Mining and functional characterization of NADPH-cytochrome P450 reductases of the DNJ biosynthetic pathway in mulberry leaves.

BACKGROUND: 1-Deoxynojirimycin (DNJ), the main active ingredient in mulberry leaves, with wide applications in the medicine and food industries due to its significant functions in lowering blood sugar, and lipids, and combating viral infections. Cytochrome P450 is a key enzyme for DNJ biosynthesis, its activity depends on the electron supply of NADPH-cytochrome P450 reductases (CPRs). However, the gene for MaCPRs in mulberry leaves remains unknown. RESULTS: In this study, we successfully cloned and functionally characterized two key genes, MaCPR1 and MaCPR2, based on the transcriptional profile of mulberry leaves. The MaCPR1 gene comprised 2064 bp, with its open reading frame (ORF) encoding 687 amino acids. The MaCPR2 gene comprised 2148 bp, and its ORF encoding 715 amino acids. The phylogenetic tree indicates that MaCPR1 and MaCPR2 belong to Class I and Class II, respectively. In vitro, we found that the recombinant enzymes MaCPR2 protein could reduce cytochrome c and ferricyanide using NADPH as an electron donor, while MaCPR1 did not. In yeast, heterologous co-expression indicates that MaCPR2 delivers electrons to MaC3'H hydroxylase, a key enzyme catalyzing the production of chlorogenic acid from 3-O-p-coumaroylquinic acid. CONCLUSIONS: These findings highlight the orchestration of hydroxylation process mediated by MaCPR2 during the biosynthesis of secondary metabolite biosynthesis in mulberry leaves. These results provided a foundational understanding for fully elucidating the DNJ biosynthetic pathway within mulberry leaves.

Effect of Frost on the Different Metabolites of Two Mulberry (Morus nigra L. and Morus alba L.) Leaves.

Mulberry leaves are a well-known traditional Chinese medicine herb, and it has been observed since ancient times that leaves collected after frost have superior medicinal properties. Therefore, understanding the changes in critical metabolic components of mulberry leaves, specifically Morus nigra L., is essential. In this study, we conducted widely targeted metabolic profiling analyses on two types of mulberry leaves, including Morus nigra L. and Morus alba L., harvested at different times. In total, we detected over 100 compounds. After frost, 51 and 58 significantly different metabolites were identified in the leaves of Morus nigra L. and Morus alba L., respectively. Further analysis revealed a significant difference in the effect of defrosting on the accumulation of metabolites in the two mulberries. Specifically, in Morus nigra L., the content of 1-deoxynojirimycin (1-DNJ) in leaves decreased after frost, while flavonoids peaked after the second frost. In Morus alba L., the content of DNJ increased after frost, reaching its peak one day after the second frost, whereas flavonoids primarily peaked one week before frost. In addition, an analysis of the influence of picking time on metabolite accumulation in two types of mulberry leaves demonstrated that leaves collected in the morning contained higher levels of DNJ alkaloids and flavonoids. These findings provide scientific guidance for determining the optimal harvesting time for mulberry leaves.

1-Deoxynojirimycin promotes cardiac function and rescues mitochondrial cristae in mitochondrial hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is the most prominent cause of sudden cardiac death in young people. Due to heterogeneity in clinical manifestations, conventional HCM drugs have limitations for mitochondrial hypertrophic cardiomyopathy. Discovering more effective compounds would be of substantial benefit for further elucidating the pathogenic mechanisms of HCM and treating patients with this condition. We previously reported the MT-RNR2 variant associated with HCM that results in mitochondrial dysfunction. Here, we screened a mitochondria-associated compound library by quantifying the mitochondrial membrane potential of HCM cybrids and the survival rate of HCM-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) in galactose media. 1-Deoxynojirimycin (DNJ) was identified to rescue mitochondrial function by targeting optic atrophy protein 1 (OPA1) to promote its oligomerization, leading to reconstruction of the mitochondrial cristae. DNJ treatment further recovered the physiological properties of HCM iPSC-CMs by improving Ca2+ homeostasis and electrophysiological properties. An angiotensin II-induced cardiac hypertrophy mouse model further verified the efficacy of DNJ in promoting cardiac mitochondrial function and alleviating cardiac hypertrophy in vivo. These results demonstrated that DNJ could be a potential mitochondrial rescue agent for mitochondrial hypertrophic cardiomyopathy. Our findings will help elucidate the mechanism of HCM and provide a potential therapeutic strategy.

