Structure-based interface engineering methodology in designing a thermostable amylose-forming transglucosylase.

Many drugs and prebiotics derive their activities from sugar substituents. Due to the prevalence and complexity of these biologically active compounds, enzymatic glycodiversification that facilitates easier access to these compounds can make profound contributions to the pharmaceutical, food, and feed industries. Amylosucrases (ASases) are attractive tools for glycodiversification because of their broad acceptor substrate specificity, but the lack of structural information and their poor thermostability limit their industrial applications. Herein, we reported the crystal structure of ASase from Calidithermus timidus, which displays a homotetrameric quaternary organization not previously observed for other ASases. We employed a workflow composed of five common strategies, including interface engineering, folding energy calculations, consensus sequence, hydrophobic effects enhancement, and B-factor analysis, to enhance the thermostability of C. timidus ASase. As a result, we obtained a quadruple-point mutant M31 ASase with a half-life at 65 °C increased from 22.91 h to 52.93 h, which could facilitate biosynthesis of glucans with a degree of polymerization of more than 20 using sucrose as a substrate at 50 °C. In conclusion, this study provides a structural basis for understanding the multifunctional biocatalyst ASase and presents a powerful methodology to effectively and systematically enhance protein thermostability.

Recent development of phenyllactic acid: physicochemical properties, biotechnological production strategies and applications.

Phenyllactic acid (PLA) is capable of inhibiting the growth of many microorganisms, showing a broad-spectrum antimicrobial property, which allows it to hold vast applications in the: food, feed, pharmaceutical, and cosmetic industries, especially in the field of food safety. Recently, the production of PLA has garnered considerable attention due to the increasing awareness of food safety from the public. Accordingly, this review mainly updates the recent development for the production of PLA through microbial fermentation and whole-cell catalysis (expression single-, double-, and triple-enzyme) strategies. Firstly, the: physicochemical properties, existing sources, and measurement methods of PLA are systematically covered. Then, the inhibition spectrum of PLA is summarized, and synchronously, the antimicrobial and anti-biofilm mechanisms of PLA on commonly pathogenic microorganisms in foods are described in detail, thereby clarifying the reason for extending the shelf life of foods. Additionally, the factors affecting the production of PLA are summarized from the biosynthesis and catabolism pathway of PLA in microorganisms, as well as external environmental parameters insights. Finally, the downstream treatment process and applications of PLA are discussed and outlined. In the future, clinical data should be supplemented with the metabolic kinetics of PLA in humans and to evaluate animal toxicology, to enable regulatory use of PLA as a food additive. A food-grade host, such as Bacillus subtilis and Lactococcus lactis, should also be developed as a cell vector expressing enzymes for PLA production from a food safety perspective.

Spatial organization of pathway enzymes via self-assembly to improve 2'-fucosyllactose biosynthesis in engineered Escherichia coli.

As one of the most abundant components in human milk oligosaccharides, 2'-fucosyllactose (2'-FL) possesses versatile beneficial health effects. Although most studies focused on overexpressing or fine-tuning the expression of pathway enzymes and achieved a striking increase of 2'-FL production, directly facilitating the metabolic flux toward the key intermediate GDP-l-fucose seems to be ignored. Here, multienzyme complexes consisting of sequential pathway enzymes were constructed by using specific peptide interaction motifs in recombinant Escherichia coli to achieve a higher titer of 2'-FL. Specifically, we first fine-tuned the expression level of pathway enzymes and balanced the metabolic flux toward 2'-FL synthesis. Then, two key enzymes (GDP-mannose 4,6-dehydratase and GDP- l-fucose synthase) were self-assembled into enzyme complexes in vivo via a short peptide interaction pair RIAD-RIDD (RI anchoring disruptor-RI dimer D/D domains), resulting in noticeable improvement of 2'-FL production. Next, to further strengthen the metabolic flux toward 2'-FL, three pathway enzymes were further aggregated into multienzyme assemblies by using another orthogonal protein interaction motif (Spycatcher-SpyTag or PDZ-PDZlig). Intracellular multienzyme assemblies remarkably enlarged the flux toward 2'-FL biosynthesis and showed a 2.1-fold increase of 2'-FL production compared with a strain expressing free-floating and unassembled enzymes. The optimally engineered strain EZJ23 accumulated 4.8 g/L 2'-FL in shake flask fermentation and was capable of producing 25.1 g/L 2'-FL by fed-batch cultivation. This work provides novel approaches for further improvement and large-scale production of 2'-FL and demonstrates the effectiveness of spatial assembly of pathway enzymes to improve the production of valuable products in the engineered host strain.

