Protein Engineering of a Novel ß-Galactosidase from Thermus scotoductus for Efficient Synthesis of Lacto-N-Neotetraose from Chitin Powder.

A novel ß-galactosidase (TsGal48) from Thermus scotoductus was cloned, and the enzyme was biochemically characterized. TsGal48 catalyzed the synthesis of lacto-N-neotetraose (LNnT) from lactose via the transglycosylation reaction with a maximal yield of 20%, which is the highest yield for the synthesis of LNnT so far. To further improve the yield of LNnT, TsGal48 was successfully engineered by directed evolution and site-saturation mutagenesis. A mutated ß-galactosidase (mTsGal48) was selected and characterized. mTsGal48 produced LNnT with a yield of 27.7 g/L, which is 1.4-fold higher than that of TsGal48 (19.7 g/L). Then, a developed strategy for LNnT synthesis from chitin powder was provided in a 30 L bioreactor. The reaction process included chitin powder hydrolysis, lacto-N-triose II (LNT2) synthesis, and LNnT synthesis. The reaction time was reduced from 44 to 17 h in chitin powder hydrolysis and LNT2 synthesis. The content of LNnT was up to 25 g/L in the multienzyme system. The green and efficient route may be suitable for large-scale production of LNnT from chitin powder.

Simultaneous improvement of thermostability and maltotriose-forming ability of a fungal α-amylase for bread making by directed evolution.

For applications in food industries, a fungal α-amylase from Malbranchea cinnamomea was engineered by directed evolution. Through two rounds of screening, a mutant α-amylase (mMcAmyA) was obtained with higher optimal temperature (70 °C, 5 °C increase) and better hydrolysis properties (18.6 % maltotriose yield, 2.5-fold increase) compared to the wild-type α-amylase (McAmyA). Site-directed mutations revealed that Threonine (Thr) 226 Serine (Ser) substitution was the main reason for the property evolution of mMcAmyA. Through high cell density fermentation, the highest expression level of Thr226Ser was 3951 U/mL. Thr226Ser was further used for bread baking with a dosage of 1000 U/kg flour, resulting in a 17.8 % increase in specific volume and a 35.6 % decrease in hardness compared to the control. The results were a significant improvement on those of McAmyA. Moreover, the mutant showed better anti-staling properties compared to McAmyA, as indicated by the improved sensory evaluation after 4 days of storage at 4 and 25 °C. These findings provide insights into the structure-function relationship of fungal α-amylase and introduce a potential candidate for bread-making industry.

Efficient Biosynthesis of Difucosyllactose via De Novo GDP-l-Fucose Pathway in Metabolically Engineered Escherichia coli.

Difucosyllactose (DFL) is an important component of human milk oligosaccharides (HMOs) and has significant benefits for the growth and development of infants. So far, a few microbial cell factories have been constructed for the production of DFL, which still have problems of low production and high cost. Herein, a high-level de novo pathway DFL-producing strain was constructed by multistep optimization strategies in Escherichia coli BL21star(DE3). We first efficiently synthesized the intermediate 2'-fucosyllactose (2'-FL) in E. coli BL21star(DE3) by the advisable stepwise strategy. The truncated α-1,3/4-fucosyltransferase (Hp3/4FT) was then introduced into the engineered strain to achieve de novo biosynthesis of DFL. ATP-dependent protease (Lon) and GDP-mannose hydrolase (NudK) were deleted, and mannose-6-phosphate isomerase (ManA) was overexpressed to improve GDP-l-fucose accumulation. The regulator RcsA was overexpressed to fine-tune the expression level of pathway genes, thereby increasing the synthesis of DFL. The final strain produced 6.19 g/L of DFL in the shake flask and 33.45 g/L of DFL in the 5 L fermenter, which were the highest reported titers so far. This study provides a more economical, sustainable, and effective strategy to produce the fucosylated human milk oligosaccharides (HMOs).

Structural and functional analysis of SpGlu64A: a novel glycoside hydrolase family 64 laminaripentaose-producing ß-1,3-glucanase from Streptomyces pratensis.