A comprehensive review on the production, pharmacokinetics and health benefits of mulberry leaf iminosugars: Main focus on 1-deoxynojirimycin, d-fagomine, and 2-O-ɑ-d-galactopyranosyl-DNJ.

Mulberry leaves are rich in biologically active compounds, including phenolics, polysaccharides, and alkaloids. Mulberry leaf iminosugars (MLIs; a type of polyhydroxylated alkaloids), in particular, have been gaining increasing attention due to their health-promoting effects, including anti-diabetic, anti-obesity, anti-hyperglycemic, anti-hypercholesterolemic, anti-inflammatory, and gut microbiota-modulatory activities. Knowledge regarding the in vivo bioavailability and bioactivity of MLIs are crucial to understand their role and function and human health. Therefore, this review is aimed to comprehensively summarize the existing studies on the oral pharmacokinetics and the physiological significance of selected MLIs (i.e.,1-deoxynojirimycin, d-fagomine, and 2-O-ɑ-d-galactopyranosyl-DNJ). Evidence have suggested that MLIs possess relatively good uptake and safety profiles, which support their prospective use for oral intake; the therapeutic potential of these compounds against metabolic and chronic disorders and the underlying mechanisms behind these effects have also been studied in in vitro and in vivo models. Also discussed are the biosynthetic pathways of MLIs in plants, as well as the agronomic and processing factors that affect their concentration in mulberry leaves-derived products.

Molecular cloning, expression, and functional analysis of copper amine oxidase gene from mulberry (Morus alba L.).

In this study, we investigated a key enzyme encoded by the gene copper amine oxidase (MaCAO), which is involved in the biosynthetic pathway of 1-deoxynojirimycin (DNJ)1, an active ingredient in mulberry leaves. The 1680 bp long MaCAO was successfully cloned (GenBank accession no: MH205733). Subsequently, MaCAO was heterologously expressed using a recombinant plasmid, pET-22b (+)/MaCAO in Escherichia coli BL21 (DE3). A protein with a molecular mass of 62.9 kDa was obtained, whose function was validated through enzymatic reaction. Bioinformatics analysis identified that MaCAO contained the same conserved domain as that of copper amine oxidases ("NYDY"). Furthermore, the tertiary structure of the predicted protein using homology modeling revealed 46% similarity with that of copper amine oxidase (Protein Data Bank ID: 1W2Z). Gas chromatography-mass spectrometry analysis of the enzymatic reaction revealed that MaCAO could catalyze 1,5-pentanediamine to produce 5-aminopentanal. Additionally, levels of mulberry leaf DNJ content were significantly positively correlated with expression levels of MaCAO (P < 0.001). Our results conclude that MaCAO is the key enzyme involved in the biosynthetic pathway of DNJ. The function of MaCAO is validated, providing a foundation for the further analysis of biosynthetic pathways of DNJ in mulberry leaves using tools of synthetic biology.

Mulberry-Derived 1-Deoxynojirimycin Prevents Type 2 Diabetes Mellitus Progression via Modulation of Retinol-Binding Protein 4 and Haptoglobin.

