D-allulose 3-epimerase for low-calorie D-allulose synthesis: microbial production, characterization, and applications.

D-allulose, an epimer of D-fructose at C-3 position, is a low-calorie rare sugar with favorable physiochemical properties and special physiological functions, which displays promising perspectives in the food and pharmaceutical industries. Currently, D-allulose is extremely sparse in nature and is predominantly biosynthesized through the isomerization of D-fructose by D-allulose 3-epimerase (DAEase). In recent years, D-allulose 3-epimerase as the key biocatalyst for D-allulose production has received increasing interest. The current review begins by providing a summary of D-allulose regarding its characteristics and applications, as well as different synthesis pathways dominated by biotransformation. Then, the research advances of D-allulose 3-epimerase are systematically reviewed, focusing on heterologous expression and biochemical characterization, crystal structure and molecular modification, and application in D-allulose production. Concerning the constraint of low yield of DAEase for industrial application, this review addresses the various attempts made to promote the production of DAEase in different expression systems. Also, various strategies have been adopted to improve its thermotolerance and catalytic activity, which is mainly based on the structure-function relationship of DAEase. The application of DAEase in D-allulose biosynthesis from D-fructose or low-cost feedstocks through single- or multi-enzymatic cascade reaction has been discussed. Finally, the prospects for related research of D-allulose 3-epimerase are also proposed, facilitating the industrialization of DAEase and more efficient and economical bioproduction of D-allulose.

Adsorption and Assembly of Octenyl Succinic Anhydride Starch/Chitosan Electrostatic Complexes at Oil-Water Interface.

Biopolymer electrostatic complexes are popular Pickering stabilizers whose structures greatly affect their interfacial properties. This study comprehensively demonstrated the interfacial adsorption and assembly of dissolved octenyl succinic anhydride (OSA) starch (OSA-D)/chitosan (CS) electrostatic complexes with different structures through complementary characterization methods. We found that compared with single-component systems, OSA-D/CS complexes exhibited significantly increased wetting stability and adsorption rate to the interface, which was reinforced by molecular dynamics simulations. Their soft structures and the entanglement of molecular chains led to the formation of thick and highly viscoelastic multilayer adsorbed films, which greatly resisted deformation against shearing forces. The adsorption and assembly of the complexes were strongly influenced by OSA-D/CS ratios and pH, which could be related to the different interfacial interaction strengths. Overall, the electrostatic complexation, structural characteristics, and interfacial properties of OSA-D/CS complexes were well related, thereby providing valuable information for the regulation of controlled interfaces and bulk system properties.

Formation Mechanism of Starch-Based Double Emulsions from the Interfacial Perspective.

Double emulsions are of significant practical value in protecting the core material owing to their multicomponent structure and have thus been applied in various fields, such as food, cosmetics, and drugs. However, the mechanism of double emulsion formation by native starch is not well established. Herein, we demonstrate a facile route to develop type-A, type-B, and type-C double emulsions using native starch and develop an innovative design for a carrier. Interfacial interaction, enthalpy changes of starch, and interfacial properties are key factors governing the formation of double emulsions and controlling the type of double emulsions formed. Therefore, the results of this study provide a better understanding of how and what type of starch-based double emulsions are formed.

Expression, purification, characterization, and patient IgE reactivity of new macadamia nut iso-allergen.

Structural and functional information about food allergens is essential for understanding the allergenicity of food proteins. All allergens belong to a small number of protein families. Various allergens from different families have been successfully produced recombinantly in E. coli for their characterization and applications in allergy diagnosis and treatment. However, recombinant hexameric 11S seed storage protein has not been reported, although numerous 11S legumins are known to be food allergens, including the recently identified macadamia nut allergen Mac i 2. Here we report the production of a macadamia nut legumin by expressing it in E. coli with a substrate site of HRV 3C protease and cleaving the purified protein with HRV 3C protease. The protease divided the protein into two chains and left a native terminus for the C-terminal chain, resulting in a recombinant hexameric 11S allergen for the first time after the residues upstream to the cleavage site flipped out of the way of the trimer-trimer interaction. The 11S allergens are known to have multiple isoforms in many species. The present study removed an obstacle in obtaining homogeneous allergens needed for studying allergens and mitigating allergenicity. Immunoreactivity of the protein with serum IgE confirmed it to be a new isoform of Mac i 2.

