applications.

Characteristics of Crosslinking Polymers Play Major Roles in Improving the Stability and Catalytic Properties of Immobilized Thermomyces lanuginosus Lipase.

This study aimed to improve the stability and catalytic properties of Thermomyces lanuginosus lipase (TLL) adsorbed on a hydrophobic support. At the optimized conditions (pH 5 and 25 °C without any additions), the Sips isotherm model effectively fitted the equilibrium adsorption data, indicating a monolayer and the homogenous distribution of immobilized lipase molecules. To preserve the high specific activity of adsorbed lipase, the immobilized lipase (IL) with a moderate loading amount (approximately 40% surface coverage) was selected. Polyethylenimine (PEI) and chitosan (CS) were successfully applied as bridging units to in situ crosslink the immobilized lipase molecules in IL. At the low polymer concentration (0.5%, w/w) and with 1 h incubation, insignificant changes in average pore size were detected. Short-chain PEI and CS (MW ≤ 2 kDa) efficiently improved the lipase stability, i.e., the lipase loss decreased from 40% to <2%. Notably, CS performed much better than PEI in maintaining lipase activity. IL crosslinked with CS-2 kDa showed a two- to three-fold higher rate when hydrolyzing p-nitrophenyl butyrate and a two-fold increase in the catalytic efficiency in the esterification of hexanoic acid with butanol. These in situ crosslinking strategies offer good potential for modulating the catalytic properties of TLL for a specific reaction.

Flow-through enzymatic reactors using polymer monoliths: From motivation to application.

The application of monolithic materials as carriers for enzymes has rapidly expanded to the realization of flow-through analysis and bioconversion processes. This expansion is partly attributed to the absence from diffusion limitation in many monoliths-based enzyme reactors. Particularly, the relatively ease of introducing functional groups renders polymer monoliths attractive as enzyme carriers. After summarizing the motivation to develop enzymatic reactors using polymer monoliths, this review reports the most recent applications of such reactors. Besides, the present review focuses on the crucial characteristics of polymer monoliths affecting the immobilization of enzymes and the processing parameters dictating the performance of the resulting enzymatic reactors. This review is intended to provide a guideline for designing and applying flow-through enzymatic reactors using polymer monoliths.

Analyses of transcriptomics and metabolomics reveal pathway of vacuolar Sur7 contributed to biocontrol potential of entomopathogenic Beauveria bassiana.

Beauveria bassiana is a critical entomopathogenic fungus for pest biocontrol, whose efficiency depends on fungal development and stress resistance. Unlike its revealed location in plasma membrane patches in other organisms, B. bassiana Sur7 specifically localized in vacuoles. This vacuolar Sur7 was previously demonstrated to affect stress tolerance, hyphal development and virulence. There, however, remain more mechanistic details to be explored. In this study, transcriptomics and metabolomics were applied to investigate the mechanism of vacuolar Sur7. Analyses of transcriptomics and metabolomics displayed many differentially expressed genes and abundant metabolites in response to Sur7 loss, respectively. Together with genes associated with vacuolar biofunction (including transportation and hydrolysis), the altered metabolites contributed to cell wall construction and stress resistance. Particularly, an N-acetylglucosamine-associated Brg1/Nrg1 pathway was enriched and partially affected by Sur7. Absence of Sur7 changed the expression level of Brg1/Nrg1 pathway-related transcript factors, which interfered with downstream phenotype of sporulation. In addition, Sur7 was involved in the accumulation of sphingoid bases, which may affect sphingolipid-related signaling pathway. Although experimental evidence is further required, our studies provide a preliminary framework for future exploring the regulatory mechanism of Sur7, and give a new version of metabolic agency connecting Sur7 and downstream signaling pathway.

Correction to: Screening of pectinase-producing bacteria from citrus peel and characterization of a recombinant pectate lyase with applied potential.

In the original article.

Archives of microbiology: screening of pectinase-producing bacteria from citrus peel and characterization of a recombinant pectate lyase with applied potential.

