Microbial community structure and interactions between Aspergillus oryzae and bacteria in traditional solid-state fermentation of Jiangqu.

Microbial interactions play an important role in the formation, stabilization and functional performance of natural microbial communities. However, little is known about how the microbes present interactions to build a stable natural microbial community. Here, we developed Jiangqu, the solid-state fermented starters of thick broad-bean sauce formed naturally in factory, as model microbial communities by characterizing its diversity of microbial communities and batch stability. The dominant microbial strains and their fungi-bacteria interactions during solid-state fermentation of Jiangqu were characterized. In all batches of Jiangqu, Aspergillus oryzae, Bacillus, Staphylococcus and Weissella dominated in the communities and such a community structure could almost reduplicate between batches. Direct adsorption and competition were identified as the main interactions between A. oryzae and dominant bacteria during solid-state fermentation, which were quite different from liquid co-cultivation of A. oryzae and dominant bacteria. These results will help us better understand the intrinsic mechanism in the formation and stabilization of microbial communities from traditional solid-state qu-making and fermentation.

Promoter engineering for efficient production of sucrose phosphorylase in Bacillus subtilis and its application in enzymatic synthesis of 2-O-α-D-glucopyranosyl-L-ascorbic acid.

2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G), a stable glucoside derivative of L-ascorbic acid (L-AA), can be one-step synthesized by sucrose phosphorylase (SPase). In this study, we attempted to produce extracellular SPase in Bacillus subtilis WB800 for the food-grade production of AA-2G. The results showed that the secretion of SPases did not require signal peptide. Promoter and its compatibility to target SPase gene were proved to be the key factors for high-level secretion. The strong promoter P43 and synthetic SPase gene derived from Bifidobacterium longum (BloSPase) were selected due to generate a relatively high extracellular activity (0.94 U/mL) for L-AA glycosylation. A highly active dual-promoter system PsigH-100-P43 was further constructed, which produced the highest extracellular and intracellular activity were 5.53 U/mL and 6.85 U/mL in fed-batch fermentation, respectively. Up to 113.58 g/L of AA-2G could be achieved by the supernatant of fermentation broth and a higher yield of 146.42 g/L was obtained by whole-cells biotransformation. Therefore, the optimal dual-promoter system in B. subtilis is suitable for the food-grade scale-up production of AA-2G.

Efficient production of α-monoglucosyl hesperidin by cyclodextrin glucanotransferase from Bacillus subtilis.

α-Monoglucosyl hesperidin is a promising food additive with various activities. However, there are a few reports about the production of α-monoglucosyl hesperidin. Here, to develop a practical and safe process for α-monoglucosyl hesperidin synthesis, we used nonpathogenic Bacillus subtilis as a host to express cyclodextrin glucanotransferase (CGTase) from Bacillus sp. A2-5a. The promoters and signal peptides were screened to optimize the transcription and secretion of CGTase in B. subtilis. The results of optimization showed that the best signal peptide and promoter were YdjM and PaprE, respectively. Finally, the enzyme activity increased to 46.5 U mL-1, 8.7 times that of the enzyme expressed from the strain containing pPHpaII-LipA, and the highest yield of α-monoglucosyl hesperidin was 2.70 g L-1 by enzymatic synthesis using the supernatant of the recombinant B. subtilis WB800 harboring the plasmid pPaprE-YdjM. This is the highest α-monoglucosyl hesperidin production level using recombinant CGTase to date. This work provides a generally applicable method for the scaled-up production of α-monoglucosyl hesperidin. KEY POINTS: • A three-step procedure was created for high throughput signal peptide screening. • YdjM and PaprE were screened from 173 signal peptides and 13 promoters. • α-Monoglucosyl hesperidin was synthesized by CGTase with a yield of 2.70 g L-1.

Design, synthesis, and antifungal activity of novel dithiin tetracarboximide derivatives as potential succinate dehydrogenase inhibitors.

