Characterization of a GH20 ß-N-Acetylhexosaminidase from Flavobacterium algicola Suitable to Synthesize Lacto-N-triose II.

ß-N-Acetylhexosaminidases have attracted much attention in the enzymatic synthesis of lacto-N-triose II (LNT2) as a backbone precursor of human milk oligosaccharides (HMOs). In this study, a novel glycoside hydrolase (GH) 20 family ß-N-acetylhexosaminidase, FlaNag2353, from Flavobacterium algicola was biochemically characterized and applied to synthesize LNT2. FlaNag2353 displayed optimal activity to p-nitrophenyl N-acetyl-ß-d-glucosaminide (pNP-GlcNAc) at 40 °C and pH 8.0. In addition to its excellent hydrolysis activity toward pNP-GlcNAc and chitooligosaccharides, FlaNag2353 showed trans-glycosylation activity. Under conditions of pH 9.0 and 55 °C for 2 h and utilizing 200 mM lactose and 10 mM pNP-GlcNAc, FlaNag2353 synthesized LNT2 with a conversion ratio of 4.15% calculated from pNP-GlcNAc. Moreover, when applied to LNT2 synthesis with 10 mM pNP-GlcNAc and 9.7% (w/v) industrial waste whey powder, FlaNag2353 achieved a conversion ratio of 2.39%. This study has significant implications for broadening the applications of GH20 ß-N-acetylhexosaminidases and promoting the high-value utilization of whey powder.

Chimeric versus isolated proteins: Biochemical characterization of the NADP+-dependent formate dehydrogenase from Pseudomonas sp. 101 fused with the Baeyer-Villiger monooxygenase from Thermobifida fusca.

The expression of multifunctional proteins can facilitate the setup of a biotechnology process that requires multiple functions absolved by different proteins. Herein the functional and conformational characterization of a formate dehydrogenase-monooxygenase chimera enzyme is presented. The fused enzyme (FDH-PAMO) was prepared by linking the C-terminus of the mutant NADP+-dependent formate dehydrogenase from Pseudomonas sp. 101 (FDH) to the N-terminus of the NADPH-dependent monooxygenase from Thermobifida fusca (PAMO) through a peptide linker of 9 amino acids (ASGGGGSGT) generating a chimera protein of 107,056 Da. The catalytic properties (e.g., kinetic parameters kcat and Km), stability, fluorescence and circular dichroism spectra showed that the so-obtained chimera enzyme FDH-PAMO retains the same functional and conformational properties of the two parental enzymes. Furthermore, SEC chromatographic analysis indicated that, in solution (pH 7.4), FDH-PAMO assembles to tetramers (up to 4.2 %) due to the propensity of FDH and PAMO to form dimers, up to 96.6 % and 6.2 %, respectively. This study provides valuable insights into the structural stability of a thermostable protein (e.g., PAMO) after increasing its size through fusion with another similarly sized thermostable protein (e.g., FDH).

Structure-based mining of a chitosanase with distinctive degradation mode and product specificity.

Chitosan derivates with varying degrees of polymerization (DP) have attracted great concern due to their excellent biological activities. Increasing the abundance of chitosanases with different degradation modes contributes to revealing their catalytic mechanisms and facilitating the production of chitosan derivates. However, the identification of endo-chitosanases capable of producing chitobiose and D-glucosamine (GlcN) from chitosan substrates has remained elusive. Herein, an endo-chitosanase (CsnCA) belonging to the GH46 family was identified based on structural analysis in phylogenetic evolution. Moreover, we demonstrate that CsnCA acts in a random endo-acting manner, producing chitosan derivatives with DP ≤ 2. The in-depth analysis of CsnCA revealed that (GlcN)3 serves as the minimal substrate, undergoing cleavage in the mode that occupies the subsites - 2 to + 1, resulting in the release of GlcN. This study succeeded in discovering a chitosanase with distinctive degradation modes, which could facilitate the mechanistic understanding of chitosanases, further empowering the production of chitosan derivates with specific DP. KEY POINTS: • Structural docking and evolutionary analysis guide to mining the chitosanase. • The endo-chitosanase exhibits a unique GlcN-producing cleavage pattern. • The cleavage direction of chitosanase to produce GlcN was identified.

