Characterization of a novel multifunctional ß-glucosidase/xylanase/feruloyl esterase and its effects on improving the quality of Longjing tea.

Herein, a novel multifunctional enzyme ß-glucosidase/xylanase/feruloyl esterase (GXF) was constructed by fusion of ß-glucosidase and bifunctional xylanase/feruloyl esterase. The activities of ß-glucosidase, xylanase, feruloyl esterase and acetyl xylan esterase displayed by GXF were 67.18 %, 49.54 %, 38.92 % and 23.54 %, respectively, higher than that of the corresponding single functional enzymes. Moreover, the GXF performed better in enhancing aroma and quality of Longjing tea than the single functional enzymes and their mixtures. After treatment with GXF, the grassy and floral odors of tea infusion were significantly improved. Moreover, GXF treatment could improve concentrations of flavonoid aglycones of myricetin, kaempferol and quercetin by 68.1-, 81.42- and 77.39-fold, respectively. In addition, GXF could accelerate the release of reducing sugars, ferulic acid and xylo-oligosaccharides by 9.48-, 8.25- and 4.11-fold, respectively. This multifunctional enzyme may have potential applications in other fields such as food production and biomass degradation.

The Property of a Key Amino Acid Determines the Function of Farnesyl Pyrophosphate Synthase in Sporobolomyces pararoseus NGR.

Farnesyl pyrophosphate synthase (FPPS) catalyzes the synthesis of C15 farnesyl diphosphate (FPP) from C5 dimethylallyl diphosphate (DMAPP) and two or three C5 isopentenyl diphosphates (IPPs). FPP is an important precursor for the synthesis of isoprenoids and is involved in multiple metabolic pathways. Here, farnesyl pyrophosphate synthase from Sporobolomyces pararoseus NGR (SpFPPS) was isolated and expressed by the prokaryotic expression system. The SpFPPS full-length genomic DNA and cDNA are 1566 bp and 1053 bp, respectively. This gene encodes a 350-amino acid protein with a predicted molecular mass of 40.33 kDa and a molecular weight of 58.03 kDa (40.33 kDa + 17.7 kDa), as detected by SDS-PAGE. The function of SpFPPS was identified by induction, purification, protein concentration and in vitro enzymatic activity experiments. Structural analysis showed that Y90 was essential for chain termination and changing the substrate scope. Site-directed mutation of Y90 to the smaller side-chain amino acids alanine (A) and lysine (K) showed in vitro that wt-SpFPPS catalyzed the condensation of the substrate DMAPP or geranyl diphosphate (GPP) with IPP at apparent saturation to synthesize FPP as the sole product and that the mutant protein SpFPPS-Y90A synthesized FPP and C20 geranylgeranyl diphosphate (GGPP), while SpFPPS-Y90K hydrolyzed the substrate GGPP. Our results showed that FPPS in S. pararoseus encodes the SpFPPS protein and that the amino acid substitution at Y90 changed the distribution of SpFPPS-catalyzed products. This provides a baseline for potentially regulating SpFPPS downstream products and improving the carotenoid biosynthesis pathway.

Progress in Gene Editing and Metabolic Regulation of Saccharomyces cerevisiae with CRISPR/Cas9 Tools.

The CRISPR/Cas9 systems have been developed as tools for genetic engineering and metabolic engineering in various organisms. In this review, various aspects of CRISPR/Cas9 in Saccharomyces cerevisiae, from basic principles to practical applications, have been summarized. First, a comprehensive review has been conducted on the history of CRISPR/Cas9, successful cases of gene disruptions, and efficiencies of multiple DNA fragment insertions. Such advanced systems have accelerated the development of microbial engineering by reducing time and labor, and have enhanced the understanding of molecular genetics. Furthermore, the research progress of the CRISPR/Cas9-based systems in the production of high-value-added chemicals and the improvement of stress tolerance in S. cerevisiae have been summarized, which should have an important reference value for genetic and synthetic biology studies based on S. cerevisiae.

Rewiring Bacillus subtilis and bioprocess optimization for oxidoreductive reaction-mediated biosynthesis of D-tagatose.