An extensive review: How starch and gluten impact dough machinability and resultant bread qualities.

Wheat flour can form dough with a three-dimensional viscoelastic structure that is responsible for gas holding during fermentation and oven-rise, creating a typical fixed, open-cell foam structure of bread after baking. As the major components of dough, the continuous reticular skeleton formed by gluten proteins and the concentrated starch granules entrapped in gluten matrix predominantly determine dough rheological behaviors and bread qualities. This review surveys the latest literatures and draws out a conclusion from a plethora of information related to the filling effects of starch granules on gluten matrix and the cross-linking mechanisms between gluten proteins and starch granules, which is of great significance to provide sufficient scientific knowledge for development of bread with satisfactory attributes and quality control of end products.

A review of controlled release from cyclodextrins: release methods, release systems and application.

The controlled release of guest molecules from cyclodextrin (CD) inclusion complexes is very important for specific industrial applications in foods, medicine, cosmetics, textiles, agriculture, environmental protection, and chemical materials. The term "controlled release" encompasses several related methods, including those referred to as immediate release, sustained release and targeted release. Many different CD-based controlled release systems are currently used in practical applications. CD inclusion complexes, CD coupling, supramolecular hydrogels, and supramolecular micelles are among the most common. This review systematically introduces the principles and applications of CD-based controlled release systems, providing a theoretical basis for improving the bioavailability of effective substances and broadening their range of application.

Designing liquefaction and saccharification processes of highly concentrated starch slurry: Challenges and recent advances.

Starch-based sugars are an important group of starch derivatives used in food, medicine, chemistry, and other fields. The production of starch sugars involves starch liquefaction and saccharification processes. The production cost of starch sugars can be reduced by increasing the initial concentration of starch slurry. However, the usage of the highly concentrated starch slurry is characterized by challenges such as low reaction efficiency and poor product performance during the liquefaction and saccharification processes. In this study, we endeavored to provide a reference guide for improving high-concentration starch sugar production. Thus, we reviewed the effects of substrate concentration on the starch sugar production process and summarized several potential strategies. These regulation strategies, such as physical field pretreatment, complex enzyme-assisted, and temperature control, can significantly increase the starch concentration and mitigate the challenges of using highly concentrated starch slurry. We believe that highly concentrated starch sugar production will achieve a qualitative leap in the future. This review provides theoretical guidance and highlights the importance of high concentration in starch-based sugar production. Further studies are needed to explore the fine structure and enzyme attack mode during the liquefaction and saccharification processes to regulate the production of more targeted products.

S6K1 amplification confers innate resistance to CDK4/6 inhibitors through activating c-Myc pathway in patients with estrogen receptor-positive breast cancer.

BACKGROUND: CDK4/6 inhibitors combined with endocrine therapy has become the preferred treatment approach for patients with estrogen receptor-positive metastatic breast cancer. However, the predictive biomarkers and mechanisms of innate resistance to CDK4/6 inhibitors remain largely unknown. We sought to elucidate the molecular hallmarks and therapeutically actionable features of patients with resistance to CDK4/6 inhibitors. METHODS: A total of 36 patients received palbociclib and endocrine therapy were included in this study as the discovery cohort. Next-generation sequencing of circulating tumour DNA in these patients was performed to evaluate somatic alterations associated with innate resistance to palbociclib. Then the candidate biomarker was validated in another independent cohort of 104 patients and publicly available datasets. The resistance was verified in parental MCF-7 and T47D cells, as well as their derivatives with small interfering RNA transfection and lentivirus infection. The relevant mechanism was examined by RNA sequencing, chromatin immunoprecipitation and luciferase assay. Patient-derived organoid and patient-derived xenografts studies were utilized to evaluated the antitumor activity of rational combinations. RESULTS: In the discovery cohort, S6K1 amplification (3/35, 9%) was identified as an important reason for innate resistance to CDK4/6 inhibitors. In the independent cohort, S6K1 was overexpressed in 15/104 (14%) patients. In those who had received palbociclib treatment, patients with high-expressed S6K1 had significantly worse progression free survival than those with low S6K1 expression (hazard ratio = 3.0, P = 0.0072). Meta-analysis of public data revealed that patients with S6K1 amplification accounted for 12% of breast cancers. Breast cancer patients with high S6K1 expression had significantly worse relapse-free survival (hazard ratio = 1.31, P < 0.0001). In breast cancer cells, S6K1 overexpression, caused by gene amplification, was sufficient to promote resistance to palbociclib. Mechanistically, S6K1 overexpression increased the expression levels of G1/S transition-related proteins and the phosphorylation of Rb, mainly through the activation of c-Myc pathway. Notably, this resistance could be abrogated by the addition of mTOR inhibitor, which blocked the upstream of S6K1, in vitro and in vivo. CONCLUSIONS: S6K1 amplification is an important mechanism of innate resistance to palbociclib in breast cancers. Breast cancers with S6K1 amplification could be considered for combinations of CDK4/6 and S6K1 antagonists.