Overview of strategies for developing high thermostability industrial enzymes: Discovery, mechanism, modification and challenges.

Biocatalysts such as enzymes are environmentally friendly and have substrate specificity, which are preferred in the production of various industrial products. However, the strict reaction conditions in industry including high temperature, organic solvents, strong acids and bases and other harsh environments often destabilize enzymes, and thus substantially compromise their catalytic functions, and greatly restrict their applications in food, pharmaceutical, textile, bio-refining and feed industries. Therefore, developing industrial enzymes with high thermostability becomes very important in industry as thermozymes have more advantages under high temperature. Discovering new thermostable enzymes using genome sequencing, metagenomics and sample isolation from extreme environments, or performing molecular modification of the existing enzymes with poor thermostability using emerging protein engineering technology have become an effective means of obtaining thermozymes. Based on the thermozymes as biocatalytic chips in industry, this review systematically analyzes the ways to discover thermostable enzymes from extreme environment, clarifies various interaction forces that will affect thermal stability of enzymes, and proposes different strategies to improve enzymes' thermostability. Furthermore, latest development in the thermal stability modification of industrial enzymes through rational design strategies is comprehensively introduced from structure-activity relationship point of view. Challenges and future research perspectives are put forward as well.

Recent progress in health effects and biosynthesis of lacto-N-tetraose, the most dominant core structure of human milk oligosaccharide.

Human milk oligosaccharides (HMOs), which are a group of complex carbohydrates highly abundant in human milk, have been recognized as critical functional biomolecules for infant health. Lacto-N-tetraose (LNT) is one of the most abundant HMO members and the most dominant core structure of HMO. The promising physiological effects of LNT have been well documented, including prebiotic property, antiadhesive antimicrobial activity, and antiviral effect. Its safety has been evaluated and it has been commercially added to infant formula as a functional ingredient. Because of great commercial importance of LNT, increasing attention has been paid to its highly efficient biological production. In particular, microbial synthesis based on metabolic engineering displays obvious advantages in large-scale production of LNT. This review contains important information about the recent progress in physiological effects, safety evaluation, and biosynthesis of LNT.

Occurrence, functional properties, and preparation of 3-fucosyllactose, one of the smallest human milk oligosaccharides.

Human milk oligosaccharides (HMOs) are receiving wide interest and high attention due to their health benefits, especially for newborns. The HMOs-fortified products are expected to mimic human milk not only in the kinds of added oligosaccharides components but also the appropriate proportion between these components, and further provide the nutrition and physiological effects of human milk to newborns as closely as possible. In comparison to intensively studied 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL) has less attention in almost all respects. Nerveless, 3-FL naturally occurs in breast milk and increases roughly over the course of lactation with a nonnegligible content, and plays an irreplaceable role in human milk and delivers functional properties to newborns. According to the safety evaluation, 3-FL shows no acute oral toxicity, genetic toxicity, and subchronic toxicity. It has been approved as generally recognized as safe (GRAS). Biological production of 3-FL can be realized by enzymatic and cell factory approaches. The α1,3- or α1,3/4-fucosyltransferase is the key enzyme for 3-FL biosynthesis. Various metabolic engineering strategies have been applied to enhance 3-FL yield using cell factory approach. In conclusion, this review gives an overview of the recent scientific literatures regarding occurrence, bioactive properties, safety evaluation, and biotechnological preparation of 3-FL.

Glycosylation of flavonoids by sucrose- and starch-utilizing glycoside hydrolases: A practical approach to enhance glycodiversification.