BACKGROUND: Succinate dehydrogenase inhibitor (SDHI) fungicides are an important class of agricultural fungicides with the advantages of high efficiency and a broad bactericidal spectrum. To pursue novel SDHIs, a series of N-substituted dithiin tetracarboximide derivatives were designed, synthesized, and characterized by 1 H NMR, 13 C NMR, and high resolution mass spectrum (HRMS). RESULTS: These engineered compounds displayed potent fungicidal activity against phytopathogens, including Sclerotinia sclerotiorum, Botrytis cinerea, and Rhizoctonia solani, comparable with that of the commercial SDHI fungicide boscalid. In particular, compound 18 stood out with prominent activity against S. sclerotiorum with a half-maximal effective concentration (EC50 ) value of 1.37 µg ml-1 . Compound 1 exhibited the most potent antifungal activity against B. cinerea with EC50 values of 5.02 µg ml-1 . As for R. solani, 12 and 13 exhibited remarkably inhibitory activity with EC50 values of 4.26 and 5.76 µg ml-1 , respectively. In the succinate dehydrogenase (SDH) inhibition assay, 13 presented significant inhibitory activity with a half-maximal inhibitory concentration (IC50 ) value of 15.3 µm, which was approximately equivalent to that of boscalid (14.2 µm). Furthermore, molecular docking studies revealed that 13 could anchor in the binding site of SDH. CONCLUSION: Taken together, results suggested that the dithiin tetracarboximide scaffold possessed a huge potential to be developed as novel fungicides and SDHIs. © 2023 Society of Chemical Industry.

Stereoselective analysis of chiral succinate dehydrogenase inhibitors (SDHIs) in foods of plant origin and animal origin by supercritical fluid chromatography-tandem mass spectrometry (SFC-MS/MS).

The stereoisomers of chiral SDHIs were prepared using Autoprep HPLC and chiral columns. The method of combining theoretical calculation with experimental determination was used to confirm the absolute configuration of stereoisomer. SFC-MS/MS and four kinds of chiral columns were used to separate the eight chiral SDHIs, and they could be separated simultaneously using OD-3 column in 6.5 min. The integrated QuEChERS strategy was used to analyse the chiral SDHIs in foods of plant and animal origin, and the average recoveries ranged from 71 % to 119 % with RSD ≤ 18 %, and the LOQ was 1 ng/g. There were 99.2 % and 63.6 % matrix effects were in the range of 0.8-1.2 in foods of plant and animal origin, respectively, showing weak matrix effects. The study provided methods for monitoring chiral SDHIs stereoisomers residues, which were crucial for stereoselective evaluations and improving risk assessments.

Comprehensive evaluation of chiral penflufen metabolite (penflufen-3-hydroxy-butyl): Identification, synthesis, enantioseparation, toxicity and enantioselective metabolism.

Identification and evaluations of pesticide metabolites are necessary for risk assessment and toxicological research. In this study, the metabolites of penflufen (a widely used chiral pesticide) in rat liver microsomes were identified using liquid chromatography Q-Exactive Plus mass spectrometry. In total, 17 penflufen metabolites were identified, and most of them were hydroxylation products, which were generated by oxygenation at different candidate sites of penflufen. The relative abundance of metabolite M12 (penflufen-3-hydroxy-butyl, 32 %) was the largest, followed by M8 (15.6 %) and M2 (12.8 %). The major metabolite penflufen-3-hydroxy-butyl was first synthesized by 11 reactions with a 99.73 % purity. The absolute configuration of M12 enantiomers were confirmed after preparing enantiomers, and establishing the enantioseparation method. The M12 enantiomers toxicity to Danio rerio (LC50, >10 mg/L) and four kinds of phytopathogens (EC50, 148-34969 mg/L) were significantly lower than parents (LC50, 0.449-24.3 mg/L; EC50, 0.027-92.0 mg/L). In rat liver microsomes, approximately 40-47 % of the penflufen enantiomers were metabolized to M12 enantiomers, and R-penflufen was preferentially metabolized. The generation concentrations of S-M12 were higher than R-M12 after 10 min, and the metabolic half-lives of R-M12 (29.0-32.5 min) were shorter than S-M12 (35.2-38.1 min), and were approximately 4 times longer than parent penflufen enantiomers (4.5-9.5 min). Simultaneously, the generated contents (relative contents) of M8 (27.1-57 %) and M10 (2.22-8.36 %) from S-penflufen were lower than those from R-penflufen (M8, 24.7-92.4 %; M10, 27.4-69.5 %). The enantioselective evaluations of M12, M10 and M8 deserve further study. These findings were helpful in understanding the fate and risks of chiral penflufen.

Optimization of fermentation medium and conditions for enhancing valinomycin production by Streptomyces sp. ZJUT-IFE-354.