OsciDrop: A Versatile Deterministic Droplet Generator.

This paper describes OsciDrop, a versatile chip-free droplet generator used to produce size-tunable droplets on demand. Droplet generation is fundamental to miniaturized analysis. We designed OsciDrop to segment the fluid flowing out of the orifice of a disposable pipette tip into droplets by oscillating its distal end underneath an immiscible continuous phase. We described the theoretical model and investigated the effect of flow rate, oscillating amplitude, frequency, and waveform on droplet generation. Our study revealed a previously underexplored Weber number-dominated regime that leverages inertial force instead of viscous force to generate droplets. The same pipette tip allowed robust and deterministic generation of monodisperse droplets with programmable sizes ranging from 200 pL to 2 µL by asymmetrical oscillation. We validated this platform with two droplet-based nucleic acid amplification tests: a digital loop-mediated isothermal amplification assay for absolute quantification of African swine fever virus and a multi-volume digital polymerase chain reaction assay for the high dynamic range measurement of human genomic DNA. The OsciDrop method opens a facile avenue to miniaturization, integration, and automation, exhibiting full accessibility for digital molecular diagnostics.

The modulatory roles and regulatory strategy of starch in the textural and rehydration attributes of dried noodle products.

Noodles are popular staple foods globally, and dried noodle products (DNPs) have gained increasing attention due to recent changes in consumer diet behavior. Rapid rehydration and excellent texture quality are the two major demands consumers make of dried noodle products. Unfortunately, these two qualities conflict with each other: the rapid rehydration of DNPs generally requires a loose structure, which is disadvantageous for good texture qualities. This contradiction limits further development of the noodle industry, and overcoming this limitation remains challenging. Starch is the major component of noodles, and it has two main roles in DNPs. It serves as a skeleton for the noodle in gel networks form or acts as a noodle network filler in granule form. In this review, we comprehensively investigate the different roles of starch in DNPs, and propose strategies for balancing the conflicts between texture and rehydration qualities of DNPs by regulating the gel network and granule structure of starch. Current strategies in regulating the gel network mainly focused on the hydrogen bond strength, the orientation degree, and the porosity; while regulating granule structure was generally performed by adjusting the integrity and the gelatinization degree of starch. This review assists in the production of instant dried noodle products with desired qualities, and provides insights into promising enhancements in the quality of starch-based products by manipulating starch structure.

Influence of different kinds of fatty acids on the behavior, structure and digestibility of high amylose maize starch-fatty acid complexes.