Flavonoids are ubiquitous and diverse in plants and inseparable from the human diet. However, in terms of human health, their further research and application in functional food and pharmaceutical industries are hindered by their low water solubility. Therefore, flavonoid glycosylation has recently attracted research attention because it can modulate the physicochemical and biochemical properties of flavonoids. This review represents a comprehensive overview of the O-glycosylation of flavonoids catalyzed by sucrose- and starch-utilizing glycoside hydrolases (GHs). The characteristics of this feasible biosynthesis approach are systematically summarized, including catalytic mechanism, specificity, reaction conditions, and yields of the enzymatic reaction, as well as the physicochemical properties and bioactivities of the product flavonoid glycosides. The cheap glycosyl donor substrates and high yields undoubtedly make it a practical flavonoid modification approach to enhance glycodiversification.

Synthesis of bioactive oligosaccharides and their potential health benefits.

Oligosaccharides, a low polymerization degree of carbohydrate, possess various physiological activities, such as anti-diabetes, anti-obesity, anti-aging, anti-viral, and gut microbiota regulation, having a widely used in food and medical fields. However, due to the limited natural oligosaccharides, many un-natural oligosaccharides from complex polysaccharides are being studied for amplifying the available pool of oligosaccharides. More recently, various oligosaccharides were developed by using several artificial strategies, such as chemical degradation, enzyme catalysis, and biosynthesis, then they can be applied in various sectors. Moreover, it has gradually become a trend to use biosynthesis to realize the synthesis of oligosaccharides with clear structure. Emerging research has found that un-natural oligosaccharides exert more comprehensive effects against various human diseases through multiple mechanisms. However, these oligosaccharides from various routes have not been critical reviewed and summarized. Therefore, the purpose of this review is to present the various routes of oligosaccharides preparations and healthy effects, with a focus on diabetes, obesity, aging, virus, and gut microbiota. Additionally, the application of multi-omics for these natural and un-natural oligosaccharides has also been discussed. Especially, the multi-omics are needed to apply in various disease models to find out various biomarkers to respond to the dynamic change process of oligosaccharides.

Recent progress in fucosylated derivatives of lacto-N-tetraose and lacto-N-neotetraose.

Human milk oligosaccharides (HMOs) have attracted considerable attention owing to their unique physiological functions. Two important tetrasaccharides, lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT), are core structures of HMOs. Their safety has been evaluated and they can be added to infant formula as functional ingredients. The fucosylated derivatives of LNT and LNnT, mainly lacto-N-fucopentaose (LNFP) I, LNFP II, LNFP III, and lacto-N-difucohexaose I, exhibit prominent physiological characteristics, including modificating the intestinal microbiota, immunomodulation, anti-bacterial activities, and antiviral infection. However, they have received lesser attention than 2'-fucosyllactose. As precursors, LNT and LNnT are connected to one or two fucosyl units through α1,2/3/4 glycosidic bonds, forming a series of compounds with complex structures. These complex fucosylated oligosaccharides can be biologically synthesized using enzymatic and cell factory approaches. This review summarizes the occurrence, physiological effects, and biosynthesis of fucosylated LNT and LNnT derivatives and their future development.

A comprehensive review on the properties, production, and applications of functional glucobioses.

Glucobiose is a range of disaccharides consisting of two glucose molecules, generally including trehalose, kojibiose, sophorose, nigerose, laminaribiose, maltose, cellobiose, isomaltose, and gentiobiose. The difference glycosidic bonds of two glucose molecules result in the diverse molecular structures, physiochemical properties and physiological functions of these glucobioses. Some glucobioses are abundant in nature but have unconspicuous roles on health like maltose, whereas some rare glucobioses display remarkable biological effects. It is unpractical process to extract these rare glucobioses from natural resources, while biological synthesis is a feasible approach. Recently, the production and application of glucobiose have attracted considerable attention. This review provides a comprehensive overview of glucobioses, including their natural sources and physicochemical properties like structure, sweetness, digestive performance, toxicology, and cariogenicity. Specific enzymes used for the production of various glucobioses and fermentation production processes are summarized. Additionally, their versatile functions and broad applications are also introduced.

