Engineering Bacillus subtilis for highly efficient production of functional disaccharide lactulose from lactose.

Bioconversion of lactose to functional lactose derivatives attracts increasing attention. Lactulose is an important high-value lactose derivative, which has been widely used in pharmaceutical, nutraceutical, and food industries. Lactulose can be enzymatically produced from lactose by cellobiose 2-epimerase (CEase). Several studies have already focused on the food-grade expression of CEase, but they are all aimed at the biosynthesis of epilactose. Herein, we reported for the first time the biosynthesis of lactulose using the recombinant food-grade Bacillus subtilis. Lactulose biosynthesis was optimized by varying lactulose-producing CEases and expression vectors. Caldicellulosiruptor saccharolyticus CEase and pP43NMK were determined to be the optimal CEase and expression vector. Fine-tuning of CEase expression was investigated by screening a beneficial N-terminal coding sequence. After fed-batch cultivation, the highest fermentation isomerization activity reached 11.6 U/mL. Lactulose was successfully produced by the broth of the engineered B. subtilis with a yield of 52.1 %.

Enhancement of the d-Allulose 3-Epimerase Expression in Bacillus subtilis through Both Transcriptional and Translational Regulations.

d-Allulose, a functional bulk sweetener, has recently attracted increasing attention because of its low-caloric-ness properties and diverse health effects. d-Allulose is industrially produced by the enzymatic epimerization of d-fructose, which is catalyzed by ketose 3-epimerase (KEase). In this study, the food-grade expression of KEase was studied using Bacillus subtills as the host. Clostridium sp. d-allulose 3-epimerase (Clsp-DAEase) was screened from nine d-allulose-producing KEases, showing better potential for expression in B. subtills WB600. Promoter-based transcriptional regulation and N-terminal coding sequence (NCS)-based translational regulation were studied to enhance the DAEase expression level. In addition, the synergistic effect of promoter and NCS on the Clsp-DAEase expression was studied. Finally, the strain with the combination of a PHapII promoter and gln A-Up NCS was selected as the best Clsp-DAEase-producing strain. It efficiently produced Clsp-DAEase with a total activity of 333.2 and 1860.6 U/mL by shake-flask and fed-batch cultivations, respectively.

Minimally invasive transurethral holmium laser surgery for the management of an IUD with bladder migration.

CASE: Intrauterine device (IUD) is used worldwide as an effective contraceptive method, but the migration of IUD is a serious complication. We report the case of IUD migration leading to bladder calculus formation and a minimally invasive transurethral surgical approach was performed for treatment. Holmium laser was used to break up the bladder calculus and cut through the bladder mucosa where the IUD was attached, finally the IUD was removed through the urethra. This minimally invasive procedure is a safe and effective treatment for IUD migration, and similar cases have not been reported in the literature. CONCLUSION: That the secondary bladder calculus were smashed by intense pulse mode of holmium laser, and the bladder tissue around the attached IUD was opened by cutting mode of holmium laser, and finally the IUD was completely removed from urethra, this surgical method is safe and effective, and there is no case report on IUD removal of transurethral cystoscope in the literature.

A review of fructosyl-transferases from catalytic characteristics and structural features to reaction mechanisms and product specificity.

Carbohydrate-active enzymes are accountable for the synthesis and degradation of glycosidic bonds among diverse carbohydrates. Fructosyl-transferases represent a subclass of these enzymes, employing sucrose as a substrate to generate fructooligosaccharides (FOS) and fructan polymers. This category primarily includes levansucrase (LS, EC 2.4.1.10), inulosucrase (IS, EC 2.4.1.9), and ß-fructofuranosidase (Ffase, EC 3.2.1.26). These three enzymes possess a similar five-bladed ß-propeller fold and employ an anomer-retaining reaction mechanism mediated by nucleophiles, transition state stabilizers, and general acids/bases. However, they exhibit distinct product profiles, characterized by variations in linkage specificity and molecular mass distribution. Consequently, this article comprehensively explores recent advancements in the catalytic characteristics, structural features, reaction mechanisms, and product specificity of levansucrase, inulosucrase, and ß-fructofuranosidase (abbreviated as LS, IS, and Ffase, respectively). Furthermore, it discusses the potential for modifying catalytic properties and product specificity through structure-based design, which enables the rational production of custom fructan and FOS.

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.

Rational design for thermostability improvement of a novel PL-31 family alginate lyase from Paenibacillus sp. YN15.