D-tagatose holds significant importance as a functional monosaccharide with diverse applications in food, medicine, and other fields. This study aimed to explore the oxidoreductive pathway for D-tagatose production, surpassing the contemporary isomerization-mediated biosynthesis approach in order to enhance the thermodynamic equilibrium of the reactions. Initially, a novel galactitol dehydrogenase was discovered through biochemical and bioinformatics analyses. By co-expressing the galactitol dehydrogenase and xylose reductase, the oxidoreductive pathway for D-tagatose synthesis was successfully established in Bacillus subtilis. Subsequently, pathway fine-tuning was achieved via promoter regulation and dehydrogenase-mediated cofactor regeneration, resulting in 6.75-fold higher D-tagatose compared to that produced by the strain containing the unmodified promoter. Finally, optimization of fermentation conditions and medium composition produced 39.57 g/L D-tagatose in a fed-batch experiment, with a productivity of 0.33 g/L/h and a yield of 0.55 mol/mol D-galactose. These findings highlight the potential of the constructed redox pathway as an effective approach for D-tagatose production.

Research progress of engineering microbial cell factories for pigment production.

Pigments are widely used in people's daily life, such as food additives, cosmetics, pharmaceuticals, textiles, etc. In recent years, the natural pigments produced by microorganisms have attracted increased attention because these processes cannot be affected by seasons like the plant extraction methods, and can also avoid the environmental pollution problems caused by chemical synthesis. Synthetic biology and metabolic engineering have been used to construct and optimize metabolic pathways for production of natural pigments in cellular factories. Building microbial cell factories for synthesis of natural pigments has many advantages, including well-defined genetic background of the strains, high-density and rapid culture of cells, etc. Until now, the technical means about engineering microbial cell factories for pigment production and metabolic regulation processes have not been systematically analyzed and summarized. Therefore, the studies about construction, modification and regulation of synthetic pathways for microbial synthesis of pigments in recent years have been reviewed, aiming to provide an up-to-date summary of engineering strategies for microbial synthesis of natural pigments including carotenoids, melanins, riboflavins, azomycetes and quinones. This review should provide new ideas for further improving microbial production of natural pigments in the future.

An experimental study of magnetic compression technique for ureterovesical anastomosis in rabbits.

This study aimed to explore the feasibility of the magnetic compression technique (MCT) for ureterovesical anastomosis in a rabbit model with ureteral obstruction. The distal ureteral obstruction model using female New Zealand rabbits was induced by ligating the distal end of the right ureter with silk thread for four weeks. A pair of cylindrical NdFeB magnets (daughter magnet and parent magnet) with a hole in the center was used for the ureterovesical anastomosis. The daughter magnet and the parent magnet were respectively placed close to the obstruction site through the dilated proximal ureter and urethra, and then the daughter-parent magnets pair was attracted together automatically. Postoperative X-rays were taken to confirm the position of the magnets. The anastomotic stoma specimens were obtained two weeks postoperatively, and the anastomotic stoma formation was observed by the naked eye and histological staining. The operation time and the anastomotic burst pressure were measured. The ureter was significantly dilated in the fourth week after ligation, which satisfied the placement of the daughter magnet. The ureterovesical magnet placements were successfully performed in ten experimental rabbits, with an operation time of 36.5 ± 6.09 min. The parent and daughter magnets attracted each other well and were subsequently removed through the urethra two weeks postoperatively, resulting in the establishment of ureterovesical anastomosis. The anastomotic burst pressure was 147.5 ± 14.59 mmHg. Gross specimens and histological examination of the anastomotic stoma showed that the anastomotic stoma healed well. MCT is feasible and simple for ureterovesical anastomosis.

The strategy of cell extract based metal organic frameworks (CE-MOF) for improved enzyme characteristics.

A convenient cell extract based metal organic frameworks (CE-MOF) strategy was used to produce self-assembled hybrid microparticles of enzymes with improved characteristics. It was shown that many metal ions and enzymes could be used to construct catalytically active CE-MOF microparticles. As a proof-of-principle study, the ß-xylosidase BH3683 was used to prepare FeSO4-CE-MOF-BH3683 microparticles to explore the factors influencing preparation of the microparticles. As a result, DNA, RNA, polysaccharides and proteins were found to play important roles in the formation of the microparticles and affected enzyme activities through interaction with enzyme molecules. Compared with the free BH3683, the optimum temperature of FeSO4-CE-MOF-BH3683 increased 5 °C, and the relative activity at 70 °C increased two times. Moreover, FeSO4-CE-MOF-BH3683 have stronger tolerance to different concentrations of various organic solvents and high-concentration xylose than the free BH3683, and the CE-MOF microparticles prepared by BH3683 and xylanase XynII could catalyze high-concentration xylan more efficiently than their free counterparts. In addition, FeSO4-CE-MOF-BH3683 exhibited about 40 % of its initial activity after reused for 10 times, showing satisfactory reusability. To sum up, this strategy might have wide application potential in the fields of biocatalysis, biofuel production, fertilizer industry, etc.