Application of molecular dynamics simulation in the field of food enzymes: improving the thermal-stability and catalytic ability.

Enzymes can produce high-quality food with low pollution, high function, high acceptability, and medical aid. However, most enzymes, in their native form, do not meet the industrial requirements. Sequence-based and structure-based methods are the two main strategies used for enzyme modification. Molecular Dynamics (MD) simulation is a sufficiently comprehensive technology, from a molecular perspective, which has been widely used for structure information analysis and enzyme modification. In this review, we summarize the progress and development of MD simulation, particularly for software, force fields, and a standard procedure. Subsequently, we review the application of MD simulation in various food enzymes for thermostability and catalytic improvement was reviewed in depth. Finally, the limitations and prospects of MD simulation in food enzyme modification research are discussed. This review highlights the significance of MD simulation and its prospects in food enzyme modification.

Recent developments of mycotoxin-degrading enzymes: identification, preparation and application.

Mycotoxins are secondary metabolites produced by fungi during their growth. They not only seriously affect the yield of food crops but also pose a threat to human and animal health. Physical and chemical methods have been widely used to reduce the production and accumulation of mycotoxins in the field or after harvest, but these methods have difficulty in completely removing mycotoxins while keeping the nutrients at the same time. Biodegradation methods using isolated enzymes have shown superiority and potential for modest reaction conditions, high degradation efficiency and degradation products with low toxicity. Therefore, the occurrence, chemical structures, and toxicology of six prevalent mycotoxins (deoxynivalenol, zearalenone, aflatoxin, patulin, fumonisin, and ochratoxin) were described in this manuscript. The identification and application of mycotoxin-degrading enzymes were thoroughly reviewed. It is believed that in the near future, mycotoxin-degrading enzymes are expected to be commercially developed and used in the feed and food industries.

D-allulose, a versatile rare sugar: recent biotechnological advances and challenges.

D-Allulose is the C-3 epimer of D-fructose, and widely regarded as a promising substitute for sucrose. It's an excellent low-calorie sweetener, with 70% sweetness of sucrose, 0.4 kcal/g dietary energy, and special physiological functions. It has been approved as GRAS by the U.S. Food and Drug Administration, and is allowed to be excluded from total and added sugar counts on the food labels. Therefore, D-allulose gradually attracts more public attention. Owing to scarcity in nature, the bioproduction of D-allulose by using ketose 3-epimerase (KEase) has become the research hotspot. Herein, we give a summary of the physicochemical properties, physiological function, applications, and the chemical and biochemical synthesis methods of D-allulose. In addition, the recent progress in the D-allulose bioproduction using KEases, and the possible solutions for existing challenges in the D-allulose industrial production are comprehensively discussed, focusing on the molecular modification, immobilization, food-grade expression, utilizing low-cost biomass as feedstock, overcoming thermodynamic limitation, as well as the downstream separation and purification. Finally, Prospects for further development are also proposed.

Low-calorie bulk sweeteners: Recent advances in physical benefits, applications, and bioproduction.

At present, with the continuous improvement of living standards, people are paying increasing attention to dietary nutrition and health. Low sugar and low energy consumption have become important dietary trends. In terms of sugar control, more and more countries have implemented sugar taxes in recent years. Hence, as the substitute for sugar, low-calorie sweeteners have been widely used in beverage, bakery, and confectionary industries. In general, low-calorie sweeteners consist of high-intensity and low-calorie bulk sweeteners (some rare sugars and sugar alcohols). In this review, recent advances and challenges in low-calorie bulk sweeteners are explored. Bioproduction of low-calorie bulk sweeteners has become the focus of many researches, because it has the potential to replace the current industrial scale production through chemical synthesis. A comprehensive summary of the physicochemical properties, physiological functions, applications, bioproduction, and regulation of typical low-calorie bulk sweeteners, such as D-allulose, D-tagatose, D-mannitol, sorbitol, and erythritol, is provided.