Currently, alginate oligosaccharides (AOS) become attractive due to their excellent physiological effects. AOS has been widely used in food, pharmaceutical, and cosmetic industries. Generally, AOS can be produced from alginate using alginate lyase (ALyase) as the biocatalyst. However, most ALyase display poor thermostability. In this study, a thermostable ALyase from Paenibacillus sp. YN15 (Payn ALyase) was characterized. It belonged to the polysaccharide lyase (PL) 31 family and displayed poly ß-D-mannuronate (Poly M) preference. Under the optimum condition (pH 8.0, 55 °C, 50 mM NaCl), it exhibited maximum activity of 90.3 U/mg and efficiently degraded alginate into monosaccharides and AOS with polymerization (DP) of 2-4. Payn ALyase was relatively stable at 55 °C, but the thermostability dropped rapidly at higher temperatures. To further improve its thermostability, rational design mutagenesis was carried out based on a combination of FireProt, Consensus Finder, and PROSS analysis. Finally, a triple-point mutant K71P/Y129G/S213G was constructed. The optimum temperature was increased from 55 to 70 °C, and the Tm was increased from 62.7 to 64.1 °C. The residual activity after 30 min incubation at 65 °C was enhanced from 36.0 % to 83.3 %. This study provided a promising ALyase mutant for AOS industrial production.

Integrin-targeting disulfide-crosslinked micellar docetaxel eradicates lung and prostate cancer patient-derived xenografts.

Actively targeted nanomedicines though conceptually attractive for tumor therapy are extremely hard to realize due to problems of premature drug leakage, excessive liver accretion, inadequate tumor uptake, and/or retarded drug release inside tumor cells. Here, we systemically studied the influence of disulfide crosslinking on the in vitro and in vivo performance of integrin-targeting micellar docetaxel (t-MDTX). Of note, t-M5DTX with a high disulfide content was clearly advantageous in terms of stability, intracellular drug release, anti-tumor activity toward αVß3-overexpressing A549 cells, blood circulation and therapeutic efficacy in orthotopic A549-luc lung tumor-bearing mice. t-MDTX induced extraordinary tumor targetability with tumor-to-normal tissue ratios of 1.7-8.3. Further studies indicated that t-M5DTX could effectively eradicate αVß3-overexpressing lung and prostate cancer patient-derived xenografts (PDX), in which ca. 80% mice became tumor-free. This integrin-targeting disulfide-crosslinked micellar docetaxel emerges as a promising actively targeted nanoformulation for tumor therapy. STATEMENT OF SIGNIFICANCE: Nanomedicines have a great potential in treating advanced tumor patients; however, their tumor-targeting ability and therapeutic efficacy remain unsatisfactory. In addition to PEGylation and ligand selection, particle size, stability and drug release behavior are also critical to their performance in vivo. In this paper, we find that small and cRGD-guided disulfide-crosslinked micellar docetaxel (t-MDTX) induces superior tumor uptake and retention but without increasing liver burden, leading to extraordinary selectivity and inhibition of αvß3 overexpressing lung tumors. t-MDTX is further shown to effectively treat αvß3-positive patient-derived tumor models, lending it a high potential for clinical translation.

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.

Infant formulae - Key components, nutritional value, and new perspectives.

Breastfeeding is the most effective strategy for meeting the nutritional demands of infants, whilst infant formulae are manufactured foods that mimic human milk and can be safely used to replace breastfeeding. In this paper, the compositional differences between human milk and other mammalian milk are reviewed, and thus nutritional profiles and compositions of standard bovine milk-based formulae as well as special formulae are discussed. Differences between breast milk and other mammalian milk in composition and content affect their digestion and absorption in infants. Characteristics and mimicking of breast milk have been intensively studied with the objective of narrowing the gap between human milk and infant formulae. The functions of the key nutritional components in infant formulae are examined. This review detailed recent developments in the formulation of different types of special infant formulae and efforts for their humanization, and summarized safety and quality control of infant formulae.

Identification of a Thermostable Levansucrase from Pseudomonas orientalis That Allows Unique Product Specificity at Different Temperatures.