Establishment of Yan-Zhang's staging of digestive tract magnetic compression anastomosis in a rat model.

Magnetic compression anastomosis, also known as magnamosis, is a safe and feasible method for digestive tract anastomosis. However, the pathological process involved in magnamosis of the digestive tract has not been investigated. This study aimed to establish the stages of digestive tract magnamosis in a rat model. Eighty-four Sprague-Dawley albino rats (200-250 g) were randomly divided into 14 groups (n = 6 per group). All rats underwent colonic magnamosis. Starting from postoperative day (POD) 1, one group of rats was sacrificed every other day to obtain the specimens. Burst pressure at the anastomotic site of each specimen was examined. Gross and histological examination of the anastomotic site was performed to establish the stages of the digestive tract magnamosis. Colonic magnamosis was successfully performed in all rats and the mean anastomosis time was 5.62 ± 0.91 min. The postoperative survival rate was 100%. The lowest anastomotic burst pressure was 78.33 ± 3.44 mmHg on POD3. The anastomotic burst pressure gradually increased and stabilized on POD21. Macroscopic and histological examination showed that the anastomotic mucosal and serosal layer did not heal on POD1. The serosal layer of the anastomosis healed by adhesion on POD3, and the mucosal layer began to heal on POD3-11 and was established by POD21. According to the anastomotic bursting pressure, digestive tract magnamosis can be staged into the magnetic maintenance, fragile, strengthening, and stable phases, which on histology correspond to the serosal adhesion formation, serosal healing, mucosal healing, and stereotyping, respectively.

Biocatalytic conversion of a lactose-rich dairy waste into D-tagatose, D-arabitol and galactitol using sequential whole cell and fermentation technologies.

Dairy industry waste has been explored as a cheap and attractive raw material to produce various commercially important rare sugars. In this study, a lactose-rich dairy byproduct, namely cheese whey powder (CWP), was microbially converted into three low caloric sweeteners using whole-cell and fermentation technologies. Firstly, the simultaneous lactose hydrolysis and isomerization of lactose-derived D-galactose into D-tagatose was performed by an engineered Escherichia coli strain co-expressing ß-galactosidase and L-arabinose isomerase, which eventually produced 68.35 g/L D-tagatose during sequential feeding of CWP. Subsequently, the mixed syrup containing lactose-derived D-glucose and residual D-galactose was subjected to fermentation by Metschnikowia pulcherrima E1, which produced 60.12 g/L D-arabitol and 28.26 g/L galactitol. The net titer of the three rare sugars was 156.73 g/L from 300 g/L lactose (equivalent to 428.57 g/L CWP), which was equivalent to 1.12 mol product/mol lactose and 52.24% conversion efficiency in terms of lactose.

Creation of gastroenteric anastomosis through natural orifice in rats by magnetic compression technique.

BACKGROUND: Being one of the core techniques of magnetic surgery, magnetic compression technique (MCT) has been used for digestive tract anastomosis reconstruction in experimental studies. This study verified the feasibility of gastroenteric anastomosis through natural orifice using MCT in rats. METHODS: The parent and daughter magnets were designed and manufactured for oral and anal insertion in 20 Sprague-Dawley rats. After anesthesia, the parent magnet was inserted into the colon spleen area through the anus, and the daughter magnet was inserted into the stomach through the mouth. Then the two magnets were positioned to attract each other and bind together. The position of the two magnets was monitored using X-ray. The time required for the formation of the anastomosis and expulsion of the magnets were recorded. 2 weeks later, the animal was sacrificed and the anastomotic specimen was obtained which was observed under naked eye and microscope. RESULTS: The gastroenteric anastomosis was successfully performed via natural orifices in 18 out of 20 rats. The mean time to construct the anastomosis was 3.78 ± 0.88 min. X-ray examination showed that the magnets were in the appropriate position in 17 rats. The magnets were excreted in 9.47 ± 1.62 days after surgery. The gross and microscopic examination of the specimen showed that the anastomoses were patent and the mucosa at the anastomotic was smooth. The mean bursting pressure of the anastomosis was 136.94 ± 6.79 mmHg. CONCLUSION: It is feasible to perform gastroenteric anastomosis through natural orifices by MCT.