Expanding the application range of the κ­carrageenase OUC-FaKC16A when preparing oligosaccharides from κ-carrageenan and furcellaran.

Carrageenan oligosaccharides are important products that have demonstrated numerous bioactivities useful in the food, medicine, and cosmetics industries. However, the specific structure-function relationships of carrageenan oligosaccharides are not clearly described due to the deficiency of high specific carrageenases. Here, a truncated mutant OUC-FaKC16Q based on the reported κ-neocarratetrose (Nκ4)-producing κ-carrageenase OUC-FaKC16A from Flavobacterium algicola was constructed and further studied. After truncating the C-terminal Por_Secre_tail (PorS) domain (responsible for substrate binding), the catalytic efficiency and temperature stability decreased to a certain extent. Surprisingly, this truncation also enabled OUC-FaKC16Q to hydrolyze Nκ4 into κ-neocarrabiose (Nκ2). The offset of Arg265 residue in OUC-FaKC16Q may explain this change. Moreover, the high catalytic abilities, the main products, and the degradation modes of OUC-FaKC16A and OUC-FaKC16Q toward furcellaran were also demonstrated. Data suggested OUC-FaKC16A and OUC-FaKC16Q could hydrolyze furcellaran to produce mainly the desulfated oligosaccharides DA-G-(DA-G4S)2 and DA-G-DA-G4S, respectively. As a result, the spectrum of products of κ-carrageenase OUC-FaKC16A has been fully expanded in this study, indicating its promising potential for application in the biomanufacturing of carrageenan oligosaccharides with specific structures. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-023-00181-2.

Detection, production, modification, and application of arylsulfatases.

Arylsulfatase is a subset of sulfatase which catalyzes the hydrolysis of aryl sulfate ester. Arylsulfatase is widely distributed among microorganisms, mammals and green algae, but the arylsulfatase-encoding gene has not yet been found in the genomes of higher plants so far. Arylsulfatase plays an important role in the sulfur flows between nature and organisms. In this review, we present the maturation and catalytic mechanism of arylsulfatase, and the recent literature on the expression and production of arylsulfatase in wild-type and engineered microorganisms, as well as the modification of arylsulfatase by genetic engineering are summarized. We focus on arylsulfatases from microbial origin and give an overview of different assays and substrates used to determine the arylsulfatase activity. Furthermore, the researches about arylsulfatase application on the field of agar desulfation, soil sulfur cycle and soil evaluation are also discussed. Finally, the perspectives concerning the future research on arylsulfatase are prospected.

Response of Salmonella enterica serovar Typhimurium to alginate oligosaccharides fermented with fecal inoculum: integrated transcriptomic and metabolomic analyses.

Alginate oligosaccharides (AOS), extracted from marine brown algae, are a common functional feed additive; however, it remains unclear whether they modulate the gut microbiota and microbial metabolites. The response of Salmonella enterica serovar Typhimurium, a common poultry pathogen, to AOS fermented with chicken fecal inocula was investigated using metabolomic and transcriptomic analyses. Single-strain cultivation tests showed that AOS did not directly inhibit the growth of S. Typhimurium. However, when AOS were fermented by chicken fecal microbiota, the supernatant of fermented AOS (F-AOS) exhibited remarkable antibacterial activity against S. Typhimurium, decreasing the abundance ratio of S. Typhimurium in the fecal microbiota from 18.94 to 2.94%. Transcriptomic analyses showed that the 855 differentially expressed genes induced by F-AOS were mainly enriched in porphyrin and chlorophyll metabolism, oxidative phosphorylation, and Salmonella infection-related pathways. RT-qPCR confirmed that F-AOS downregulated key genes involved in flagellar assembly and the type III secretory system of S. Typhimurium, indicating metabolites in F-AOS can influence the growth and metabolism of S. Typhimurium. Metabolomic analyses showed that 205 microbial metabolites were significantly altered in F-AOS. Among them, the increase in indolelactic acid and 3-indolepropionic acid levels were further confirmed using HPLC. This study provides a new perspective for the application of AOS as a feed additive against pathogenic intestinal bacteria. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-023-00176-z.