The biological production of levan by levansucrase (LS, EC 2.4.1.10) has aroused great interest in the past few years. Previously, we identified a thermostable levansucrase from Celerinatantimonas diazotrophica (Cedi-LS). A novel thermostable LS from Pseudomonas orientalis (Psor-LS) was successfully screened using the Cedi-LS template. The Psor-LS showed maximum activity at 65 °C, much higher than the other LSs. However, these two thermostable LSs showed significantly different product specificity. When the temperature was decreased from 65 to 35 °C, Cedi-LS tended to produce high-molecular-weight (HMW) levan. By contrast, Psor-LS prefers to generate fructooligosaccharides (FOSs, DP ≤ 16) rather than HMW levan under the same conditions. Notably, at 65 °C, Psor-LS would produce HMW levan with an average Mw of 1.4 × 106 Da, indicating that a high temperature might favor the accumulation of HMW levan. In summary, this study allows a thermostable LS suitable for HMW levan and levan-type FOSs production simultaneously.

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.

Successful Manipulation of the Product Spectrum of the Erwinia amylovora Levansucrase by Modifying the Residues around loop1, Loop 3, and Loop 4.

Levansucrase (LS, EC 2.4.1.10) catalyzes the synthesis of levan by successively transferring the fructosyl moiety from sucrose to an elongated fructan chain. Although the product distribution of LS from Erwinia amylovora (Ea-LS) was studied under different sucrose concentrations, the effect of residues on the product formation is yet unknown. The first levanhexaose-complexed structure of LS from Bacillus subtilis (Bs-SacB) provided information on the oligosaccharide binding sites (OB sites), from +1 to +4 subsites. Since Ea-LS would efficiently produce fructooligosaccharides, a substitution mutation of OB sites in Bs-SacB and the corresponding residues of Ea-LS were conducted to investigate how these mutants would influence the product distribution. As a result, a series of mutants with different product spectrum were obtained. Notably, the mutants of G98E, V151F, and N200T around loop 1, loop 3, and loop 4 all showed a significant increase in both the molecular mass and the yield of high-molecular-mass levan, suggesting that the product profile of Ea-LS was significantly modified.

Characterization of a processive inulosucrase from Lactobacillus mulieris for efficient biosynthesis of high-molecular-weight inulin.

Inulin has been determined to have many exceptional properties and functions and has been used in the food and pharmaceutical fields. Recently, microbial high-molecular-weight inulin synthesized from sucrose by inulosucrase attracted much attention. In this study, a novel inulosucrase from Lactobacillus mulieris was constructed, overexpressed, purified, and identified. The recombinant enzyme displayed the maximum activity at pH 6.0 and 55 °C, and it exhibited high thermostability below 45 °C. After optimizing the production conditions, the conversion rate from 100 g/L sucrose to inulin reached 31 %, meanwhile, the maximum molecular weight of produced inulin reached 3.21 × 106 g/mol. The truncated IS showed a "processive" transfructosylation process, only synthesizing a small number of short-chain oligosaccharides with polymerization degrees below 6, which was in favor of the accumulation of high-molecular-weight inulin. Given this, L. mulieris inulosucrase might be a good potential candidate for the industrial production of high-molecular-weight inulin.

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.

Mutations in the Different Residues between Dextransucrase Gtf-DSM and Reuteransucrase GtfO for the Investigation of Linkage Specificity Determinants.

The dextransucrase Gtf-DSM has 99.3% sequence identity with the reuteransucrase GtfO, and only 11 out of 1045 residues are different between their N-terminally truncated recombinant forms. Gtf-DSM is capable of synthesizing a dextran with 1% (α1 → 2), 6% (α1 → 4), 24% (α1 → 3), and 69% (α1 → 6) linkages, while GtfO produces a reuteran with 21% (α1 → 6) and 79% (α1 → 4) linkages. In this work, using recombinant Gtf-DSM and GtfO as templates, parallel substitutions targeting these 11 distinguishing residues were performed to investigate their linkage specificity determinants. The combinatorial mutation (I937L/D977A/D1083V/Q1086K/K1087G) at the acceptor binding subsites +1 and +2 nearly converted the linkage specificity of Gtf-DSM to that of GtfO. Surprisingly, all of the individual or combinatorial mutations in four residues from domains IV and V of Gtf-DSM significantly altered the linkage specificity of Gtf-DSM. Additionally, all mutations in the 11 distinguishing residues of Gtf-DSM resulted in a dramatically reduced transferase/hydrolysis activity ratio, which was closer to that of GtfO. These mutation results suggested that the linkage specificity differences between Gtf-DSM and GtfO are determined by the distinct micro-physicochemical environments, formed by the concerted action of a series of residues not only from the acceptor binding subsites +1 and +2 but also from domains IV and V.

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.

Directionally modulating the product chain length of an inulosucrase by semi-rational engineering for efficient production of 1-kestose.