Novel biocatalytic strategy of levan: His-ELP-intein-tagged protein purification and biomimetic mineralization.

Here a versatile fusion tag composed of His-tag, intein, and elastin-like polypeptide (ELP) tag was prepared for the first time to be fused with levansucrase SacB to construct a recombinant His-ELP-intein-SacB (HEIS) protein to realize nonchromatographic purification of SacB. The efficient biomimetic mineralization of CaHPO4 and HEIS-based hybrid-hydrangea (CaHPO4-HEIS-HH) with good reusability, excellent storage stability and 254.3% improved relative levan yield was prepared with the biomimetic mineralization method. Additionally, the CaHPO4-HEIS-HH showed outstanding operation activity when catalyzing sucrose in solution and up to 75% sucrose conversion rate in fruit juices. The mechanism of biomimetic mineralization was analyzed to show that the HEIS protein might serve as a "binder" to assemble the nanoflakes during biomimetic mineralization. The CaHPO4-HEIS-HH was applicable for efficient production of the levan-type prebiotic polysaccharides, and this approach should be highly valuable for nonchromatographic purification and convenient preparation of various encapsulated enzymes for more efficient catalysis.

Biochemical characterization of an engineered bifunctional xylanase/feruloyl esterase and its synergistic effects with cellulase on lignocellulose hydrolysis.

Herein, the xylanase and feruloyl esterase domains of the xylanase/feruloyl esterase bifunctional enzyme (Xyn-Fae) from Prevotella ruminicola 23 were identified using N- and C-terminal truncation mutagenesis. In addition, a novel and more efficient xylanase/feruloyl esterase bifunctional enzyme XynII-Fae was constructed, and its synergistic action with a commercial cellulase for lignocellulose hydrolysis was studied. When 40% cellulase was replaced by XynII-Fae, the production of reducing sugars increased by 65% than that with the cellulase alone, and the conversions of xylan and glucan were increased by 125.1% and 54.3%, respectively. When 80% cellulase was substituted by XynII-Fae, up to 43.5 µg/mL ferulic acid and 418.7 µg/mL acetic acid were obtained. The XynII-Fae could also accelerate the hydrolysis of wheat straw and sugarcane bagasse with commercial cellulase. These results indicated that the synergistic action of XynII-Fae with cellulase could dramatically improve the hydrolysis efficiency of lignocellulose, showing the great potential for industrial applications.

Enhanced Biosynthesis of D-Arabitol by Metschnikowia reukaufii Through Optimizing Medium Composition and Fermentation Conditions.

D-Arabitol is an important functional sugar alcohol, which can be used in the preparation of foods, chemicals, and medicines. Despite biological production of D-arabitol from low-cost substrates has recently been the focus of research, low yield of this technology has limited its large-scale exploitation. Optimization of this bioprocess could be a promising option to improve the yield of D-arabitol. In this study, one-factor-at-a-time (OFAT) strategy and Box-Behnken design (BBD) were used to increase D-arabitol production by Metschnikowia reukaufii CICC 31,858 through optimizing the fermentation conditions and medium composition. The OFAT optimization provided the optimal conditions for temperature, agitation speed, and fermentation time of 30℃, 220 rpm, and 6 days, respectively. Likewise, the optimum concentrations of peptone, ammonium sulfate, KH2PO4, MgSO4·7H2O, and fumaric acid in the fermentation medium were (g/L) 7.5, 1, 2, 0.5, and 7.5, respectively. Under these optimum conditions, 80.43 g/L of D-arabitol was produced from 200 g/L of glucose, with a productivity of 0.56 g/L/h. The BBD optimization with three important components of fermentation medium (KH2PO4, MgSO4·7H2O, and fumaric acid) showed that the predicted titer of D-arabitol varied from 47.21 to 89.27 g/L, and the actual titer of D-arabitol ranged from 47.36 to 89.83 g/L. The optimum concentrations (g/L) of KH2PO4, MgSO4·7H2O, and fumaric acid in the fermentation medium were found to be 1.0, 0.5, and 4.7 g/L, respectively. Under the optimum conditions, 92.45 g/L of D-arabitol was finally produced with the yield and productivity of 0.46 g/g and 0.64 g/L/h, respectively.