Characterization of l-fucose isomerase from Paenibacillus rhizosphaerae to produce l-fuculose from hydrolyzed fucoidan and commercial fucose.

AIMS: l-Fuculose is a valuable rare sugar that is used to treat a variety of ailments, including HIV, cancer, Hepatitis B, human lysosomal disease (fucosidosis), and cardio-protective medications. The enzymatic approach for the production of l-fuculose using l-fucose as a substrate would be an advantageous method with a wide range of industrial applications. The objective of this study is the characterization of recombinant l-fucose isomerase from Paenibacillus rhizosphaerae (Pa-LFI) for the production of l-fuculose from an inexpensive and natural source (fucoidan) as well as its comparison with commercial l-fucose (Sigma-Aldrich). METHODS AND RESULTS: Fucoidan, a fucose-containing polysaccharide (FPs), was isolated from Undaria pinnatifida, subsequently hydrolyzed, and characterized before the enzymatic production of l-fuculose. The results elaborate that FPs contain 35.9% of fucose along with other kinds of monosaccharides. The purified Pa-LFI exhibited a single band at 65 kDa and showed it as a hexamer with a native molecular mass of 396 kDa. The highest activity of 104.5 U mg-1 of Pa-LFI was perceived at a temperature of 50°C and pH 6.5 in the presence of 1 mM of Mn2+. The Pa-LFI revealed a melting temperature (Tm) of 75°C and a half-life of 12.6 h at 50°C. It exhibited that Pa-LFI with aldose substrate (l-fucose), has a stronger isomerizing activity, disclosing Km,kcat, and kcat/Km 86.2 mM, 32 831 min-1, and 335 min-1 mM-1, respectively. After reaching equilibrium, Pa-LFI efficiently catalyzed the reaction to convert l-fucose into l-fuculose and the conversion ratios of l-fuculose from 100 g L-1 of FPs and commercial fucose were around 6% (5.6 g L-1) and 30% (30.2 g L-1), respectively. CONCLUSIONS: According to the findings of the current study, the Pa-LFI will be useful in the manufacturing of l-fuculose using an effective and easy approach that produces no by-products.

Physiological effects, biosynthesis, and derivatization of key human milk tetrasaccharides, lacto-N-tetraose, and lacto-N-neotetraose.

Human milk oligosaccharides (HMOs) have recently attracted ever-increasing interest because of their versatile physiological functions. In HMOs, two tetrasaccharides, lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT), constitute the essential components, each accounting 6% (w/w) of total HMOs. Also, they serve as core structures for fucosylation and sialylation, generating functional derivatives and elongation generating longer chains of core structures. LNT, LNnT, and their fucosylated and/or sialylated derivatives account for more than 30% (w/w) of total HMOs. For derivatization, LNT and LNnT can be modified into a series of complex fucosylated and/or sialylated HMOs by transferring fucose residues at α1,2-, α1,3-, and α1,3/4-linkage and/or sialic acid residues at α2,3- and α2,6-linkage. Such structural diversity allows these HMOs to possess great commercial value and an application potential in the food and pharmaceutical industries. In this review, we first elaborate the physiological functions of these tetrasaccharides and derivatives. Next, we extensively review recent developments in the biosynthesis of LNT, LNnT, and their derivatives in vitro and in vivo by employing advanced enzymatic reaction systems and metabolic engineering strategies. Finally, future perspectives in the synthesis of these HMOs using enzymatic and metabolic engineering approaches are presented.

Difructose anhydride III: a 50-year perspective on its production and physiological functions.

Production and applications of difructose anhydride III (DFA-III) have attracted considerable attention because of its versatile physiological functions. Recently, large-scale production of DFA-III has been continuously explored, which opens a horizon for applications in the food and pharmaceutical industries. This review updates recent advances involving DFA-III, including: biosynthetic strategies, purification, and large-scale production of DFA-III; physiological functions of DFA-III and related mechanisms; DFA-III safety evaluations; present applications in food systems, existing problems, and further research prospects. Currently, enzymatic synthesis of DFA-III has been conducted both industrially and in academic research. Two biosynthetic strategies for DFA-III production are summarized: single- and double enzyme-mediated. DFA-III purification is achieved via yeast fermentation. Enzyme membrane bioreactors have been applied to meet the large-scale production demands for DFA-III. In addition, the primary physiological functions of DFA-III and their underlying mechanisms have been proposed. However, current applications of DFA-III are limited. Further research regarding DFA-III should focus on commercial production and purification, comprehensive study of physiological properties, extensive investigation of large-scale human experiments, and expansion of industrial applications. It is worthy to dig deep into potential application and commercial value of DFA-III.