Microbial inulosucrase as a transfructosylation tool has been used to produce inulin and inulin-type fructooligosaccharides with various polymerization degrees. Tailor-made oligosaccharides could be generated by inulosucrase via chain length modulation. In this study, a semi-rational design based on the modeled structure of Lactobacillus reuteri 121 inulosucrase was carried out to screen and construct variants. The residues Arg541 and Arg544 were determined to be significant to the product chain elongation of L. reuteri 121 inulosucrase. The variant R544W altered the product specificity of inulosucrase and produced short-chain fructooligosaccharides with 1-kestose as the main component. Molecular dynamic simulations verified an increased binding free energy of variant R544W with 1-kestose than the wild-type enzyme with 1-kestose. After optimization, 1-kestose and total short-chain fructooligosaccharides production reached approximately 206 g/L and 307 g/L, respectively. This study suggests the great potential of variant R544W in the biotransformation from sucrose to functional sugar.

Comprehensive utilization of sucrose resources via chemical and biotechnological processes: A review.

Sucrose, one of the most widespread disaccharides in nature, has been available in daily human life for many centuries. As an abundant and cheap sweetener, sucrose plays an essential role in our diet and the food industry. However, it has been determined that many diseases, such as obesity, diabetes, hyperlipidemia, etc., directly relate to the overconsumption of sucrose. It arouses many explorations for the conversion of sucrose to high-value chemicals. Production of valuable substances from sucrose by chemical methods has been studied since a half-century ago. Compared to chemical processes, biotechnological conversion approaches of sucrose are more environmentally friendly. Many enzymes can use sucrose as the substrate to generate functional sugars, especially those from GH68, GH70, GH13, and GH32 families. In this review, enzymatic catalysis and whole-cell fermentation of sucrose for the production of valuable chemicals were reviewed. The multienzyme cascade catalysis and metabolic engineering strategies were addressed.

Crystal Structure of Levansucrase from the Gram-Negative Bacterium Brenneria Provides Insights into Its Product Size Specificity.

Microbial levansucrases (LSs, EC 2.4.1.10) have been widely studied for the synthesis of ß-(2,6)-fructans (levan) from sucrose. LSs synthesize levan-type fructo-oligosaccharides, high-molecular-mass levan polymer or combinations of both. Here, we report crystal structures of LS from the G--bacterium Brenneria sp. EniD 312 (Brs-LS) in its apo form, as well as of two mutants (A154S, H327A) targeting positions known to affect LS reaction specificity. In addition, we report a structure of Brs-LS complexed with sucrose, the first crystal structure of a G--LS with a bound substrate. The overall structure of Brs-LS is similar to that of G-- and G+-LSs, with the nucleophile (D68), transition stabilizer (D225), and a general acid/base (E309) in its active site. The H327A mutant lacks an essential interaction with glucosyl moieties of bound substrates in subsite +1, explaining the observed smaller products synthesized by this mutant. The A154S mutation affects the hydrogen-bond network around the transition stabilizing residue (D225) and the nucleophile (D68), and may affect the affinity of the enzyme for sucrose such that it becomes less effective in transfructosylation. Taken together, this study provides novel insights into the roles of structural elements and residues in the product specificity of LSs.

Immunotherapy of Malignant Glioma by Noninvasive Administration of TLR9 Agonist CpG Nano-Immunoadjuvant.

Immunotherapy with toll like receptor 9 (TLR9) agonist CpG ODN offers an emergent strategy to treat life-threatening malignant glioma. CpG is typically applied invasively by intracranial and intrathecal administration which induces not only poor compliance and lessened potency but also possibly strong adverse effects and immunotoxicity. Here, it is reported that immunotherapy of murine LCPN glioma is greatly boosted by polymersome-steered intravenous and intranasal brain delivery of CpG. CpG is efficiently loaded in apolipoprotein E peptide-directed polymersomes to give blood-brain barrier permeable and glioma and cervical lymph node-homing CpG nano-immunoadjuvant (t-NanoCpG) which strongly stimulates the maturation of dendritic cells, antigen cross-presentation, and production of proinflammatory cytokines in vivo. Intriguingly, both intravenous and intranasal administration of t-NanoCpG brings about significant survival benefits in murine LCPN glioma-bearing mice while free CpG and nontargeted CpG nano-immunoadjuvant (NanoCpG) afford modest therapeutic effects. Moreover, combination of t-NanoCpG with radiotherapy further boosts the immunotherapeutic effects leading to more improved survival rate of mice. This intelligent brain-permeable nano-immunoadjuvant provides a new, minimally invasive and highly potent strategy for immunotherapy of glioma.