Recent developments in the production and utilization of photosynthetic microorganisms for food applications.

The growing use of photosynthetic microorganisms for food and food-related applications is driving related biotechnology research forward. Increasing consumer acceptance, high sustainability, demand of eco-friendly sources for food, and considerable global economic concern are among the main factors to enhance the focus on the novel foods. In the cases of not toxic strains, photosynthetic microorganisms not only provide a source of sustainable nutrients but are also potentially healthy. Several published studies showed that microalgae are sources of accessible protein and fatty acids. More than 400 manuscripts were published per year in the last 4 years. Furthermore, industrial approaches utilizing these microorganisms are resulting in new jobs and services. This is in line with the global strategy for bioeconomy that aims to support sustainable development of bio-based sectors. Despite the recognized potential of the microalgal biomass value chain, significant knowledge gaps still exist especially regarding their optimized production and utilization. This review highlights the potential of microalgae and cyanobacteria for food and food-related applications as well as their market size. The chosen topics also include advanced production as mixed microbial communities, production of high-value biomolecules, photoproduction of terpenoid flavoring compounds, their utilization for sustainable agriculture, application as source of nutrients in space, and a comparison with heterotrophic microorganisms like yeast to better evaluate their advantages over existing nutrient sources. This comprehensive assessment should stimulate further interest in this highly relevant research topic.

A novel microbial-derived family 19 endochitinase with exochitinase activity and its immobilization.

A novel chitinase gene of 888 bp from Streptomyces bacillaris was cloned and expressed in Escherichia coli BL21. The purified recombinant enzyme (SbChiAJ103) was identified as the first microbial-derived family 19 endochitinase that showed exochitinase activity. SbChiAJ103 exhibited the substrate preference for N-acetylchitooligosaccharides with even degrees of polymerization and the capability to specifically hydrolyze colloidal chitin into (GlcNAc)2. Mono-methyl adipate was employed as a novel linker for the efficient covalent immobilization of chitinase on magnetic nanoparticles (MNPs). The immobilized SbChiAJ103, SbChiAJ103@MNPs, exhibited superior pH tolerance, temperature stability, and storage stability than free SbChiAJ103. Even after incubation at 45 °C for 24 h, SbChiAJ103@MNPs could retain more than 60.0% initial activity. As a result, the enzymatic hydrolysis yield of SbChiAJ103@MNPs increased to 1.58 times that of free SbChiAJ103. Moreover, SbChiAJ103@MNPs could be reused by convenient magnetic separation. After 10 recycles, SbChiAJ103@MNPs could retain almost 80.0% of its initial activity. The immobilization of the novel chitinase SbChiAJ103 paves the way to the efficient and eco-friendly commercial production of (GlcNAc)2. KEY POINTS: • The first microbial GH19 endochitinase with exochitinase activity was reported. • Mono-methyl adipate was first employed to immobilize chitinase. • SbChiAJ103@MNPs showed excellent pH stability, thermal stability, and reusability.

Synthesis and Conformational Analysis of Hydantoin-Based Universal Peptidomimetics.

The synthesis of a collection of enantiomerically pure, systematically substituted hydantoins as structural privileged universal mimetic scaffolds is presented. It relies on a chemoselective condensation/cyclization domino process between isocyanates of quaternary or unsubstituted α-amino esters and N-alkyl aspartic acid diesters followed by standard hydrolysis/coupling reactions with amines, using liquid-liquid acid/base extraction protocols for the purification of the intermediates. Besides the nature of the α carbon on the isocyanate moiety, either a quaternary carbon or a more flexible methylene group, conformational studies in silico (molecular modeling), in solution (NMR, circular dichroism (CD), Fourier transform infrared (FTIR)), and in solid state (X-ray) showed that the presented hydantoin-based peptidomimetics are able to project their substituents in positions superimposable to the side chains of common protein secondary structures such as α-helix and ß-turn, being the open α-helix conformation slightly favorable according to molecular modeling, while the closed ß-turn conformation preferred in solution and in solid state.