Laminaripentaose (L5)-producing ß-1,3-glucanases can preferentially cleave the triple-helix curdlan into ß-1,3-glucooligosaccharides, especially L5. In this study, a newly identified member of the glycoside hydrolase family 64, ß-1,3-glucanase from Streptomyces pratensis (SpGlu64A), was functionally and structurally characterized. SpGlu64A shared highest identity (30%) with a ß-1,3-glucanase from Streptomyces matensis. The purified SpGlu64A showed maximal activity at pH 7.5 and 50 °C, and exhibited strict substrate specificity toward curdlan (83.1 U·mg-1 ). It efficiently hydrolyzed curdlan to produce L5 as the end product. The overall structure of SpGlu64A consisted of a barrel domain and a mixed (α/ß) domain, which formed an unusually wide groove with a crescent-like structure. In the two complex structures (SpGlu64A-L3 and SpGlu64A-L4), two oligosaccharide chains were captured and the triple-helical structure was relatively compatible with the wide groove, which suggested the possibility of binding to the triple-helical ß-1,3-glucan. A catalytic framework (ß6-ß9-ß10) and the steric hindrance formed by the side chains of residues Y161, N163, and H393 in the catalytic groove were predicted to complete the exotype-like cleavage manner. On the basis of the structure, a fusion protein with the CBM56 domain (SpGlu64A-CBM) and a mutant (Y161F; by site-directed mutation) were obtained, with 1.2- and 1.7-fold increases in specific activity, respectively. Moreover, the combined expression of SpGlu64A-CBM and -Y161F improved the enzyme activity by 2.63-fold. The study will not only be helpful in understanding the reaction mechanism of ß-1,3-glucanases but will also provide a basis for further enzyme engineering.

Construction of an engineered Escherichia coli for effective synthesis of 2'-fucosyllactose via the salvage pathway.

2'-Fucosyllactose (2'-FL) is one of the important functional oligosaccharides in breast milk. So far, few attempts on biosynthesis of 2'-FL by the salvage pathway have been reported. Herein, the salvage pathway enzyme genes were introduced into the E. coli BL21star(DE3) for synthesis of 2'-FL. The 2'-FL titer increased from 1.56 to 2.13 g/L by deleting several endogenous genes on competitive pathways. The α-1,2-fucosyltransferase (WbgL) was selected, and improved the 2'-FL titer to 2.88 g/L. Additionally, the expression level of pathway enzyme genes was tuned through optimizing the plasmid copy number. Furthermore, the spatial distribution of WbgL was enhanced by fusing with the MinD C-tag. After optimizing the fermentation conditions, the 2'-FL titer reached to 7.13 g/L. The final strain produced 59.22 g/L of 2'-FL with 95% molar conversion rate of lactose and 92% molar conversion rate of fucose in a 5 L fermenter. These findings will contribute to construct a highly efficient microbial cell factory to produce 2'-FL or other HMOs.

Characterization of a novel aspartic protease from Trichoderma asperellum for the preparation of duck blood peptides.

A novel aspartic protease gene (TaproA1) from Trichoderma asperellum was successfully expressed in Komagataella phaffii (Pichia pastoris). TaproA1 showed 52.8% amino acid sequence identity with the aspartic protease PEP3 from Coccidioides posadasii C735. TaproA1 was efficiently produced in a 5 L fermenter with a protease activity of 4092 U/mL. It exhibited optimal reaction conditions at pH 3.0 and 50 °C and was stable within pH 3.0-6.0 and at temperatures up to 45 °C. The protease exhibited broad substrate specificity with high hydrolysis activity towards myoglobin and hemoglobin. Furthermore, duck blood proteins (hemoglobin and plasma protein) were hydrolyzed by TaproA1 to prepare bioactive peptides with high ACE inhibitory activity. The IC50 values of hemoglobin and plasma protein hydrolysates from duck blood proteins were 0.105 mg/mL and 0.091 mg/mL, respectively. Thus, the high yield and excellent biochemical characterization of TaproA1 presented here make it a potential candidate for the preparation of duck blood peptides. KEY POINTS: • An aspartic protease (TaproA1) from Trichoderma asperellum was expressed in Komagataella phaffii. • TaproA1 exhibited broad substrate specificity and the highest activity towards myoglobin and hemoglobin. • TaproA1 has great potential for the preparation of bioactive peptides from duck blood proteins.

