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.

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.

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.

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.

Zearalenone lactonase: characteristics, modification, and application.

Zearalenone (ZEN) and its derivatives are one of the most contaminated fungal toxins worldwide, posing a severe threat to food security and human life. Traditional physical and chemical detoxifying methods are unsatisfactory due to incomplete detoxification, nutrient loss, and secondary pollutants. In recent years, bioremediation for eliminating fungal toxins has been gradually investigated. ZEN lactone hydrolase (lactonase) has been widely studied because of its high activity, mild conditions, and non-toxic product property. This review comprehensively represents the gene mining, characterization, molecular modification, and application of microbial-derived ZEN lactonases. It is aimed to elucidate the advantages and challenges of ZEN lactonases in industrial application, which also provides perspectives on obtaining innovative and promising biocatalysts for ZEN degradation. KEY POINTS: • A timely and concise review related to enzymatic elimination towards ZEN is shown. • The catalytic conditions and mechanism of ZEN lactonase is presented. • The modification and application of ZEN lactonase are exhibited also.

Recent advances and future prospective of organophosphorus-degrading enzymes: identification, modification, and application.

The organophosphorus-based OPs) nerve agents and pesticides have been applied in the agriculture industry for a long time. However, they were found to have a persistent effect on the environment and threaten human health. Traditional methods, including incineration and landfilling, could not thoroughly remove these organophosphorus compounds (OPs). Meanwhile, chemical hydrolysis for decontamination was also inhibited due to the presence of corrosive materials and high costs. Biological remediation for OPs employing microorganisms and organophosphorus-degrading enzymes is promising due to a mild and controllable procedure, environmental-friendly reactions, and high efficacy. A wide variety of enzymes have shown latent ability in degrading OPs hazards like organophosphorus hydrolase (OPH), organophosphorus acid anhydrolase (OPAA), the diisopropylfluorophosphatase (DFPase), and mammalian paraoxonase 1 (PON 1). To this end, increasing efforts have been made on these intriguing enzymes to increase their expression level, enhance the catalytic activity, modify the optimal substrate, and expand the practical application. In this review, the enzyme resource, crystal structure, molecular modification, and industry application were compared and discussed in detail. Moreover, the proposed ideas and positive results could be useful for the other relevant OPs-degrading enzymes.

Overview of a bioremediation tool: organophosphorus hydrolase and its significant application in the food, environmental, and therapy fields.

In the past decades, the organophosphorus compounds had been widely used in the environment and food industries as pesticides. Owing to the life-threatening and long-lasting problems of organophosphorus insecticide (OPs), an effective detection and removal of OPs have garnered growing attention both in the scientific and practical fields in recent years. Bacterial organophosphorus hydrolases (OPHs) have been extensively studied due to their high specific activity against OPs. OPH could efficiently hydrolyze a broad range of substrates both including the OP pesticides and some nerve agents, suggesting a great potential for the remediation of OPs. In this review, the microbial identification, molecular modification, and practical application of OPHs were comprehensively discussed.Key points• Microbial OPH is a significant bioremediation tool against OPs.• Identification and molecular modification of OPH was discussed in detail.• The applications of OPH in food, environmental, and therapy fields are presented.

An overview of D-galactose utilization through microbial fermentation and enzyme-catalyzed conversion.

D-Galactose is an abundant carbohydrate monomer in nature and widely exists in macroalgae, plants, and dairy wastes. D-Galactose is useful as a raw material for biomass fuel production or low-calorie sweetener production, attracting increased attention. This article summarizes the studies on biotechnological processes for galactose utilization. Two main research directions of microbial fermentation and enzyme-catalyzed conversion from galactose-rich biomass are extensively reviewed. The review provides the recent discoveries for biofuel production from macroalgae, including the innovative methods in the pretreatment process and technological development in the fermentation process. As modern people pay more attention to health, enzyme technologies for low-calorie sweetener production are more urgently needed. D-Tagatose is a promising low-calorie alternative to sugar. We discuss the recent studies on characterization and genetic modification of L-arabinose isomerase to improve the bioconversion of D-galactose to D-tagatose. In addition, the trends and critical challenges in both research directions are outlined at the end. KEY POINTS: • The value and significance of galactose utilization are highlighted. • Biofuel production from galactose-rich biomass is accomplished by fermentation. • L-arabinose isomerase is a tool for bioconversion of D-galactose to D-tagatose.

Mannitol: physiological functionalities, determination methods, biotechnological production, and applications.