Preparation and characterization of phosphatidyl-agar oligosaccharide liposomes for astaxanthin encapsulation.

Surface modification of liposomes is an effective way to maintain the physicochemical activity of encapsulated substances. A novel astaxanthin (Ast)-based vesicle carrier system, namely, phosphatidyl-agar oligosaccharide (Ptd-AOS) liposomes (Lip), was prepared to improve the structural stability and in vitro digestibility of astaxanthin. During the transphosphatidylation reaction of synthesizing Ptd-AOS from phosphatidylcholine (PC) and AOS with different degrees of polymerization, phosphatidyl galactose (Ptd-Gal) and phosphatidyl neoagarobiose (Ptd-NA2) showed higher yields (85 and 96%, respectively). In terms of morphology, modified liposomes exhibited smaller particle sizes and more uniform dispersion compared with PC-Ast-Lip. In addition, the astaxanthin in the modified liposomes showed enhanced stability during liposome characterization and in vitro digestion. The transformations of astaxanthin in the modified liposomes were distributed in the range of 57-74% compared with free astaxanthin (25%). These findings suggest that the modification of liposomes by Ptd-AOS has potential applications in the delivery of functional ingredients.

Effects of intestinal microbiota on physiological metabolism and pathogenicity of Vibrio.

Vibrio species are disseminated broadly in the marine environment. Some of them can cause severe gastroenteritis by contaminating seafood and drinking water, such as Vibrio parahaemolyticus, Vibrio cholerae, and Vibrio vulnificus. However, their pathogenic mechanism still needs to be revealed to prevent and reduce morbidity. This review comprehensively introduces and discusses the common pathogenic process of Vibrio including adhesion, cell colonization and proliferation, and resistance to host immunity. Vibrio usually produces pathogenic factors including hemolysin, type-III secretion system, and adhesion proteins. Quorum sensing, a cell molecular communication system between the bacterial cells, plays an important role in Vibrio intestinal invasion and colonization. The human immune system can limit the virulence of Vibrio or even kill the bacteria through different responses. The intestinal microbiota is a key component of the immune system, but information on its effects on physiological metabolism and pathogenicity of Vibrio is seldom available. In this review, the effects of intestinal microorganisms and their metabolites on the invasion and colonization of common pathogenic Vibrio and VBNC status cells are discussed, which is conducive to finding the next-generation prebiotics. The strategy of dietary intervention is discussed for food safety control. Finally, future perspectives are proposed to prevent Vibrio infection in aquaculture.

Advances in enzyme biocatalysis for the preparation of functional lipids.

Functional lipids, mainly ω-3 polyunsaturated fatty acids (n-3 PUFAs) such as eicosapentaenoic (EPA; 20:5n-3) and docosahexaenoic (DHA; 22:6n-3), are known to have a variety of health benefits. Lipases and phospholipases are widely used to prepare different forms of structured lipids, since biocatalytic methods can be carried out under mild conditions, preserving the quality of the products. On the other hand, many processes still are conducted at high temperatures and with organic solvents, which are conditions unfavorable for the production of nutritional products. This article gives an updated overview of enzyme biocatalysis methods for the preparation of different derivatives containing n-3 PUFAs, including specific reactions, enzyme immobilization research for high-efficiency catalysis, and enzyme engineering technologies (higher selectivity, stability, and activity). Furthermore, advanced control strategies of biocatalytic processes and reactors are presented. The future prospect and opportunities for marine functional lipids are also discussed. Therefore, the obtainment of enzymes endowed with superior properties and the development of optimized processes, still have to be pursued to achieve greener bio-catalyzed processes.

Chiral Fibers Formation Upon Assembly of Tetraphenylalanine Peptide Conjugated to a PNA Dimer.