Pre-diabetic or early-stage type 2 diabetes patients may develop an adverse diabetic progression, leading to several complications and increasing hospitalization rates. Mulberry leaves, which contain 1-deoxynojirimycin (DNJ), have been used as a complementary medicine for diabetes prevention and treatment. Our recent study demonstrated that mulberry leaf powder with 12 mg of DNJ improves postprandial hyperglycemia, fasting plasma glucose, and glycated hemoglobin. However, the detailed mechanisms are still unknown. This study investigates the effect of long-term (12-week) supplementation of mulberry leaves in obese people with prediabetes and patients with early-stage type 2 diabetes. Participants' blood was collected before and after supplementation. The protein profile of the plasma was examined by proteomics. In addition, the mitochondrial function was evaluated by energetic and homeostatic markers using immunoelectron microscopy. The proteomics results showed that, from a total of 1291 proteins, 32 proteins were related to diabetes pathogenesis. Retinol-binding protein 4 and haptoglobin protein were downregulated, which are associated with insulin resistance and inflammation, respectively. For mitochondrial function, the haloacid dehalogenase-like hydrolase domain-containing protein 3 (HDHD-3) and dynamin-related protein 1 (Drp-1) displayed a significant increment in the after treatment group. In summary, administration of mulberry leaf powder extract in prediabetes and the early stage of diabetes can alleviate insulin resistance and inflammation and promote mitochondrial function in terms of energy production and fission.

Screening, cloning and functional characterization of key methyltransferase genes involved in the methylation step of 1-deoxynojirimycin alkaloids biosynthesis in mulberry leaves.

MAIN CONCLUSION: The novel C-methyltransferase, MaMT1, could catalyze the conversion of piperidine to 2-methylpiperidine, which may be involved in the methylation step of DNJ biosynthesis in mulberry leaves. Mulberry (Morus alba L.) is a worldwide crop with medicinal, feeding and nutritional value, and 1-deoxynojirimycin ((2R, 3R, 4R, 5S)-2-hydroxymethyl-3, 4, 5-trihydroxypiperidine, DNJ) alkaloid, a potent α-glucosidase inhibitor, is its main active ingredient. Our previous researches clarified the biosynthetic pathway of DNJ from lysine to Δ1-piperideine, but its downstream pathway is unclear. Herein, eight differential methyltransferases (MTs) genes were screened from transcriptome profiles of mulberry leaves with significant differences in DNJ content (P < 0.01). Subsequently, MaMT1 (OM140666) and MaMT2 (OM140667) were hypothesized as candidate genes related to DNJ biosynthesis by correlation analysis of genes expression levels and DNJ content of mulberry leaves at different dates. Functional characterization of MaMT1 and MaMT2 were performed by cloning, prokaryotic expression and enzymatic reaction in vitro, and it showed that MaMT1 protein could catalyze the conversion of piperidine to 2-methylpiperidine. Moreover, molecular docking confirmed the interaction of MaMT1 protein with piperidine and S-adenosyl-L-methionine (SAM), indicating that MaMT1 had C-methyltransferase activity, while MaMT2 did not. The above results suggested that MaMT1 may be involved in the methylation step of DNJ alkaloid biosynthesis in mulberry leaves, which is a breakthrough in the analysis of DNJ alkaloid biosynthetic pathway. It is worth mentioning that the novel MaMT1, annotated as serine hydroxymethyltransferase, could rely on SAM to perform C-methyltransferase function. Therefore, our findings contribute new insights into the research of DNJ alkaloid biosynthesis and C-methyltransferase family.

α-Glucosidase Inhibitory Activity of Fermented Okara Broth Started with the Strain Bacillus amyloliquefaciens SY07.

In this work, a new strain of Bacillus amyloliquefaciens SY07 isolated from a traditional fermented soybean food was reported to possess remarkable α-glucosidase inhibitor-producing ability. Different culture media were applied for the proliferation of B. amyloliquefaciens SY07, and it was found that fermented okara broth presented the highest α-glucosidase inhibitory activity, while Luria-Bertani medium showed a negative effect. The extract from fermented okara broth acted in a dose-dependent manner to inhibit α-glucosidase activity, with an IC50 value of 0.454 mg/mL, and main inhibitors in the fermentation extract presented a reversible, uncompetitive pattern according to Lineweaver-Burk plots. Moreover, 1-deoxynojirimycin, a recognized α-glucosidase inhibitor, was found in the extract. Results indicated that B. amyloliquefaciens SY07 could utilize okara, a by-product from the soy processing industry, to generate α-glucosidase inhibitors effectively, and be regarded as a novel excellent microbial candidate for safe, economical production of potential functional foods or ingredients with hypoglycemic effect.