Evaluating Recalibrating AI Models for Breast Cancer Diagnosis in a New Context: Insights from Transfer Learning, Image Enhancement and High-Quality Training Data Integration.

This paper investigates the adaptability of four state-of-the-art artificial intelligence (AI) models to the Australian mammographic context through transfer learning, explores the impact of image enhancement on model performance and analyses the relationship between AI outputs and histopathological features for clinical relevance and accuracy assessment. A total of 1712 screening mammograms (n = 856 cancer cases and n = 856 matched normal cases) were used in this study. The 856 cases with cancer lesions were annotated by two expert radiologists and the level of concordance between their annotations was used to establish two sets: a 'high-concordances subset' with 99% agreement of cancer location and an 'entire dataset' with all cases included. The area under the receiver operating characteristic curve (AUC) was used to evaluate the performance of Globally aware Multiple Instance Classifier (GMIC), Global-Local Activation Maps (GLAM), I&H and End2End AI models, both in the pretrained and transfer learning modes, with and without applying the Contrast Limited Adaptive Histogram Equalization (CLAHE) algorithm. The four AI models with and without transfer learning in the high-concordance subset outperformed those in the entire dataset. Applying the CLAHE algorithm to mammograms improved the performance of the AI models. In the high-concordance subset with the transfer learning and CLAHE algorithm applied, the AUC of the GMIC model was highest (0.912), followed by the GLAM model (0.909), I&H (0.893) and End2End (0.875). There were significant differences (p < 0.05) in the performances of the four AI models between the high-concordance subset and the entire dataset. The AI models demonstrated significant differences in malignancy probability concerning different tumour size categories in mammograms. The performance of AI models was affected by several factors such as concordance classification, image enhancement and transfer learning. Mammograms with a strong concordance with radiologists' annotations, applying image enhancement and transfer learning could enhance the accuracy of AI models.

Development of a two-enzyme system in Aspergillus niger for efficient production of N-acetyl-ß-D-glucosamine from powdery chitin.

A chitinase (PbChi70) from Paenibacillus barengoltzii was engineered by directed evolution to enhance its hydrolysis efficiency towards powder chitin. Through two rounds of screening, a mutant (mPbChi70) with a maximum specific activity of 73.21 U/mg was obtained, which is by far the highest value ever reported. The mutant gene was further transformed into Aspergillus niger FBL-B (ΔglaA) which could secrete high level of endogenously ß-N-acetylglucosaminidase (GlcNAcase), thus a two-enzyme expression system was constructed. The highest chitinase activity of 61.33 U/mL with GlcNAcase activity of 353.1 U/mL was obtained in a 5-L fermentor by high-cell density fermentation. The chitin-degrading enzyme cocktail was used for the bioconversion of GlcNAc from powder chitin directly, and the highest conversion ratio reached high up to 71.9 % (w/w) with GlcNAc purity ≥95 % (w/w). This study may provide an excellent chitinase as well as a double enzyme cocktail system for efficient biological conversion of chitin materials.

Manno-oligosaccharides from Cassia Seed Gum Attenuate Atherosclerosis through Inflammation Modulation and Intestinal Barrier Integrity Improvement in ApoE-/- Mice.