Valinomycin is a cyclodepsipeptide antibiotic with a broad spectrum of biological activities, such as antiviral, antitumor, and antifungal activities. However, the low yield of valinomycin often limits its applications in medicine, agriculture, and industry. In our previous report, Streptomyces sp. ZJUT-IFE-354 was identified as a high-yielding strain of valinomycin. In this study, Plackett-Burman design (PBD) and response surface methodology (RSM) were used to optimize components of medium. The optimal medium contained 31 g/L glucose, 22 g/L soybean meal, and 1.6 g/L K2HPO4·3H2O, which could generate 262.47 ± 4.28 mg/L of valinomycin. Then, the culture conditions were optimized by a one-factor-at-a-time (OFAT) approach. The optimal conditions for the strain included a seed age of 24 h, an inoculum size of 8% (v/v), an incubation temperature of 28 °C, an initial pH of 7.2, an elicitor of 0.1% Bacillus cereus feeding at 24 h cultivation, and the feeding of 0.6% L-valine at 36 h cultivation. The final valinomycin production increased to 457.23 ± 9.52 mg/L, which was the highest yield ever reported. It highlights that RSM and OFAT may be efficient methods to enhance valinomycin production by Streptomyces sp. ZJUT-IFE-354.

Engineering of Cyclodextrin Glycosyltransferase through a Size/Polarity Guided Triple-Code Strategy with Enhanced α-Glycosyl Hesperidin Synthesis Ability.

Hesperidin, a flavonoid enriched in citrus peel, can be enzymatically glycosylated using CGTase with significantly improved water solubility. However, the reaction catalyzed by wild-type CGTase is rather inefficient, reflected in the poor production rate and yield. By focusing on the aglycon attacking step, seven residues were selected for mutagenesis in order to improve the transglycosylation efficiency. Due to the lack of high-throughput screening technology regarding to the studied reaction, we developed a size/polarity guided triple-code strategy in order to reduce the library size. The selected residues were replaced by three rationally chosen amino acids with either changed size or polarity, leading to an extremely condensed library with only 32 mutants to be screened. Twenty-five percent of the constructed mutants were proved to be positive, suggesting the high quality of the constructed library. Specific transglycosylation activity of the best mutant Y217F was assayed to be 935.7 U/g, and its kcat/KmA is 6.43 times greater than that of the wild type. Homology modeling and docking computation suggest the source of notably enhanced catalytic efficiency is resulted from the combination of ligand transfer and binding effect. IMPORTANCE Size/polarity guided triple-code strategy, a novel semirational mutagenesis strategy, was developed in this study and employed to engineer the aglycon attacking site of CGTase. Screening pressure was set as improved hesperidin glucoside synthesis ability, and eight positive mutants were obtained by screening only 32 mutants. The high quality of the designed library confirms the effectiveness of the developed strategy is potentially valuable to future mutagenesis studies. Mechanisms of positive effect were explained. The best mutant exhibits 6.43 times enhanced kcat/KmA value and confirmed to be a superior whole-cell catalyst with potential application value in synthesizing hesperidin glucosides.

Effects of Different Pesticides on the Brewing of Wine Investigated by GC-MS-Based Metabolomics.

The application of pesticides is critical during the growth of high-quality grape for wine making. However, pesticide residues have significant influence on the wine flavor. In this study, gas chromatography-mass spectrometry (GC-MS) was performed and the obtained datasets were analyzed with multivariate statistical methods to investigate changes in flavor substances in wine during fermentation. The principal component analysis (PCA) score plot showed significant differences in the metabolites of wine treated with various pesticides. In trials using five pesticides (hexaconazole, difenoconazole, flutriafol, tebuconazole, and propiconazole), more than 86 metabolites were changed. Most of these metabolites were natural flavor compounds, like carbohydrates, amino acids, and short-chain fatty acids and their derivatives, which essentially define the appearance, aroma, flavor, and taste of the wine. Moreover, the five pesticides added to grape pulp exhibited different effects on the metabolic pathways, involving mainly alanine, aspartate and glutamate metabolism, butanoate metabolism, arginine, and proline metabolism. The results of this study will provide new insight into the potential impact of pesticide residues on the metabolites and sensory profile of wine during fermentation.