Comparative Transcriptome Analysis of Key Reductase Genes Involved in the 1-Deoxynojirimycin Biosynthetic Pathway in Mulberry Leaves and Cloning, Prokaryotic Expression, and Functional Analysis of MaSDR1 and MaSDR2.

The alkaloid 1-deoxynojirimycin (DNJ) is the main bioactive ingredient in the hypoglycemic action of mulberry leaves (Morus alba L.). Our previous research clarified the upstream pathway from lysine to Δ1-piperideine in the biosynthesis of DNJ in mulberry leaves, but the pathway and related reductase genes from Δ1-piperideine to piperidine are still unclear. Here, a comparative transcriptome was used to analyze the transcriptome data of two samples (July and November) of mulberry leaves with significant differences in the content of DNJ and screen-related reductase genes. Results showed that expression levels of MaSDR1 and MaSDR2 were significantly and positively correlated with the content of DNJ (P < 0.05) in different seasons. MaSDR1 (GenBank accession no. MT989445) and MaSDR2 (GenBank accession no. MT989446) were successfully cloned and used for prokaryotic expression and functional analysis in vitro. MaSDR1 and MaSDR2 could catalyze the reaction of Δ1-piperideine with the coenzyme NADPH to generate piperidine. The kinetic parameters of MaSDR1 and MaSDR2 indicated that MaSDR2 had a higher binding ability to Δ1-piperideine than MaSDR1. This study provided insights into the biosynthesis of DNJ in mulberry leaves.

Intestinal Absorption and Tissue Distribution of Aza-Sugars from Mulberry Leaves and Evaluation of Their Transport by Sugar Transporters.

Mulberry leaves are rich in aza-sugars, particularly 1-deoxynojirimycin (DNJ), fagomine, and 2-O-α-d-galactopyranosyl-1-deoxynojirimycin (GAL-DNJ), which have antidiabetes and antiobesity properties. To help us understand the mechanisms of action of aza-sugars, pharmacokinetic studies are necessary. Therefore, in this study, we evaluated and compared the absorption and organ distribution of these aza-sugars in rats. Following oral intake, DNJ exhibited the highest plasma concentration followed by fagomine and GAL-DNJ. Meanwhile, similar amounts of DNJ and fagomine were present in organs, while GAL-DNJ was hardly detected, suggesting the diversity in absorption and distribution characteristics of these aza-sugars. We then investigated the role of the sodium-glucose cotransporter and the glucose transporter (GLUT) in the transport of aza-sugars and found that both are involved in DNJ transport, while transport of fagomine is solely facilitated by the GLUT. These findings provide insight into the bioavailability and bioactive mechanisms of these aza-sugars.

N,O-Dialkyl deoxynojirimycin derivatives as CERT START domain ligands.

Dysregulation of the ceramide transport protein CERT is associated to diseases such as cancer. In search for new CERT START domain ligands, N-dodecyl-deoxynojirimycin (N-dodecyl-DNJ) iminosugar was found to display, as a ceramide mimic, significant protein recognition. To reinforce the lipophilic interactions and strengthen this protein binding, a docking study was carried out in order to select the optimal position on which to introduce an additional O-alkyl chain on N-dodecyl-DNJ. Analysis of the calculated poses for three different regioisomers indicated an optimal calculated interaction pattern for N,O3-didodecyl-DNJ. The two most promising regioisomers were prepared by a divergent route and their binding to the CERT START domain was evaluated with fluorescence intensity (FLINT) binding assay. N,O3-didodecyl-DNJ was confirmed to be a new binder prototype with level of protein recognition in the FLINT assay comparable to the best known ligands from the alkylated HPA-12 series. This work opens promising perspectives for the development of new inhibitors of CERT-mediated ceramide trafficking.