BACKGROUND: The formation of starch-lipid complexes is of interest to food processing and human nutrition. Fatty acid (FA) structure is important for the formation and structure of starch-FA complexes. However, there is limited research regarding the complexing behavior between amylose and different kinds of FAs, as well as the relationship between fine structures and digestibility of the formed complexes. This study aimed to investigate the behavior, fine structure, and digestibility of complexes formed between high amylose maize starch (HMS) and FA having various chain lengths and unsaturation degrees. RESULTS: Complexes containing different FA structures showed V6III -type crystals. Complexes containing 18-carbon unsaturated FAs displayed significantly higher complexing index (P < 0.05) than other complexes. Complexes containing 12-carbon FAs and 18-carbon FAs with one unsaturation degree showed a higher degree of structural order and resistant starch (RS) content than other complexes. The 12-carbon FAs exhibited a higher binding degree with helical cavity of amylose than other FAs. Additionally, 10-carbon and 18-carbon saturated FAs tended to combine with HMS outside amylose helices more than other FAs. Laser confocal micro-Raman imaging revealed that the physically embedded 10-carbon and 18-carbon saturated FAs showed heterogeneous distribution in complexes, and that the complexed 18-carbon FAs with one unsaturation degree exhibited homogeneous distribution. CONCLUSION: The behavior, structural order and digestibility of complexes could be regulated by FA structure. The 12-carbon FAs and 18-carbon FAs with one unsaturation degree were more suitable for the production of HMS-FA complexes with higher structural order and RS content than other FAs. © 2022 Society of Chemical Industry.

[Clinical features and genetic analysis of a Chinese pedigree affected with X-linked adrenoleukodystrophy].

OBJECTIVE: To analyze the clinical features and variants of ABCD1 gene in a Chinese pedigree affected with X-linked adrenoleukodystrophy. METHODS: Clinical data of the proband were collected and analyzed. Potential variant of the ABCD1 gene were analyzed by PCR and Sanger sequencing of the proband, his parents and 100 unrelated healthy individuals. RESULTS: The prominent features of the proband included cerebellar and brainstem lesions, along with increased serum level of very-long chain fatty acids. He was found to harbor a hemizygous c.1509delG (p.L504Sfs*54) variant of the ABCD1 gene, for which his mother was heterozygous. The same variant was not detected in his father and 100 healthy controls. CONCLUSION: X-linked adrenoleukodystrophy has a variety of clinical manifestations. Discovery of the c.1509delG (p.L504Sfs*54), as a novel pathogenic variant of the ABCD1 gene, has enabled diagnosis and genetic counseling for this pedigree.

A temperature-mediated two-step saccharification process enhances maltose yield from high-concentration maltodextrin solutions.

BACKGROUND: Designing a high-concentration (50%, w/w) maltodextrin saccharification process is a green method to increase the productivity of maltose syrup. RESULTS: In this study, a temperature-mediated two-step process using ß-amylase and pullulanase was investigated as a strategy to improve the efficiency of saccharification. During the saccharification process, both pullulanase addition time and temperature adjustment greatly impacted the final maltose yield. These results indicated that an appropriate ß-amylolysis in the first stage (the first 8 h) was required to facilitate saccharification process, with the maltose yield of 8.46% greater than that of the single step saccharification. Molecular structure analysis further demonstrated that a relatively low temperature (50 °C), as compared with a normal temperature (60 °C), in the first stage resulted in a greater number of chains polymerized by at least seven glucose units and a less heterogeneity system within the residual substrate. The molecular structure of the residual substrate might be beneficial for the subsequent cooperation between ß-amylase and pullulanase in the following 40 h (second stage). CONCLUSION: Over a 48 h saccharification, the temperature-mediated two-step process dramatically increased the conversion rate of maltodextrin and yielded significantly more maltose and less byproduct, as compared with a constant-temperature process. The two-step saccharification process therefore offered an efficient and green strategy for maltose syrup production in industry. © 2020 Society of Chemical Industry.

Bacterial 1,4-α-glucan branching enzymes: characteristics, preparation and commercial applications.