The Characterization of a Novel D-allulose 3-Epimerase from Blautia produca and Its Application in D-allulose Production.

D-allulose is a natural rare sugar with important physiological properties that is used in food, health care items, and even the pharmaceutical industry. In the current study, a novel D-allulose 3-epimerase gene (Bp-DAE) from the probiotic strain Blautia produca was discovered for the production and characterization of an enzyme known as Bp-DAE that can epimerize D-fructose into D-allulose. Bp-DAE was strictly dependent on metals (Mn2+ and Co2+), and the addition of 1 mM of Mn2+ could enhance the half-life of Bp-DAE at 55 °C from 60 to 180 min. It exhibited optimal activity in a pH of 8 and 55 °C, and the Km values of Bp-DAE for the different substrates D-fructose and D-allulose were 235.7 and 150.7 mM, respectively. Bp-DAE was used for the transformation from 500 g/L D-fructose to 150 g/L D-allulose and exhibited a 30% of conversion yield during biotransformation. Furthermore, it was possible to employ the food-grade microbial species Bacillus subtilis for the production of D-allulose using a technique of whole-cell catalysis to circumvent the laborious process of enzyme purification and to obtain a more stable biocatalyst. This method also yields a 30% conversion yield.

[Neuroprotective effect of cinnamaldehyde in MPTP-induced mouse model of subacute Parkinson's disease].

This paper aims to explore the neuroprotective effect of cinnamaldehyde(CA) in mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)-induced subacute Parkinson's disease(PD) and the mechanism. To be specific, male C57 BL/6 mice(n=72, SPF) were randomized into control group, model group, positive control(madopar 0.1 mg·g~(-1)) group, and low-dose, me-dium-dose, and high-dose CA groups(0.15, 0.30, 0.60 mg·g~(-1)). MPTP(intraperitoneal injection, 0.03 mg·g~(-1), once a day for 5 days) was used to induce subacute PD in mice except for the control group. The administration began from the day of modeling and lasted 19 days. On the 0 th, 12 th, and 19 th day, the open field test, pole test, and rotarod test were carried out. After the tests, the mice were killed and brains were separated. In addition, the organ index was measured. The number of cells in substantia nigra(SN) in the midbrain of MPTP-induced PD model mice was detected based on hematoxylin and eosin(HE) staining. The levels of tyrosine hydroxylase(TH)-and α-synuclein(α-Syn)-positive cells in SN were determined by immunohistochemical staining, and the protein levels of TH and α-Syn in SN by Western blot. The results showed that the MPTP-stimulated mice had abnormal behaviors such as erect hair, arched back, rigidity of the tail, slow movement, and tremor, decreased number of crossings and rearing, increased frequency of urination and defecation, longer time of pole climbing, and shorter time of staying on the rotating rod. In addition, the mice showed obvious damage of neurons in the SN and reduced neuron cells in irregular arrangement with some shrinking. In addition, the average optical density of TH in SN decreased and that of α-Syn increased. All these suggested the successful modeling. CA displayed obvious therapeutic effect on the PD mice, as manifested by the increased number of crossings and rearing, decreased frequency of urination and defecation, shorter time of climbing pole, longer time of staying on the rotating rod, and more neuron cells in the SN with a few pykno-tic cells. Moreover, CA significantly alleviated the decrease of TH and the overexpression of α-Syn in SN. As a result, the MPTP-induced injury of dopaminergic neurons was alleviated. The performance of 0.3 mg·g~(-1) CA was the best. This study is expected to lay a scientific basis for the development of CA products.

Construction of novel curdlan-based and Ca2+-chelated magnetic microspheres (CCMM) for efficient protein purification and oriented immobilization.