SCOPE: Manno-oligosaccharides from cassia seed gum (CMOS) have demonstrated anti-inflammatory and regulatory effects on cholesterol metabolism. However, their protective effects against the progression of atherosclerosis (AS) and underlying molecular mechanisms have not been investigated. This study investigates the anti-atherosclerotic effects of CMOS on ApoE-/- mice. METHODS AND RESULTS: CMOS are supplemented in atherosclerotic male ApoE-/- mice fed with a high-fat-high-cholesterol diet (HFHCD). After the 12-week intervention, CMOS at 1200 mg kg-1 ·bw d-1 significantly decrease the atherosclerotic lesion area by 0.63-fold and the aortic arch lesion size by 0.63-fold when compared to the HFHCD group. Moreover, inflammation in atherosclerotic lesions is reduced by CMOS intervention, and the levels of serum lipids and inflammatory cytokines are decreased. The number of goblet cells and the expression of intestinal epithelial tight junction proteins in the H-CMOS group increase, thus indicating that CMOS can restore intestinal barrier integrity in atherosclerotic mice. Furthermore, CMOS reshape the unbalanced gut microbiota in ApoE-/- mice caused by HFHCD, and reduce the relative abundance of Desulfovibrio and Faecalibaculum that exhibits positive relationships with inflammation. CONCLUSION: CMOS inhibit inflammation, alter intestinal barrier integrity, and regulate gut microbiota to attenuate AS in ApoE-/- mice.

High-level production of a novel alginate lyase (FsAly7) from Flammeovirga sp. for efficient production of low viscosity soluble dietary fiber from sodium alginate.

Sodium alginate is one of the most abundant sustainable gum source for dietary fiber production. However, the preparation efficiencies of low viscosity soluble dietary fiber from sodium alginate remain low. Here, a novel alginate lyase gene (FsAly7) from Flammeovirga sp. was identified and high-level expressed in Pichia pastoris for low viscosity soluble dietary fiber production. The highest enzyme production of 3050 U mL-1 was achieved, which is by far the highest yield ever reported. FsAly7 was used for low viscosity soluble dietary fiber production from sodium alginate, and the highest degradation rate of 85.5 % was achieved under a high substrate content of 20 % (w/v). The molecular weight of obtained soluble dietary fiber converged to 10.75 kDa. FsAly7 catalyzed the cleavage of glycosidic bonds in alginate chains with formation of unsaturated non-reducing ends simultaneously in the degradation process, thus altered the chemical structures of hydrolysates. The soluble dietary fiber exhibited excellent properties, including low viscosity, high oil adsorption capacity activity (2.20 ± 0.03 g g-1) and high emulsifying activity (60.05 ± 2.96 mL/100 mL). This investigation may provide a novel alginate lyase catalyst as well as a solution for the efficient production of low viscosity soluble dietary fiber from sodium alginate.

CRISPR/Cas9 System-Mediated Multi-copy Expression of an Alkaline Serine Protease in Aspergillus niger for the Production of XOD-Inhibitory Peptides.

CRISPR/Cas9 system-mediated multi-copy expression of an alkaline serine protease (AoproS8) from Aspergillus oryzae was successfully built in Aspergillus niger. Furthermore, AoproS8 was continuously knocked in the glaA, amyA, and aamy gene loci in A. niger to construct multi-copy expression strains. The yield of the AoproS8 3.0 strain was 2.1 times higher than that of the AoproS8 1.0 strain. Then, a high protease activity of 11,023.2 U/mL with a protein concentration of 10.8 mg/mL was obtained through fed-batch fermentation in a 5 L fermenter. This is the first report on the high-level expression of alkaline serine proteases in A. niger. AoproS8 showed optimal activity at pH 9.0 and 40 °C. It was used for the production of xanthine oxidase (XOD)-inhibitory peptides from eight food processing protein by-products. Among them, the duck hemoglobin hydrolysates showed the highest XOD-inhibitory activity with an IC50 value of 2.39 mg/mL. Thus, our work provides a useful way for efficient expression of proteases in A. niger and high-value utilization of protein by-products.

Biochemical characterization of a novel ß-galactosidase from Lacticaseibacillus zeae and its application in synthesis of lacto-N-tetraose.