An overview of the biological production of 1-deoxynojirimycin: current status and future perspective.

1-Deoxynojirimycin (DNJ), a representative iminopyranose, is widely used in anti-diabetic, antioxidant, anti-inflammatory, and anti-obesity applications. It naturally exists in plants, insects, and the culture broth of some microbes as a secondary metabolite product. However, the content of DNJ in plants and insects is relatively low, which is an obstacle to investigating DNJ in terms of structure-function relationships and restricts large-scale industrial production. With the development of micro-biotechnology, the production of DNJ during microbial fermentation has potential for industrial applications. In this review, we primarily focus on the microbial production of DNJ. The review covers sources of DNJ, determination methods, and physiological functions and applications. In a discussion of the efficient production of DNJ microorganisms, we summarize the current methods for DNJ screening and how to guide industrialized large-scale production of DNJ through fermentation regulation strategies. In addition, differences in the biosynthetic pathways of DNJ in different sources are also summarized. Finally, a comparison of DNJ content derived from different sources is discussed.

Synergistic improvement of PQQ-dependent D-sorbitol dehydrogenase activity from Gluconobacter oxydans for the biosynthesis of miglitol precursor 6-(N-hydroxyethyl)-amino-6-deoxy-α-L-sorbofuranose.

6-(N-hydroxyethyl) amino-6-deoxy-l-sorbofuranose (6NSL) is the direct precursor of miglitol for diabetes therapy. The regio- and stereo-selective dehydrogenation offered by the membrane-bound d-sorbitol dehydrogenase (mSLDH) from Gluconobacter oxydans provides an elegant enzymatic method for 6NSL production. In this study, two subunits sldA and sldB of mSLDH were introduced into G. oxydans ZJB-605, and the specific enzyme activity of mSLDH towards NHEG was enhanced by 2.15-fold. However, the endogenous PQQ level was dramatically reduced in the recombinant strain and became a bottleneck to support the holo-enzyme activity. A combined supplementation of four amino acids (Glu, Ile, Ser, Arg) involved in biosynthesis of PQQ in conventional media effectively increased extracellular accumulation of PQQ by 1.49-fold, which further enhanced mSLDH activity by 1.33-fold. The synergic improvement of mSLDH activity provided in this study supports the superior high dehydrogenate activity towards substrate N-2-hydroxyethyl-glucamine, 184.28 g·L-1 of 6NSL was produced after a repeated bioconversion process catalyzed by the resting cells of G. oxydans/pBB-sldAB, all of which presenting a great potential of their industrial application in 6NSL biosynthesis.

Physiological Effects and Organ Distribution of Bacillus amyloliquefaciens AS385 Culture Broth Powder Containing 1-Deoxynojirimycin in C57BL/6J Mice.

1-Deoxynojirimycin (DNJ) has been known as a potent α-glucosidase inhibitor from mulberry leaves and considered beneficial in prevention of type 2 diabetes. Due to limited amount of DNJ in mulberry leaves, recent studies have focused in finding alternative source that can produce higher amount of DNJ. Previously, we produced a high DNJ-containing culture medium from Bacillus amyloliquefaciens AS385 and constructed a concentration method of bacterial culture medium using cation exchange column. However, less complicated concentration procedure is necessary to save time and cost during the large-scale production. Therefore, we developed a simpler concentration method using anion exchange resin to yield B. amyloliquefaciens AS385 culture broth powder (CBP; 1% DNJ) and evaluated the physiological effects of 5-wk dietary CBP intake in C57BL/6J mice. CBP intake tended to suppress the elevation of blood glucose level during oral glucose tolerance test. Moreover, CBP intake significantly lowered the fasting plasma glucose level and white adipose tissue mass. Next, we evaluated the absorption and distribution of DNJ in mice organs after daily CBP intake. We found detectable amount of DNJ in organs with intestine and kidney as the major targeted organs. We concluded that the DNJ content in CBP is absorbed from digestive tract, distributed and accumulated in organs, which most likely to contribute to the alteration of blood glucose regulation and adiposity in C57BL/6J mice. Our study was the first to report the physiological effects of CBP produced from B. amyloliquefaciens AS385 and the organ distribution of DNJ from CBP.