Pectinase is widely used in numerous industrial fields, including the food, wine, and paper industries. In this work, seven bacteria were isolated from orange peel and their pectinase production activity was assayed. One bacterium (OR-B2) identified as a Bacillus sp. showed the highest enzyme activity towards others. A gene encoding a pectate lyase designed as PelB-B2 in this work was amplified and heterogeneous expressed in E.coli. PelB-B2 was defined as a member of the PelB pectate lyase family after phylogenic tree analysis. 3D model of PelB-B2 was constructed by SWISS-MODEL and PelB-B2 showed conserved para-ß structure. After inducing culture and purified by Ni-affinity chromatography, the properties of the purified PelB-B2 were assayed. Optimal pH and temperature for PelB-B2 was pH 8.0 and 50 °C, respectively. PelB-B2 showed excellent pH stability and thermostability. It was stable within pH range 3.0-11.0 and retained more than 51% activity after incubation at 40 °C, 50 °C, or 60 °C for 1 h. Furthermore, we determined that PelB-B2 was a Ca2+-dependent pectinase and the pectin extracted from citrus was the benefit substrate for PelB-B2. The Km and Vmax of PelB-B2 were 1.64 g/L and 232.56 mol/(L min), respectively. The OR-B2 can be a new resource for pectinase production and the PelB-B2 has potential for industrial application. 7 bacteria were isolated from orange peel, namely OR-B1 to OR-B7 and their pectinase activities were assayed. One pectate lyase belongs to PelB family was cloned from OR-B2 and heterogeneous expressed in E. coli. Purified PelB-B2 was further studied with its properties. Effects of pH, temperature, chemicals, substrate on the enzyme activity were assayed and the enzyme kinetic was also measured.

Mushroom extracts and compounds with suppressive action on breast cancer: evidence from studies using cultured cancer cells, tumor-bearing animals, and clinical trials.

This article reviews mushrooms with anti-breast cancer activity. The mushrooms covered which are better known include the following: button mushroom Agaricus bisporus, Brazilian mushroom Agaricus blazei, Amauroderma rugosum, stout camphor fungus Antrodia camphorata, Jew's ear (black) fungus or black wood ear fungus Auricularia auricula-judae, reishi mushroom or Lingzhi Ganoderma lucidum, Ganoderma sinense, maitake mushroom or sheep's head mushroom Grifola frondosa, lion's mane mushroom or monkey head mushroom Hericium erinaceum, brown beech mushroom Hypsizigus marmoreus, sulfur polypore mushroom Laetiporus sulphureus, Lentinula edodes (shiitake mushroom), Phellinus linteus (Japanese "meshimakobu," Chinese "song gen," Korean "sanghwang," American "black hoof mushroom"), abalone mushroom Pleurotus abalonus, king oyster mushroom Pleurotus eryngii, oyster mushroom Pleurotus ostreatus, tuckahoe or Fu Ling Poria cocos, and split gill mushroom Schizophyllum commune. Antineoplastic effectiveness in human clinical trials and mechanism of anticancer action have been reported for Antrodia camphorata, Cordyceps sinensis, Coriolus versicolor, Ganoderma lucidum, Grifola frondosa, and Lentinula edodes.

High-throughput amplicon sequencing demonstrates extensive diversity of xylanase genes in the sediment of soda lake Dabusu.

OBJECTIVE: To explore the diversity of glycoside hydrolase family 10 xylanase genes in the sediment of soda lake Dabusu by using high-throughput amplicon sequencing based on the Illumina HiSeq2500 platform. RESULTS: A total of 227,420 clean reads, representing approximately 49.5 M bp, were obtained. Operational taxonomic unit (OTU) classification, with a 95% sequence identity cut-off, resulted in 467 OTUs with 392 annotated as GH10 xylanase, exhibiting 35-99% protein sequence identity with their closest-related xylanases in GenBank. Above 75% of the total OTUs demonstrated less than 80% identity with known xylanases. In addition, xylanases derived from the sediment were found to be affiliated to 12 different phyla, with Bacteroidetes, Proteobacteria, Actinobacteria, Firmicutes, Verrucomicrobia, and Basidiomycota being the dominant phyla. Moreover, barcode sequence had a major effect on abundance with only a minor effect on diversity. CONCLUSIONS: High-throughput amplicon sequencing offers insight into xylanase gene diversity at a substantially higher resolution and lesser cost than library cloning and Sanger sequencing, facilitating a more thorough understanding of xylanase distribution and ecology.

Toward Achieving Highly Ordered Fluorinated Surfaces of Spin-Coated Polymer Thin Films by Optimizing the Air/Liquid Interfacial Structure of the Casting Solutions.