Self-assembly of biomolecules such as peptides, nucleic acids or their analogues affords supramolecular objects, exhibiting structures and physical properties dependent on the amino-acid or nucleobase composition. Conjugation of the peptide diphenylalanine (FF) to peptide nucleic acids triggers formation of self-assembled structures, mainly stabilized by interactions between FF. In this work we report formation of homogeneous chiral fibers upon self-assembly of the hybrid composed of the tetraphenylalanine peptide (4F) conjugated to the PNA dimer adenine-thymine (at). In this case nucleobases seem to play a key role in determining the morphology and chirality of the fibers. When the PNA "at" is replaced by guanine-cytosine dimer "gc", disordered structures are observed. Spectroscopic characterization of the self-assembled hybrids, along with AFM and SEM studies is reported. Finally, a structural model consistent with the experimental evidence has also been obtained, showing how the building blocks of 4Fat arrange to give helical fibers.

Application of enzymes as a feed additive in aquaculture.

Modern aquaculture must be sustainable in terms of energy consumption, raw materials used, and environmental impact, so alternatives are needed to replace fish feed with other raw materials. Enzyme use in the agri-food industry is based on their efficiency, safety, and protection of the environment, which aligns with the requirements of a resource-saving production system. Enzyme supplementation in fish feed can improve digestibility and absorption of both plant- and animal-derived ingredients, increasing the growth parameters of aquacultural animals. Herein we summarized the recent literature that reported the use of digestive enzymes (amylases, lipases, proteases, cellulases, and hemicellulases) and non-digestive enzymes (phytases, glucose oxidase, and lysozyme) in fish feed. In addition, we analyzed how critical steps of the pelleting process, including microencapsulation and immobilization, can interfere with enzyme activity in the final fish feed product. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-022-00128-z.

Enhanced whole-cell biotransformation of 3-chloropropiophenone into 1-phenyl-1-propanone by hydrogel entrapped Chlorella emersonii (211.8b).

OBJECTIVES: This study focuses on dehalogenation of halogenated organic substrate (3-Chloropropiophenone) using both free and hydrogel entrapped microalgae Chlorella emersonii (211.8b) as biocatalyst. We aimed at successful immobilization of C. emersonii (211.8b) cells and to assess their biotransformation efficiency. RESULTS: Aquasorb (entrapping material in this study) was found to be highly biocompatible with the cellular growth and viability of C. emersonii. A promising number of entrapped cells was achieved in terms of colony-forming units (CFUs = 2.1 × 104) per hydrogel bead with a comparable growth pattern to that of free cells. It was determined that there is no activity of hydrogenase that could transform 1-phenyl-2-propenone into 1-phenyl-1-propanone because after 12 h the ratio between two products (0.36 ± 0.02) remained constant throughout. Furthermore, it was found that the entrapped cells have higher biotransformation of 3-chloropropiophenone to 1-phenyl-1-propanone as compared to free cells at every interval of time. 1-phenyl-2-propenone was excluded from the whole-cell biotransformation as it was also found in the control group (due to spontaneous generation). CONCLUSION: Hence, enhanced synthesis of 1-phenyl-1-propanone by entrapped Chlorella (211.8b) can be ascribed to either an enzymatic activity (dehalogenase) or thanks to the antioxidants from 211-8b, especially when they are in immobilized form. The aquasorb based immobilization of microalgae is highly recommended as an effective tool for exploiting microalgal potentials of biocatalysis specifically when free cells activities are seized due to stress.

Biochemical characterization of two ß-N-acetylglucosaminidases from Streptomyces violascens for efficient production of N-acetyl-d-glucosamine.

Chitin, one of the most abundant renewable biopolymers on Earth, is commercially available from crustacean wastes. One critical step in converting chitin to high-value products is its degradation by chitinolytic enzymes to N-acetyl-d-glucosamine (GlcNAc), which plays a significant role in functional food and pharmaceutical industries. Here, we cloned and biochemically characterized two novel ß-N-acetylglucosaminidases named SvNag2557 (family-84) and SvNag4755 (family-3) from Streptomyces violascens ATCC 27968. Both SvNag2557 and SvNag4755 exhibited strict substrate specificity toward N-acetyl chitooligosaccharides with GlcNAc as the sole product. Thus, a one-pot production for pure GlcNAc from chitin by an enzyme cocktail reaction was further developed. Under the co-action of an endo-type chitinase SaChiA4 and SvNag2557 (mass ratio 1:2), the final conversion rates of colloidal chitin and ionic liquid pretreated chitin to GlcNAc were 80.2% and 73.8% with GlcNAc purities of 99.7% and 96.8%, respectively.