Lacto-N-tetraose (LNT) is one of the most important components of human milk oligosaccharides, which has various beneficial health effects. ß-Galactosidase is an important enzyme used in dairy processing. The transglycosylation activity of ß-galactosidases offers an attractive approach for LNT synthesis. In this study, we reported for the first time the biochemical characterization of a novel ß-galactosidase (LzBgal35A) from Lacticaseibacillus zeae. LzBgal35A belongs to glycoside hydrolases (GH) family 35 and shared the highest identity of 59.9% with other reported GH 35 members. The enzyme was expressed as soluble protein in Escherichia coli. The purified LzBgal35A displayed optimal activity at pH 4.5 and 55°C. It was stable within the pH range of 3.5 to 7.0 and up to 60°C. Moreover, LzBgal35A could catalyze the synthesis of LNT via transferring the galactose residue from o-nitrophenyl-ß-galactopyranoside to lacto-N-triose II. Under optimal conditions, the conversion rate of LNT reached 45.4% (6.4 g/L) within 2 h, which was by far the highest yield of LNT synthesized through a ß-galactosidase-mediated transglycosylation reaction. This study demonstrated that LzBgal35A has great potential application in LNT synthesis.

N-acetyl-d-glucosamine-based oligosaccharides from chitin: Enzymatic production, characterization and biological activities.

Chitin, the second most abundant biopolymer, possesses diverse applications in the food, agricultural, and pharmaceutical industries due to its functional properties. However, the potential applications of chitin are limited owing to its high crystallinity and low solubility. N-acetyl chitooligosaccharides and lacto-N-triose II, the two types of GlcNAc-based oligosaccharides, can be obtained from chitin by enzymatic methods. With their lower molecular weights and improved solubility, these two types of GlcNAc-based oligosaccharides display more various beneficial health effects when compared to chitin. Among their abilities, they have exhibited antioxidant, anti-inflammatory, anti-tumor, antimicrobial, and plant elicitor activities as well as immunomodulatory and prebiotic effects, which suggests they have the potential to be utilized as food additives, functional daily supplements, drug precursors, elicitors for plants, and prebiotics. This review comprehensively covers the enzymatic methods used for the two types of GlcNAc-based oligosaccharides production from chitin by chitinolytic enzymes. Moreover, current advances in the structural characterization and biological activities of these two types of GlcNAc-based oligosaccharides are summarized in the review. We also highlight current problems in the production of these oligosaccharides and trends in their development, aiming to offer some directions for producing functional oligosaccharides from chitin.

Synthesis of 2'-fucosyllactose from apple pomace-derived xyloglucan oligosaccharides by an α-L-fucosidase from Pedobacter sp. CAU209.

2'-Fucosyllactose (2'-FL) is known for its ability to provide various health benefits to infants, such as gut maturation, pathogen resistance, improved immunity, and nervous system development. However, the production of 2'-FL using α-L-fucosidases is hindered by the lack of low-cost natural fucosyl donors and high-efficiency α-L-fucosidases. In this work, a recombinant xyloglucanase from Rhizomucor miehei (RmXEG12A) was applied to produce xyloglucan-oligosaccharide (XyG-oligos) from apple pomace. Then, an α-L-fucosidase gene (PbFucB) was screened from the genomic DNA of Pedobacter sp. CAU209 and expressed in Escherichia coli. The capability of purified PbFucB to catalyze XyG-oligos and lactose to synthesize 2'-FL was further evaluated. The deduced amino acid sequence of PbFucB shared the highest identity (38.4%) with that of other reported α-L-fucosidases. PbFucB showed the highest activity at pH 5.5 and 35 °C. It catalyzed the hydrolysis of 4-nitrophenyl-α-L-fucopyranoside (pNP-Fuc, 20.3 U mg-1), 2'-FL (8.06 U mg-1), and XyG-oligos (0.43 U mg-1). Furthermore, PbFucB demonstrated a high enzymatic conversion rate in 2'-FL synthesis with pNP-Fuc or apple pomace-derived XyG-oligos as donors and lactose as acceptor. Under the optimized conditions, PbFucB converted 50% of pNP-Fuc or 31% of the L-fucosyl residue in XyG-oligos into 2'-FL. This work elucidated an α-L-fucosidase that mediates the fucosylation of lactose and provided an efficient enzymatic strategy to synthesize 2'-FL either from artificial pNP-Fuc or natural apple pomace-derived XyG-oligos. KEY POINTS: • Xyloglucan-oligosaccharide (XyG-oligos) was produced from apple pomace by a xyloglucanase from Rhizomucor miehei. • An α-L-fucosidase (PbFucB) from Pedobacter sp. CAU209 shared the highest identity (38.4%) with reported α-L-fucosidases. •PbFucB synthesized 2'-FL using apple pomace-derived XyG-oligos and lactose with a conversion ratio of 31%.