Inhibitory effect of Bacillus velezensis on biofilm formation by Streptococcus mutans.

Streptococcus mutans plays a key role in the development of dental caries and promotes the formation of oral biofilm produced by glucosyltransferases (GTFs). Bacillus velezensis K68 was isolated from traditional fermented foods and inhibits biofilm formation mediated by S. mutans. Gene amplification results demonstrated that B. velezensis K68 contained genes for the biosynthesis of 1-deoxynojirimycin (1-DNJ), a known GTF expression inhibitor. The presence of the GabT1, Yktc1, and GutB1 genes required for 1-DNJ synthesis in B. velezensis K68 was confirmed. Supernatant from B. velezensis K68 culture medium inhibited biofilm formation by 84% when S. mutans was cultured for 48 h, and inhibited it maximally when 1% glucose was added to the S. mutans culture medium as a GTF substrate. In addition, supernatant from B. velezensis K68 medium containing 3 ppb 1-DNJ decreased S. mutans cell surface hydrophobicity by 79.0 ± 0.8% compared with that of untreated control. The supernatant containing 1-DNJ decreased S. mutans adherence by 99.97% and 98.83% under sugar-dependent and sugar-independent conditions, respectively. S. mutans treated with the supernatant exhibited significantly reduced expression of the essential GTF genes gtfB, gtfC, and gtfD compared to that in the untreated group. Thus, B. velezensis inhibits biofilm formation, adhesion, and GTF gene expression of S. mutans through 1-DNJ production.

Use of a deoxynojirimycin-fluorophore conjugate as a cell-specific imaging probe targeting α-glucosidase on cell membranes.

Molecules designed for cell-specific imaging were studied, taking advantage of an enzyme-inhibitor interaction. 1-Deoxynojirimycin (DNJ) can be actively captured by cells which express the surface membrane protein α-glucosidase. New probes composed of DNJ for recognition linked to a fluorophore signal portion were prepared (DNJ-CF31, DNJ-Dans 2 and DNJ-DEAC 3). Docking simulations revealed that the inhibitors acarbose and miglitol and the inhibitor portion of the probes bind at the same position in the pocket of α-glucosidase (human-derived PDB: 3TON). The ability of probes 1-3 to detect the difference between HeLa cells (from human cervical cancer tissue), Neuro-2a cells (from a mouse neuroblastoma C1300 tumor), N1E-115 cells (from a mouse brain neuroblastoma C1300 tumor), A1 cells (from the astrocyte of a newborn mouse brain), and Caco-2 cells (from a human colon carcinoma) was evaluated, and cell-specific fluorescence imaging was possible for conjugate probes 1 and 2. Caco-2 cells treated with probes 1 and 2 showed blue and green fluorescence, respectively, from the cell membrane, and did not stain the Caco-2 cells inside. These results show that DNJ-CF31 and DNJ-Dans 2 recognize an α-glucosidase protein on the surface of Caco-2 cells. Probes 1 and 2 did not stain any part of the other cells. This cell-specific imaging strategy is applicable for a variety of therapeutic agents for many diseases.

Supramolecular azasugar clusters based on an amphiphilic fatty-acid-deoxynojirimycin derivative as multivalent glycosidase inhibitors.