The 1,4-α-glucan branching enzymes (GBEs) are ubiquitously distributed in animals, microorganisms and plants. These enzymes modify the structure of both starch and glycogen; changing the frequency and position of branches by forming new α-1,6-glucosidic linkages. In organisms, controlling the number and distribution of branches is an irreplaceable process that maintains the physiological state of starch and glycogen in the cell. The process is also the foundation for the industrial applications of GBEs. So far, a number of GBEs have been identified in eukaryotes and prokaryotes as researchers searched for GBEs with optimal properties. Among them, bacterial GBEs have received particular attention due to the convenience of heterologous expression and industrial applications of GBEs from bacteria than GBEs from other sources. The advantages of bacterial GBEs in potential applications stimulated the investigations of bacterial GBEs in terms of their structure and properties. However, full exploitation of GBEs in commercial applications is still in its infancy because of the disadvantages of currently available enzymes and of limited imagination with respect to future possibilities. Thus, in this review, we present an overview of the bacterial GBEs including their structure, biochemical properties and commercial applications in order to depict the whole picture of bacterial GBEs.

Insights into the thermostability and product specificity of a maltooligosaccharide-forming amylase from Bacillus stearothermophilus STB04.

OBJECTIVES: Analyze the thermostability, mode of action, and product specificity of a maltooligosaccharide-forming amylase from Bacillus stearothermophilus STB04 (Bst-MFA) from the biochemical and structural point of view. RESULTS: Using three-dimensional co-crystal structure of Bst-MFA with acarbose as a guide, experiments were performed to analyze the thermostability, mode of action and product specificity of Bst-MFA. The results showed that the Ca2+-Ca2+-Ca2+ metal triad of Bst-MFA is responsible for its high thermostability. Multiple substrate binding modes, rather than one productive binding mode determined by non-reducing end recognition, are in accordance with an endo-type mode of action. Significant interactions between subsites - 5 and - 6 and glucosyl residues at the non-reducing end explain the maltopentaose (G5) and maltohexaose (G6) specificity of Bst-MFA. CONCLUSIONS: Bst-MFA is a thermostable enzyme that preferentially produces G5 and G6, with an endo-type mode. The understanding of structure-function relationships provides the foundation for future efforts to the modification of Bst-MFA.

Assembled Step Emulsification Device for Multiplex Droplet Digital Polymerase Chain Reaction.

Digital PCR is a powerful method for absolute nucleic acid quantification with unprecedented accuracy and precision. To promote the wider use and application of digital PCR, several major challenges still exist, including reduction of cost, integration of the instrumental platform, and simplification of operations. This paper describes a reusable microfluidic device that generates nanoliter droplet arrays based on step emulsification for the on-chip multiplex digital PCR of eight samples simultaneously. The device contains two glass plates that can be quickly assembled with prefilled mineral oil. Droplets are simply generated through the arrays of step emulsification nozzles driven by a single pressure controller and are self-assembled into monolayer droplet arrays in U-shaped chambers. The use of mineral oil eliminates bubble generation; thus, no overpressure is required during thermocycling. Moreover, the device can be reused many times after disassembly and a brief cleaning procedure, which significantly reduces the cost of the device per dPCR assays. The device was able to detect template DNA at concentrations as low as 10 copies/µL with a dynamic range of approximately 4 logs. We applied this device in the quantitative assessment of HER2 copy number variation, which is important for targeted therapy and prognosis of breast cancer. The performance was validated by 16 clinical samples, obtaining similar results to commercial digital PCR. We envision that this low-cost, reusable, and user-friendly device can be broadly used in various applications.

Expression and characterization of an extremely thermophilic 1,4-α-glucan branching enzyme from Rhodothermus obamensis STB05.

A gene encoding 1,4-α-glucan branching enzyme (GBE, EC 2.4.1.18) from the extremely thermophilic bacterium Rhodothermus obamensis STB05 was successfully cloned and expressed in Escherichia coli. Extracellular expression of the recombinant enzyme (R.o-GBE) was achieved with a yield of 1080 mg/L. Then it was purified and further characterized biochemically. R.o-GBE was optimally active at pH 7.0 and 65 °C. It remained stable at temperatures up to 80 °C and had a half-life at 85 °C of approximately 31 min. Far-UV circular dichroism and intrinsic fluorescence analyses revealed that high temperatures reduced its activity by changing the secondary and tertiary structure of R.o-GBE. The enzyme had broad pH stability between pH 3.0 and 11.0 at 4 °C, and preferred weakly acidic conditions at high temperatures. None of the metal ions enhanced the activity of R.o-GBE, but Ca2+ may be required for its activity. Its specific activity with amylopectin was 6651 U/mg, which is much higher than that reported for other GBEs. Its excellent thermostability, broad pH stability, and high specific activity make R.o-GBE highly suitable for industrial applications.