Thin polymer films with well-assembled fluorinated groups on their surfaces are not easily achieved via spin-coating film-fabrication methods because the solution solidifies very rapidly during spin-coating, which hinders the fluorinated moieties from segregating and organizing on the film surface. In this contribution, we have proposed a comprehensive strategy toward achieving well-ordered fluorinated thin films surfaces by optimizing the molecular organization at air/liquid interface of the film-formation solutions. To validate such a route, poly(methyl methacrylate) (PMMA) end-capped with several 2-perfluorooctylethyl methacrylate (FMA) units was employed as the model polymer for investigations. The air/solution interfacial structures were optimized by systematically changing the polymer chain structures and properties of the casting solvents. It was found that the polymers that form loosely associated aggregates (e.g., FMA1- ec-PMMA65- ec-FMA1) and a solvent with better solubility to FMA while having not too low surface tension (i.e., toluene) can combine to produce solutions with well-assembled FMA at the interfaces. By spin-coating the solutions with well-organized interfaces, an ultrathin film with perfluorinated groups that were highly oriented toward the film surface was readily achieved, exhibiting surface energies as low as 7.2 mJ/m2, which is among the lowest reported so far for the spin-coated thin films, and a very high F/C ratio (i.e., 0.98).

Functional metabolomics approach reveals the reduced biosynthesis of fatty acids and TCA cycle is required for pectinase activity in Bacillus licheniformis.

Increase of pectinase activity is especially important in fermentation industry. Understanding of the metabolic mechanisms can find metabolic modulation approach to promote high yield of pectinase. Higher activity of pectinase was detected in DY1 than DY2, two strains of Bacillus licheniformis. GC-MS-based metabolomics identified differential metabolome of DY2 compared with DY1, characterizing the increased TCA cycle and biosynthesis of fatty acids. Elevated activity of pyruvate dehydrogenase (PDH), α-ketoglutaric dehydrogenase (KGDH) and succinate dehydrogenase (SDH) showed global elevation of carbon metabolism, which is consistent with the result that lowers glucose in DY2 than DY1. Inhibitors malonate, furfural and triclosan, of PDH, SDH and biosynthesis of fatty acids, promoted pectinase activity, where triclosan increased pectinase activity by 179%. These results indicate that functional metabolomics is an effective approach to understand metabolic mechanisms of fermentation production and provides clues to develop new methods for changing bacterial physiology and production.

The disruption of two salt bridges of the cold-active xylanase XynGR40 results in an increase in activity, but a decrease in thermostability.

Cold-active xylanases are of great interest due to their large potential for application in the food industry. In this study, salt bridges of the eight glycoside hydrolase (GH) family 10 cold-active xylanases reported to date were predicted and the salt bridges specific to the cold-active xylanase XynGR40 were identified. Seven mutants were constructed to disrupt salt bridges specific to XynGR40. The results suggested that five mutants lost their xylanase activity, while the other two mutants, D30N and D83N, displayed different properties when compared with the wild-type XynGR40. First, both mutations showed an obvious decrease in thermostability, with the T1/2 of D30N and D83N at 50 °C being about one half and one sixth of the wild-type, respectively. Second, both D30N and D83N had a higher specific activity than the wild-type, with activities about 13 and 163% higher, respectively. Third, both D30N and D83N had high kcat and Km values, which resulted in a higher catalytic efficiency of the mutant D83N, but a lower catalytic efficiency of the mutant D30N compared to the wild-type. Our results suggested that salt bridges play important roles in both the activity and thermostability of the cold-active xylanase XynGR40. The mutant D83N had a higher kcat and higher relative activity at low temperatures than the wild-type, and is a good candidate for application in the food industry.

Destaining of Coomassie Brilliant Blue R-250-stained polyacrylamide gels with fungal laccase.

An enzyme-based method for destaining polyacrylamide gels stained with Coomassie Brilliant Blue R-250 is described. Distilled water supplemented with diluted fermentation broth of a laccase-producing white-rot fungus, Cerrena sp., was used for gel destaining, and a clear gel background was obtained in 2 h at 37 °C. Sensitivity of protein detection was 10 ng. The method did not require organic solvents or changing the destaining solution. Due to simultaneous gel destaining and dye decolorization, the colorless destaining solution can be disposed of directly. Laccase destaining of polyacrylamide gels was simple, efficient, and environmentally friendly.