Mannitol is a naturally occurring six-carbon sugar alcohol that has wide applications in the food and pharmaceutical industry because of its many properties, namely being a natural sweetener with a low metabolism and no glycemic index. The increasing demand for mannitol has spurred many studies of its production. Compared with its chemical synthesis and extraction from plants, both of which are difficult to satisfy for industrial requirements, biotechnological production of mannitol has received considerably more attention and interest from scientists because of its known advantages over those two methods. Accordingly, in this review, we summarize recent advances made in the production of mannitol through various biotechnological methods. The physicochemical properties, sources, and physiological functionalities and applications of mannitol are systematically covered and presented. Then, different determination methods for mannitol are also described and compared. Furthermore, different biotechnological strategies for the production of mannitol via fermentation engineering, protein engineering, and metabolic engineering receive a detailed overview in terms of mannitol-producing strains, enzymes, and their key reaction parameters and conditions. KEY POINTS: • Physiological functionalities and applications of mannitol are presented in detail. • Different determination methods for mannitol are also described and compared. • Various biotechnological strategies for the production of mannitol are reviewed.

Recent advances in Levansucrase and Inulosucrase: evolution, characteristics, and application.

Levan and inulin are two types of fructan. Levan is composed of ß-(2, 6) fructosyl linkage and inulin is composed of ß-(2, 1) linkage. Both levan and inulin have been accepted and applied in the food, medicinal and chemical industries for their outstanding physicochemical properties in recent years. Microbial levansucrase and inulosucrase are key enzymes responsible for the synthesis of fructan from sucrose. In this review, levansucrase and inulosucrase are discussed together for the first time regarding the evolutionary relationships, bacteria origin, crystal structure, product-forming mechanism and commercial applications. Particularly, some insights into the product specificity about levansucrase and inulosucrase as well as the mechanism for product elongation for fructan are also discussed in the article.

An overview on biological production of functional lactose derivatives.

Lactose is a natural disaccharide obtained from the milk of most mammals and a waste product of cheese and casein manufacturing. Over the past decades, lactose in whey has increasingly been promoted as an important resource, and an increasing number of significant advances have been made to investigate its healthy and functional properties. Lactose can be biotransformed into many kinds of derivatives, including galacto-oligosaccharides, epilactose, lactulose, lactosucrose, and D-tagatose. Biological efficiency and safety are critical for the enzymatic production of lactose derivatives from lactose. These lactose derivatives show a range of prominent physiological features and effects, such as prebiotic properties, indigestibility, and obesity prevention, which can be utilized in the pharmaceutical, health, and food industries. In this review, we present the properties and physiological effects of lactose derivatives, detailing their biological production by various enzymes and their applications in dairy products, especially directly in the milk industry.

Polyol dehydrogenases: intermediate role in the bioconversion of rare sugars and alcohols.

Polyol dehydrogenases (PDHs) play a pivotal role in the biotransformation between rare sugar and alcohol. Among these PDHs, mannitol 2-dehydrogenase (MDH, EC 1.1.1.67), galactitol 2-dehydrogenase (GDH, EC 1.1.1.16), ribitol 2-dehydrogenase (RDH, EC 1.1.1.56), xylitol 4-dehydrogenase (XDH, EC 1.1.1.14), and arabitol 2-dehydrogenase (ArDH, EC 1.1.1.12) are the most studied. MDH can catalyze the transformation between D-fructose and mannitol as well as the transformation between D-arabitol and D-xylulose. In addition to MDH, the other PDHs including RDH, GDH, ArDH, and XDH are also important tools for the production of rare sugars including D-tagatose, allitol, D-xylulose, and L-xylulose. Concerning the intermediate function of PDH in the linkage of rare sugar and sugar alcohols, this review attempts to conclude their catalytic properties and potential applications.

An overview of levan-degrading enzyme from microbes.

Functional carbohydrates are ideal substitutes for table sugar and make up a large share of the worldwide functional food market because of their numerous physiological benefits. Growing attention has been focused on levan, a ß-(2,6) fructan that possesses more favorable physicochemical properties, such as lower intrinsic viscosity and greater colloidal stability, than ß-(2,1) inulin. Levan can be used not only as a functional carbohydrate but also as feedstock for the production of levan-type fructooligosaccharides (L-FOSs). Three types of levan-degrading enzymes (LDEs), including levanase (EC 3.2.1.65), ß-(2,6)-fructan 6-levanbiohydrolase (LF2ase, EC 3.2.1.64), and levan fructotransferase (LFTase, EC 4.2.2.16), play significant roles in the biological production of L-FOSs. These three enzymes convert levan into different L-FOSs, levanbiose, and difructose anhydride IV (DFA IV), respectively. The prebiotic properties of both L-FOSs and DFA IV have been confirmed in recent years. Although levanase, LF2ase, and LFTase belong to the same O-glycoside hydrolase 32 family (GH32), their catalytic properties and product spectra differ significantly. In this paper, recent studies on these LDEs are reviewed, including those investigating microbial source and catalytic properties. Additionally, comparisons of LDEs, including those of their differing cleavage behavior and applications for different L-FOSs, are presented in detail.

A close look on the effect of polyethylene glycol on the levansucrase thermostability: a case study of Brenneria sp. levansucrase.