Releasing Bioactive Compounds from Brown Seaweed with Novel Cold-Adapted Alginate Lyase and Alcalase.

Seaweeds are considered to be third-generation renewable biomasses, the comprehensive utilization of which has drawn increasing attention in recent years. A novel cold-active alginate lyase (VfAly7) was identified from Vibrio fortis and biochemically characterized for brown seaweed utilization. The alginate lyase gene was high-level expressed in Pichia pastoris, with an enzyme yield of 560 U/mL and a protein content of 9.8 mg/mL by high-cell density fermentation. The recombinant enzyme was most active at 30 °C and pH 7.5, respectively. VfAly7 was a bifunctional alginate lyase with both poly-guluronate and poly-mannuronate hydrolysis activities. On the basis of VfAly7, a bioconversion strategy for the utilization of brown seaweed (Undaria pinnatifida) was developed. The obtained AOSs showed stronger prebiotic activity towards tested probiotics when compared to that of commercial fructooligosaccharides (FOSs), while the obtained protein hydrolysates displayed strong xanthine oxidase inhibitory activity with IC50 of 3.3 mg/mL. This study provided a novel alginate lyase tool as well as a biotransformation route for the utilization of seaweeds.

Dietary neoagarotetraose extends lifespan and impedes brain aging in mice via regulation of microbiota-gut-brain axis.

INTRODUCTION: Dietary oligosaccharides can impact the gut microbiota and confer tremendous health benefits. OBJECTIVES: The aim of this study was to determine the impact of a novel functional oligosaccharide, neoagarotetraose (NAT), on aging in mice. METHODS: 8-month-old C57BL/6J mice as the natural aging mice model were orally administered with NAT for 12 months. The preventive effect of NAT in Alzheimer's disease (AD) mice was further evaluated. Aging related indicators, neuropathology, gut microbiota and short-chain fatty acids (SCFAs) in cecal contents were analyzed. RESULTS: NAT treatment extended the lifespan of these mice by up to 33.3 %. Furthermore, these mice showed the improved aging characteristics and decreased injuries in cerebral neurons. Dietary NAT significantly delayed DNA damage in the brain, and inhibited reduction of tight junction protein in the colon. A significant increase at gut bacterial genus level (such as Lactobacillus, Butyricimonas, and Akkermansia) accompanied by increasing concentrations of SCFAs in cecal contents was observed after NAT treatment. Functional profiling of gut microbiota composition indicated that NAT treatment regulated the glucolipid and bile acid-related metabolic pathways. Interestingly, NAT treatment ameliorated cognitive impairment, attenuated amyloid-ß (Aß) and Tau pathology, and regulated the gut microbiota composition and SCFAs receptor-related pathway of Alzheimer's disease (AD) mice. CONCLUSION: NAT mitigated age-associated cerebral injury in mice through gut-brain axis. The findings provide novel evidence for the effect of NAT on anti-aging, and highlight the potential application of NAT as an effective intervention against age-related diseases.

Tamarind Xyloglucan Oligosaccharides Attenuate Metabolic Disorders via the Gut-Liver Axis in Mice with High-Fat-Diet-Induced Obesity.