In this study, curdlan-based and calcium ion (Ca2+)-chelated magnetic microspheres (CCMM) were prepared for protein purification and oriented immobilization. Additional purification steps before immobilization were not required. CCMM samples were produced by reverse embedding of Fe3O4 nanoparticles with curdlan and chelated with Ca2+ in the presence of iminodiacetic acid. The ß-xylanase XynII from Trichoderma reesei QM6a was used to investigate the efficiency of CCMM preparation. The resulting CCMM-XynII was found to be very stable, showing 82 % and 60 % of initial activities after storage for 35 days and after being assayed ten times, respectively. In addition, the CCMM-XynII showed higher stabilities in the presence of organic solvents and multiple chemicals than the free XynII, suggesting that the CCMM-XynII could be efficient for applications requiring the presence of organic solvents. In addition, CCMM may be more suitable than commercially available Ni-NTA for purification of proteins intolerant of Ni2+.

Modification of DNA regions with metagenomic DNA fragments (MDRMDF): A convenient strategy for efficient protein engineering.

In this study, we have established a convenient and efficient approach named Modification of DNA Regions with Metagenomic DNA Fragments (MDRMDF) for protein engineering. Degenerate primers were designed corresponding to conserved regions of the gene of interest which were used for amplification of fragments with template of the metagenomic DNA. The resulting PCR products were used to replace the corresponding regions of the gene of interest to introduce modified gene for function-based screening. Therefore, this method can make full use of the metagenomic DNA sequences with unknown metagenomic gene information for efficient protein engineering. The ß-xylosidase BH3683 was used to construct a MDRMDF library which was screened with a newly designed p-NPX-M9 medium-based strategy. As a result, a mutant protein Xyl-M56 showing high activity, improved pH stability and higher tolerance to organic solvents was obtained which may have potential for industrial application. The MDRMDF method may find wide application in enzyme engineering, metabolic engineering and other fields, especially offering a new methodological option for the directed evolution of proteins.

Efficient Molecular Biological Manipulations with Improved Strategies Based on Novel Escherichia coli Vectors.

In this study, some novel plasmids have been constructed for flexible and zero-background molecular cloning, more efficient expression, and purification of proteins with improved strategies. The plasmids pANY4-pL18-ccdB and pANY4-pR18/pL18-ccdB have different promoters in the complementary DNA strands. Therefore, recombinant plasmids for either isopropyl-ß-d-thiogalactoside-induced or temperature-induced protein expression could be simultaneously constructed in a single molecular cloning process for parallel comparison. Intriguingly, the mutated pL18 and pR18/pL18 promoters performed similar to or even better than the T7 promoter when used for promoting the expression of the GFP or pfLamA enzyme. Moreover, the plasmid pANY8 containing the His-elastin-like polypeptide (ELP)-intein multifunctional tag was constructed, and special purification protocol was designed to obtain purified proteins without the requirement of time-consuming dialysis steps to remove imidazole and high concentration of salt ions. Additionally, the urea-based denaturation and refolding processes can be conveniently integrated into the ELP-mediated precipitation protocol for purification of insoluble inclusion bodies, omitting the time-consuming dialysis steps.

Exploring a Highly D-Galactose Specific L-Arabinose Isomerase From Bifidobacterium adolescentis for D-Tagatose Production.

D-Galactose-specific L-arabinose isomerase (L-AI) would have much potential for the enzymatic conversion of D-Galactose into D-tagatose, while most of the reported L-AIs are L-arabinose specific. This study explored a highly D-Galactose-specific L-AI from Bifidobacterium adolescentis (BAAI) for the production of D-tagatose. In the comparative protein-substrate docking for D-Galactose and L-arabinose, BAAI showed higher numbers of hydrogen bonds in D-Galactose-BAAI bonding site than those found in L-arabinose-BAAI bonding site. The activity of BAAI was 24.47 U/mg, and it showed good stability at temperatures up to 65°C and a pH range 6.0-7.5. The K m, V max, and K cat/K m of BAAI were found to be 22.4 mM, 489 U/mg and 9.3 mM-1 min-1, respectively for D-Galactose, while the respective values for L-arabinose were 40.2 mM, 275.1 U/mg, and 8.6 mM-1 min-1. Enzymatic conversion of D-Galactose into D-tagatose by BAAI showed 56.7% conversion efficiency at 55°C and pH 6.5 after 10 h.