Fungal proteinaceous compounds with multiple biological activities.

Fungi comprise organisms like molds, yeasts and mushrooms. They have been used as food or medicine for a long time. A large number of fungal proteins or peptides with diverse biological activities are considered as antibacterial, antifungal, antiviral and anticancer agents. They encompass proteases, ribosome inactivating proteins, defensins, hemolysins, lectins, laccases, ribonucleases, immunomodulatory proteins, and polysaccharopeptides. The target of the present review is to update the status of the various bioactivities of these fungal proteins and peptides and discuss their therapeutic potential.

Laccase Gene Family in Cerrena sp. HYB07: Sequences, Heterologous Expression and Transcriptional Analysis.

Laccases are a class of multi-copper oxidases with industrial potential. In this study, eight laccases (Lac1-8) from Cerrena sp. strain HYB07, a white-rot fungus with high laccase yields, were analyzed. The laccases showed moderate identities to each other as well as with other fungal laccases and were predicted to have high redox potentials except for Lac6. Selected laccase isozymes were heterologously expressed in the yeast Pichia pastoris, and different enzymatic properties were observed. Transcription of the eight laccase genes was differentially regulated during submerged and solid state fermentation, as shown by quantitative real-time polymerase chain reaction and validated reference genes. During 6-day submerged fermentation, Lac7 and 2 were successively the predominantly expressed laccase gene, accounting for over 95% of all laccase transcripts. Interestingly, accompanying Lac7 downregulation, Lac2 transcription was drastically upregulated on days 3 and 5 to 9958-fold of the level on day 1. Consistent with high mRNA abundance, Lac2 and 7, but not other laccases, were identified in the fermentation broth by LC-MS/MS. In solid state fermentation, less dramatic differences in transcript abundance were observed, and Lac3, 7 and 8 were more highly expressed than other laccase genes. Elucidating the properties and expression profiles of the laccase gene family will facilitate understanding, production and commercialization of the fungal strain and its laccases.

Highly efficient expression and characterization of a ß-mannanase from Bacillus subtilis in Pichia pastoris.

A ß-mannanase gene (Man5) from Bacillus subtilis BS5 was cloned by PCR and integrated into the genome of Pichia pastoris GS115 via pPIC9 vector. The recombinant Man5 with a molecular mass of 43 kDa was successfully expressed and secreted into the culture medium. After methanol induction in a shake flask for 96 H, the recombinant Man5 protein reached 375 µg/mL in concentration, with an enzyme activity of 892 U/mL. The recombinant Man5 was purified 3.35-fold with 60% yield by using HiTrap DEAE FF and HiTrap Phenyl FF columns. The specific activity of the purified enzyme was 7,978 U/mg. The optimum temperature and pH of the recombinant Man5 were 50 °C and 6.0, respectively. Studies of substrate specificity showed that the optimum substrate for the Man5 was konjac flour, suggesting that it has great potential as an effective additive in the food industry.

Transcriptomic and metabolomic analysis unveils a negative effect of glutathione metabolism on laccase activity in Cerrena unicolor 87613.

The white rot fungus Cerrena unicolor 87613 has been previously shown to be a promising resource in laccase production, an enzyme with significant biotechnological applications. Conventional methods face technical challenges in improving laccase activity. Attempts are still being made to develop novel approaches for further enhancing laccase activity. This study aimed to understand the regulation of laccase activity in C. unicolor 87613 for a better exploration of the novel approach. Transcriptomic and metabolomic analyses were performed to identify key genes and metabolites involved in extracellular laccase activity. The findings indicated a strong correlation between the glutathione metabolism pathway and laccase activity. Subsequently, experimental verifications were conducted by manipulating the pathway using chemical approaches. The additive reduced glutathione (GSH) dose-dependently repressed laccase activity, while the GSH inhibitors (APR-246) and reactive oxygen species (ROS) inducer (H2O2) enhanced laccase activity. Changes in GSH levels could determine the intracellular redox homeostasis in interaction with ROS and partially affect the expression level of laccase genes in C. unicolor 87613 in turn. In addition, GSH synthetase was found to mediate GSH abundance in a feedback loop. This study suggests that laccase activity is negatively influenced by GSH metabolism and provides a theoretical basis for a novel strategy to enhance laccase activity by reprogramming glutathione metabolism at a specific cultivation stage.IMPORTANCEThe production of laccase activity is limited by various conventional approaches, such as heterologous expression, strain screening, and optimization of incubation conditions. There is an urgent need for a new strategy to meet industrial requirements more effectively. In this study, we conducted a comprehensive analysis of the transcriptome and metabolome of Cerrena unicolor 87613. For the first time, we discovered a negative role played by reduced glutathione (GSH) and its metabolic pathway in influencing extracellular laccase activity. Furthermore, we identified a feedback loop involving GSH, GSH synthetase gene, and GSH synthetase within this metabolic pathway. These deductions were confirmed through experimental investigations. These findings not only advanced our understanding of laccase activity regulation in its natural producer but also provide a theoretical foundation for a strategy to enhance laccase activity by reprogramming glutathione metabolism at a specific cultivation stage.