Functional oligosaccharides exert obesity-reducing effects by acting at various pathological sites responsible for the development of obesity. In this study, tamarind xyloglucan oligosaccharides (TXOS) were used to attenuate metabolic disorders via the gut-liver axis in mice with high-fat-diet (HFD)-induced obesity, as determined through LC/MS-MS and 16S rRNA sequencing technology. A TXOS dose equivalent to 0.39 g/kg/day in humans restored the gut microbiota in obese mice, which was in part supported by the key microflora, particularly Bifidobacterium pseudolongum. Moreover, TXOS reduced the abundance of opportunistic pathogen species, such as Klebsiella variicola and Romboutsia ilealis. The bodyweight and weight gain of TXOS-treated (4.8 g/kg per day) mice began to decrease at the 14th week, decreasing by 12.8% and 23.3%, respectively. Sixteen fatty acids were identified as potential biomarkers in the liver, and B. pseudolongum and caprylic acid were found to tightly regulate each other. This was associated with reduced inflammation in the liver, circulation, and adipose tissue and protection from metabolic disorders. The findings of this study indicate that TXOS can significantly increase the gut microbiota diversity of obese mice and restore the HFD-induced dysbiosis of gut microbiota.

Nondigestible Functional Oligosaccharides: Enzymatic Production and Food Applications for Intestinal Health.

Nondigestible functional oligosaccharides are of particular interest in recent years because of their unique prebiotic activities, technological characteristics, and physiological effects. Among different types of strategies for the production of nondigestible functional oligosaccharides, enzymatic methods are preferred owing to the predictability and controllability of the structure and composition of the reaction products. Nondigestible functional oligosaccharides have been proved to show excellent prebiotic effects as well as other benefits to intestinal health. They have exhibited great application potential as functional food ingredients for various food products with improved quality and physicochemical characteristics. This article reviews the research progress on the enzymatic production of several typical nondigestible functional oligosaccharides in the food industry, including galacto-oligosaccharides, xylo-oligosaccharides, manno-oligosaccharides, chito-oligosaccharides, and human milk oligosaccharides. Moreover, their physicochemical properties and prebiotic activities are discussed as well as their contributions to intestinal health and applications in foods.

Chitin and chitin-based biomaterials: A review of advances in processing and food applications.

Chitin is the most abundant natural amino polysaccharide, showing various practical applications owing to its functional properties. However, there are barriers in the development due to the difficulty of chitin extraction and purification, regarding its high crystallinity and low solubility. In recent years, some novel technologies such as microbial fermentation, ionic liquid, electrochemical extraction have emerged for the green extraction of chitin from new sources. Furthermore, nanotechnology, dissolution systems and chemical modification were applied to develop a variety of chitin-based biomaterials. Remarkably, chitin was used in delivering active ingredients and developing functional foods for weight loss, lipid reduction, gastrointestinal health, and anti-aging. Moreover, the application of chitin-based materials was expanded into medicine, energy and the environment. This review outlined the emerging extraction methods and processing routes of different chitin sources and advances in applying chitin-based materials. We aimed to provide some direction for the multi-disciplinary production and application of chitin.

Directed evolution of a ß-N-acetylhexosaminidase from Haloferula sp. for lacto-N-triose II and lacto-N-neotetraose synthesis from chitin.

In our previous study, a ß-N-acetylhexosaminidase (HaHex74) from Haloferula sp. showing high human milk oligosaccharides (HMOs) synthesis ability was identified and characterized. In this study, HaHex74 was further engineered by directed evolution and site-saturation mutagenesis to improve its transglycosylation activity for HMOs synthesis. A mutant (mHaHex74) with improved transglycosylation activity (HaHex74-Asn401Ile/His394Leu) was obtained and characterized. mHaHex74 exhibited maximal activity at pH 5.5 and 35 °C, respectively, which were distinct from that of HaHex74 (pH 6.5 and 45 °C). Moreover, mHaHex74 showed the highest LNT2 conversion ratio of 28.2% from N,N'-diacetyl chitobiose (GlcNAc2), which is 2.2 folds higher than that of HaHex74. A three-enzyme cascade reaction for the synthesis of LNT2 and LNnT from chitin was performed in a 5-L reactor, and the contents of LNT2 and LNnT reached up to 15.0 g L1 and 4.9 g L1, respectively. Therefore, mHaHex74 maybe a good candidate for enzymatic synthesis of HMOs.