A novel supramolecular multivalent glycosidase inhibitor was constructed based on the amphiphilic deoxynojirimycin derivative FA-DNJ. FA-DNJ self-assembled into spherical assemblies with the diameters between 103 nm and 137 nm under different pH values (pH 2-7) and showed a potent glycosidase effect against α-mannosidase with a Ki value of 0.11 µM, an effect that increased approximately 330-fold compared with that of miglitol. In addition, FA-DNJ exhibited a hypoglycaemic effect in mice.

Synthesis, self-assembly behaviours and multivalent glycosidase inhibition effects of a deoxynojirimycin modified perylene bisimide derivative.

A self-assembled multivalent glycosidase inhibitor based on perylene bisimide-deoxynojirimycin conjugates was constructed, inhibited α-mannosidase and exhibited a Ki value of 38 nM, increased approximately 2763-fold compared with the control drug (miglitol). Furthermore, the postprandial blood glucose (PBG) level in mice of PBI-DNJ was firstly studied. PBI-DNJ exhibited a hypoglycaemic effect in vivo. Importantly, this work developed a new means to explore the hypoglycaemic effect in mice based on self-assembled glycosidase inhibitors.

Spectroscopic techniques investigation on the interaction of glucoamylase with 1-deoxynojirimycin: Mechanistic and conformational study.

1-Deoxynojirimycin (DNJ), a representative polyhydroxylated alkaloids, is widely used in the field of antidiabetic, antitumor, and anti-HIV. The present study tried to clarify the interaction mechanism of DNJ with glucoamylase by multi-spectroscopic techniques, dynamic light scattering in combination with molecular modeling strategies from biophysics point of view. Fluorescence and UV-vis data indicated that fluorescence quenching mechanism of glucoamylase and DNJ was a dynamic manner. The association constant, binding site and thermodynamic parameters were also obtained from fluorescence spectrum at different temperatures. Synchronous fluorescence, circular dichroism and dynamic light scattering methods demonstrated that their interaction induced microenvironment changes around tryptophan residue and protein conformational alteration. The main driving force was hydrophobic interaction and hydrogen bonding. In addition, molecular docking study indicated that 1-deoxynojirimycin could bind in the catalytic domain of glucoamylase and interact with amino acid residues Arg78, Asp79, Glu203 and Glu424 by forming hydrogen bonds. Molecular dynamics simulation demonstrated that profiles of atomic fluctuation remained the rigidity of ligand binding site. This study elucidated the detailed interaction mechanism of DNJ with glucoamylase, which will be helpful for pharmaceutical companies to design new α-glucosidase inhibitor drugs based on polyhydroxylated alkaloids compound like DNJ.

Enhanced Production of 6-(N-Hydroxyethyl)-Amino-6-Deoxy-α-L-Sorbofuranose by Immobilized Gluconobacter oxydanson Corn Stover with a pH Control Strategy in a Bubble Column Bioreactor.

6-(N-Hydroxyethyl)-amino-6-deoxy-α-L-sorbofuranose (6NSL) is a key intermediate in the synthesis of miglitol. Biotransformation of N-2-hydroxyethyl glucamine (NHEG) to 6NSL was performed by immobilized Gluconobacter oxydans, which was prepared by cultivating the cells in a home-made bubble column bioreactor where corn stover particles were loaded. The optimal carrier addition and aeration rate for 6NSL production by immobilized cells in the bioreactor were determined to be 25 g/L and 2.5 vvm respectively. The supplementation of NH4Cl was conducive to the biotransformation of NHEG and was performed by adding aqueous ammonia and HCl, which was taken as the pH controlling agents as well. An optimal pH control strategy using the mixture of aqueous ammonia and NaOH was applied, resulting in a 9.9% increased production of 6NSL, while repeated batches of biotransformation increased from three times to four times. Finally, the 6NSL concentration and the conversion rate of NHEG to 6NSLreached 44.2 ± 1.5 g/L and 88.4 ± 2.0%, respectively, in average after four cycles of biotransformation under the optimized condition.