Correlation between Protein Features and the Properties of pH-Driven-Assembled Nanoparticles: Control of Particle Size.

This study sought to understand how the features of proteins impact the properties of nanoparticles assembled using the pH-shifting approach and the mechanism behind. Four legume protein isolates from faba bean, mung bean, soy, and pea were fractionated into natural aqueous-soluble (Sup) and aqueous-insoluble (Sed) fractions, which were proved to serve as shell and core, respectively, for the pH-driven-assembled nanoparticles. Using zein instead of Sed fractions as the core improved size uniformity, and particle size can be precisely controlled by adjusting core/shell ratios. Using the proteomic technique and silico characterization, the features of identified proteins indicated that hydrophobicity rather than molecular weight, surface charge, etc., mainly determined particle size. With molecular docking, structural analysis, and dissociation tests, the assembly of zein/Sup-based nanoparticles was dominantly driven by hydrophobic interactions. This study provides constructive information on the correlation between protein features and the properties of pH-driven-assembled nanoparticles, achieving a precise control of particle size.

Characterisation of a laccase isolated from Trametes hirsuta and its application in the oligomerisation of phenolic compounds.

Phenolic compounds are widely distributed in nature and industrial environment, and their detoxification or bioactive enhancement is of great value to environmental protection and industrial development. Laccases are multicopper oxidases that catalyse the oligo- or polymerisation of phenolic compounds. Identifying new laccase producers and investigating their application potential are of great importance. In this study, a white-rot fungus, Trametes hirsuta EZ1, with significantly high laccase productivity was isolated. The optimum conditions were studied for the maximum fermentation of extracellular laccase, which was achieved at 150 U/mL with a medium containing 10% strain EZ1, 7% maltodextrin, 1.5% peptone, and 0.5 mM Cu2+, and incubation at initial pH 6.0, 32 °C, and 180 rpm for nine days. Subsequently, a 70-kDa laccase was purified that showed activity over a wide range of temperature and pH, sensitivity to many metal ions and sodium dodecyl sulphate, and high tolerance to organic solvents. Purified laccase showed a significant unreported effect by catalysing catechol or ferulic acid into dimers, trimers, and tetramers or caffeic acid into dimers, trimers, tetramers, and pentamers. The oligomeric mixtures exhibited increased antioxidative capacity compared to that of each parent monomer, except for caffeic acid derivatives. Our study offers a novel strain source for laccase production and broadens its application in the enhancement of bioactive compounds.

[An epidemiological survey of febrile convulsions among pupils in the Wenzhou region].

OBJECTIVE: To study the prevalence and clinical features of febrile convulsion (FC) among pupils in the Wenzhou region, Zhejiang Province, China. METHODS: Using a random stratified cluster sampling method, 6406 children under 12 years from two primary schools of urban areas and two primary schools of rural areas were surveyed. RESULTS: The prevalence of FC was 3.67% (235/6406). Most children (75.7%) experienced their first onset of FC at 6 months to 3 years of age (median: 16 months). The seizures were generalized (95.3%, 224/235), with a duration of less than 10 minutes (86.4%, 203/235). FC was developed into epilepsy in 13 children (5.5%) who all suffered from complex FC. Relapses were noted in 88 cases (37.4%), among whom 38 patients had only 1 recurrence and 50 patients had 2 or more relapses. EEG was performed in 200 cases, among whom 12(6.0%) showed abnormalities. CONCLUSIONS: The prevalence of FC is 3.67% among pupils in the Wenzhou region. The seizures are generalized, with a short duration. A part of complex FC can be developed into subsequent epilepsy.