Construction of a Super-Folder Fluorescent Protein-Guided Secretory Expression System for the Production of Phospholipase D in Bacillus subtilis.

Phospholipids (PLs) are one of the main ingredients in food and nutraceutical, cosmetics, agriculture, and pharmaceutical products. Phospholipase D (PLD) is a crucial enzyme for the biocatalytic synthesis or modification of PLs. Here, to prepare PLD more efficiently, we constructed a PLD expression and secretion system in Bacillus subtilis and developed an environmentally friendly reaction system. A nonclassical secretory pathway where a super-folder green fluorescent protein plays as an N-terminal guide protein was introduced. This expression system can not only achieve rapid screening of high-level expression strains but can also achieve the secretion of the target proteins. Under optimal fermentation conditions, the enzyme activity of the culture medium was 0.35 U/mL, which was 2.05-fold that of the Sec secretion pathway strains. Meanwhile, the effects of several organic solvents in the biphasic reaction media were compared. The results showed that when using cyclopentyl methyl ether as the organic phase, the final conversion rate reached 96.9%. It has shown good application potential in the synthesis of phosphatidylserine, laid the foundation for the synthesis and application of other rare and high-value PLs, and provided a reference for the production of other biocatalysts.

Co-immobilization of two-component hydroxylase monooxygenase by functionalized magnetic nanoparticles for preserving high catalytic activity and enhancing enzyme stabilty.

Cascade reactions catalyzed by two or more enzymes have been widely used in industrial production and exhibited many advantages over the single-enzyme catalytic system. In this study, two components of hydroxylase monooxygenase (HpaBC) were first co-immobilized by Ni2+-nitrilotriacetic acid (Ni-NTA) functionalized magnetic silica nanoparticles (Ni-NTA/H2N-SiO2@Fe3O4) for enhancing the stability and activity of biocatalysts with multi-components. These two components, HpaB and HpaC, were modified with histidine-tag and employed to construct a bi-enzyme catalytic system. After co-immobilization, the activity of the bi-enzyme system was 2.6 times of free enzymes. Meanwhile, the co-immobilized system was more stable against high temperature and alkaline condition, and maintained 76.6% of the initial activity for storage 12 days. Moreover, the co-immobilized HpaBC remained more than 60% catalytic activity after 7 cycles. These results showed that co-immobilized multi-component enzymes based on functionalized magnetic nanoparticles without purification would play a great potential role in the field of industrial biocatalysis.

Step-wise immobilization of multi-enzymes by zirconium-based coordination polymer in situ self-assembly and specific absorption.

The simple and quick method for co-immobilization of multiple enzymes with clear spatial distribution has presented great challenges for decades. Herein, Zr4+ and 2-methylimidazole (2MIm) coordination polymers (CPs) were used to synthetize co-immobilized nanoreactor by a simple two-step procedure in aqueous environment. The CPs was first self-assembled in situ encapsulating glucose-6-phosphate dehydrogenase (G6PD) and then utilized coordination unsaturated metal sites on the surface of CPs to selectively adsorb hexahistidine-tagged α, ß-unsaturated ketoreductase (his-tagged KRED). The obtained multi-enzymes system (G6PD@Zr-2MIm/KRED) was employed as an enzymatic reactor involving coenzymes regeneration. G6PD@Zr-2MIm/KRED still exhibited good repeatability and storage stability. The bi-enzymatic reactor could achieve more than 95% chalcone conversion ratio after 15 min and good tolerance at high temperature and different pH, retained about 70% and 80% of its initial activity after storage for 4 days and after 4 cycles, respectively. This step-wise enzyme immobilization method is easy to operate and can be used to prepare multi-enzyme systems with clear spatial distribution of the biocatalysts and allowing the coenzymes regeneration.