BACKGROUND: To increase the low residual activity of levansucrase during long-time processing, an enhancement of its weak thermostability is needed. Here, the effect of metal ions and polyethylene glycol (PEG) on the thermostability of levansucrase from Brenneria sp. EniD312 were studied and evaluated. The residual activity was determined and the protein structure was evaluated by circular dichroism spectrum, fluorescence intensity (FI), and surface hydrophobicity (S0 ). RESULTS: As a result of incubation with 10% (w/v) PEG 4000, the enzyme activity was increased by 1.24-fold. After incubation with 5% PEG 4000 for 6 h, the residual activity at 35 and 45 °C was decreased to 55% and 60% of the initial activity, with an increase of 1.2- and 3.3-fold than the wild-type enzyme. Furthermore, the random coil content of enzyme was decreased from 53% of the wild-type enzyme to 33.9% of the PEG pre-incubated enzyme. Additionally, the FI was maximally increased and the S0 was decreased from 117 to 69. CONCLUSION: All of these results suggested that after incubation with PEG 4000, the secondary and tertiary structure of wild-type enzyme could be greatly maintained and then its thermostability could be increased. This study was the first report on the enhancement of levansucrase thermostability by PEG incubation and might be a good guideline to other researches on levansucrase. © 2019 Society of Chemical Industry.

Isomerases and epimerases for biotransformation of pentoses.

Pentoses represent monosaccharides with five carbon atoms. They are organized into two main groups, aldopentoses and ketopentoses. There are eight aldopentoses and four ketopentoses and each ketopentose corresponds to two aldopentoses. Only D-xylose, D-ribose, and L-arabinose are natural sugars, but others belong to rare sugars that occur in very small quantities in nature. Recently, rare pentoses attract much attention because of their great potentials for commercial applications, especially as precursors of many important medical drugs. Pentoses Izumoring strategy provides a complete enzymatic approach to link all pentoses using four types of enzymes, including ketose 3-epimerases, aldose-ketose isomerases, polyol dehydrogenases, and aldose reductases. At least 10 types of epimerases and isomerases have been used for biotransformation of all aldopentoses and ketopentoses, and these enzymes are reviewed in detail in this article.

Recent advances on biological production of difructose dianhydride III.

Difructose dianhydride III (DFA III) is a cyclic difructose containing two reciprocal glycosidic linkages. It is easily generated with a small amount by sucrose caramelization and thus occurs in a wide range of food-stuffs during food processing. DFA III has half sweetness but only 1/15 energy of sucrose, showing potential industrial application as low-calorie sucrose substitute. In addition, it displays many benefits including prebiotic effect, low cariogenicity property, and hypocholesterolemic effect, and improves absorption of minerals, flavonoids, and immunoglobulin G. DFA III is biologically produced from inulin by inulin fructotransferase (IFTase, EC 4.2.2.18). Plenty of DFA III-producing enzymes have been identified. The crystal structure of inulin fructotransferase has been determined, and its molecular modification has been performed to improve the catalytic activity and structural stability. Large-scale production of DFA III has been studied by various IFTases, especially using an ultrafiltration membrane bioreactor. In this article, the recent findings on physiological effects of DFA III are briefly summarized; the research progresses on identification, expression, and molecular modification of IFTase and large-scale biological production of DFA III by IFTase are reviewed in detail.

Recent research on the physiological functions, applications, and biotechnological production of D-allose.

D-Allose is a rare monosaccharide, which rarely appears in the natural environment. D-Allose has an 80% sweetness relative to table sugar but is ultra-low calorie and non-toxic and is thus an ideal candidate to take the place of table sugar in food products. It displays unique health benefits and physiological functions in various fields, including food systems, clinical treatment, and the health care fields. However, it is difficult to produce chemically. The biotechnological production of D-allose has become a research hotspot in recent years. Therefore, an overview of recent studies on the physiological functions, applications, and biotechnological production of D-allose is presented. In this review, the physiological functions of D-allose are introduced in detail. In addition, the different types of D-allose-producing enzymes are compared for their enzymatic properties and for the biotechnological production of D-allose. To date, very little information is available on the molecular modification and food-grade expression of D-allose-producing enzymes, representing a very large research space yet to be explored.

Recent progress on biological production of α-arbutin.

Arbutin, a glucoside of hydroquinone, is used as a powerful skin lightening agent in the cosmeceutical industry because of its strong inhibitory effect on the human tyrosinase activity. It is a natural compound occurring in a number of plants, with a ß-anomeric form of the glycoside bond between glucose and hydroquinone. α-Arbutin, which glycoside bond is generated with α-anomeric form, is the isomer of natural arbutin. α-Arbutin is generally produced by transglucosylation of hydroquinone by microbial glycosyltransferases. It is interesting that α-arbutin is found to be over 10 times more effective than arbutin, and thus biological production of α-arbutin attracts increasing attention. Seven different microbial enzymes have been identified to be able to produce α-arbutin, including α-amylase, sucrose phosphorlase, cyclodextrin glycosyltransferase, α-glucosidase, dextransucrase, amylosucrase, and sucrose isomerase. In this work, enzymatic and microbial production of α-arbutin is reviewed in detail.