Recent advances on 2'-fucosyllactose: physiological properties, applications, and production approaches.

The trisaccharide, 2'-fucosyllactose (Fucα1-2Galß1-4Glc; 2'-FL), is the most abundant oligosaccharide in human milk. It has numerous significant biological properties including prebiotics, antibacterial, antiviral, and immunomodulating effects, and has been approved as "generally recognized as safe" (GRAS) by the Food and Drug Administration (FDA) and as a novel food (NF) by the European Food Safety Authority (EFSA). 2'-FL not only serves as a food ingredient added in infant formula, but also as a dietary supplement and medical food material in food bioprocesses. There is considerable commercial interest in 2'-FL for its irreplaceable nutritional applications. This review aims at systematically elaborating key functional properties of 2'-FL as well as its applications. In addition, several approaches for 2'-FL production are described in this review, including chemical, chemo-enzymatical, and cell factory approaches, and the pivotal research results also have been summarized. With the rapid development of metabolic engineering and synthetic biology strategies, using the engineered cell factory for 2'-FL large-scale production might be a promising approach. From an economic and safety point of view, microbial selection for cell factory engineering in 2'-FL bioprocess also should be taken into consideration.

Microbial elimination of pyrethroids: specific strains and involved enzymes.

Pyrethroids, which are synthetic organic insecticides, are widely used in agriculture and households to resist pests and control disease transmission. However, pyrethroids have inevitably caused environmental pollution, leading to concerns for food safety and human health. Bioremediation has emerged as one of the most promising methods to eliminate pyrethroids compounds. Pyrethroid-degrading microorganisms and the relevant enzymes have shown an efficient ability in degrading pyrethroids by hydrolyzing the ester linkage. In this review, a wide variety of pyrethroid-degrading strains were presented and classified from different sources, such as wastewater, soils, and oceans. In addition, the recombinant expression, enzyme identification, and molecular modification of these microbial pyrethroid-degrading enzymes were also compared and discussed in detail. Moreover, the potential applications of pyrethroid-degrading enzymes, including immobilization and biodegradation towards a series of pyrethroids, were also presented. All of the positive results obtained from this review could be a good guideline for the other research in this field. KEY POINTS: • Distribution of pyrethroid-degrading strains in different sources was summarized. • Enzymatic properties including pH, temperature, and substrate specificity were compared. • Promising molecular modification and immobilization of hydrolases were present.

Microbial elimination of carbamate pesticides: specific strains and promising enzymes.

Carbamate pesticides are widely used in the environment, and compared with other pesticides in nature, they are easier to decompose and have less durability. However, due to the improper use of carbamate pesticides, some nontarget organisms still may be harmed. To this end, it is necessary to investigate effective removal or elimination methods for carbamate pesticides. Current effective elimination methods could be divided into four categories: physical removal, chemical reaction, biological degradation, and enzymatic degradation. Physical removal primarily includes elution, adsorption, and supercritical fluid extraction. The chemical reaction includes Fenton oxidation, photo-radiation, and net electron reduction. Biological degradation is an environmental-friendly manner, which achieves degradation by the metabolism of microorganisms. Enzymatic degradation is more promising due to its high substrate specificity and catalytic efficacy. All in all, this review primarily summarizes the property of carbamate pesticides and the traditional degradation methods as well as the promising biological elimination. KEY POINTS: • The occurrence and toxicity of carbamate pesticides were shown. • Biological degradation strains against carbamate pesticides were presented. • Promising enzymes responsible for the degradation of carbamates were discussed.