GALC mutations in Chinese patients with late-onset Krabbe disease: a case report.

BACKGROUND: Krabbe disease (also known as globoid cell leukodystrophy) cause by a deficiency of the enzyme ß-galactocerebrosidase (galactosylceramidase, GALC). The deficiency of GALC leads to accumulation of galactosylceramide and psychosine, the latter GALC substrate having a potential role in triggering demyelination. Typically, the disease has an infantile onset, with rapid deterioration in the first few months, leading to death before the age of 2 years. The late onset forms (late-infantile, juvenile, and adult forms) are rare with variable clinical outcomes, presenting spastic paraplegia as the main symptom. CASE PRESENTATION: We recruited a family with two affected individuals. The proband (Patient 1), a 25-year-old male, was presented with slow progressive symptoms, including spastic gait disturbance and vision loss since the 5th year of life. His elder sister (Patient 2), became wheelchair-bound and demented at the age of 22 years. Brain magnetic resonance imaging (MRI) showed increased signal intensity in the white matter along with the involvement of the bilateral corticospinal tracts. GALC deficiency was confirmed by biochemical analysis. DNA sequencing revealed two mutations (c.865G > C: p. G289R and c.136G > T: p. D46Y) in GALC. The clinical characteristics, brain MRI, biochemical and molecular findings led to the diagnosis of Krabbe disease. CONCLUSION: Clinical and neuroimaged signs, positive enzymatic analysis and molecular data converged to definite diagnosis in this neurodegenerative disease.

Importance of Trp139 in the product specificity of a maltooligosaccharide-forming amylase from Bacillus stearothermophilus STB04.

The maltooligosaccharide-forming amylase from Bacillus stearothermophilus STB04 (Bst-MFA) randomly cleaves the α-1,4 glycosidic linkages of starch to produce predominantly maltopentaose and maltohexaose. The three-dimensional co-crystal structure of Bst-MFA with acarbose highlighted the stacking interactions between Trp139 and the substrate in subsites - 5 and - 6. Interactions like this are thought to play a critical role in maltopentaose/maltohexaose production. A site-directed mutagenesis approach was used to test this hypothesis. Replacement of Trp139 by alanine, leucine, or tyrosine dramatically increased maltopentaose production and reduced maltohexaose production. Oligosaccharide degradation indicated that these mutants also enhance productive binding of the substrate aglycone, leading to a high maltopentaose yield. Therefore, the aromatic stacking between Trp139 and substrate is suggested to control product specificity and the oligosaccharide cleavage pattern.

Effect of non-starch components on the structural properties, physicochemical properties and in vitro digestibility of waxy highland barley starch.

Highland barley (HB) endosperm with an amylose content of 0-10 % is called waxy HB (WHB). WHB is a naturally slow-digesting grain, and the interaction between its endogenous non-starch composition and the WHB starch (WHBS) has an important effect on starch digestion. This paper focuses on the mechanisms by which the components of ß-glucan, proteins and lipids affect the molecular, granular, crystalline structure and digestive properties of WHBS. After eliminating the main nutrients except for starch, the estimated glycemic index (eGI) of the samples rose from 62.56 % to 92.93 %, and the rapidly digested starch content increased from 60.81 % to 98.56 %, respectively. The resistant starch (RS) content, in contrast, dropped from 38.61 % to 0.13 %. Comparatively to lipids, ß-glucan and protein contributed more to the rise in eGI and decline in RS content. The crystalline characteristics of starch were enhanced in the decomposed samples. The samples' gelatinization properties improved, as did the order of the starch molecules. Protein and ß-glucan form a dense matrix on the surface of WHBS particles to inhibit WHBS digestion. In summary, this study revealed the mechanism influencing the digestibility of WHBS from the perspective of endogenous non-starch composition and provided a theoretical basis to develop slow-digesting foods.