Enhancing regioselectivity of sucrose phosphorylase by loop engineering for glycosylation of L-ascorbic acid.

Sucrose phosphorylase (SPase) has a remarkable capacity to synthesize numerous glucosides from abundantly available sucrose under mild conditions but suffers from specificity and regioselectivity issues. In this study, a loop engineering strategy was introduced to enhance the regioselectivity and substrate specificity of SPase for the efficient synthesis of 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G) via L-ascorbic acid (L-AA). P134, L341, and L343 were identified as "hotspots" for modulating the flexibility of loops, which significantly influenced the H-bonding network of L-AA in the active site, as well as the entrance of the substrate channel, thereby altering the regioselectivity and substrate specificity. Finally, the mutant L341V/L343F, with near-perfect control of the selectivity synthesis of the 2-OH group of L-AA (> 99%), was obtained. The AA-2G production by the mutant reached 244 g L-1 in a whole-cell biotransformation system, and the conversion rate of L-AA reached 64%, which is the highest level reported to date. Our work also provides a successful loop engineering case for modulating the regioselectivity and specificity of sucrose phosphorylase. KEY POINTS: • "Hotspots" were identified in the flexible loops of sucrose phosphorylase. • Mutants exhibited improved regioselectivity and specificity against L-ascorbic acid. • Synthesized AA-2G with high yield and regioselectivity by whole-cell of mutant.

The optimized biocatalytic synthesis of (S)-methyl 2-chlorobutanoate by Acinetobacter sp. lipase.

Epilepsy is a chronic disease caused by sudden abnormal discharge of brain neurons, leading to transient brain dysfunction. Levetiracetam, developed by the UCB company in Belgium, is an effective drug for the treatment of epilepsy. (S)-Methyl 2-chlorobutanoate is an important chiral building block of levetiracetam, which has attracted a great deal of attention. In this study, a strain of lipase-produced Acinetobacter sp. zjutfet-1 was screened from soil samples. At optimized conditions for fermentation and biocatalysis, the bacterial lipase exhibited high catalytic activity for hydrolysis and stereoselectivity toward racemic methyl 2-chlorobutanoate. When the enzymatic reaction was carried out in 6% of racemic substrate, the enantiomeric excess (e.e.s ) reached more than 95%, with a yield of over 86%. Therefore, this lipase can efficiently resolve racemic methyl 2-chlorobutanoate and obtain (S)-methyl 2-chlorobutanoate, which presents great potential in the industrial production of levetiracetam.

Identification of Process-Related Impurities and Corresponding Control Strategy in Biocatalytic Production of 2-O-α-d-Glucopyranosyl-l-ascorbic Acid Using Sucrose Phosphorylase.

2-O-α-d-Glucopyranosyl-l-ascorbic acid (AA-2G) is an ideal substitute for l-ascorbic acid because of its remarkable stability and improved biological activity, which can be easily applied in cosmetic, food, and medicine fields. However, impurity identification and control are significant procedures during the manufacturing of AA-2G. This study assessed a manufacturing routine of AA-2G synthesized by sucrose phosphorylase (SPase). First, three unknown process-related impurities were observed, which were further identified as 3-O-α-d-glucopyranosyl- l-ascorbic acid (impurity I), 2-O-α-d-glucopyranosyl-l-dehydroascorbic acid (impurity II), and 13-O-α-d-glucopyranosyl-2-O-α-d-glucopyranosyl-l-ascorbic acid (impurity III), respectively. Second, a comprehensive formation pathway of impurities was elucidated, and specific strategies corresponding to controlling each impurity were also proposed. Specifically, the content of impurity I can be reduced by 50% by fine tuning reaction conditions. The impurity II-free purification process was also achieved by applying a low concentration of alkali. Finally, a semi-rational design was introduced, and a single mutant L343F was obtained by site-directed mutagenesis, which reduced impurities I and III by 63.9 and 100%, respectively, without affecting the transglycosylation activity. It is expected that the reported impurity identification and control strategies during the AA-2G production will facilitate its industrial production.

Sucrose phosphorylase from Lactobacillus reuteri: Characterization and application of enzyme for production of 2-O-α-d-glucopyranosyl glycerol.

The enzymatic synthesis of 2-O-α-d-glucopyranosyl-glycerol (2-αGG) by transglycosylation activity of sucrose phosphorylase (SPase) is a promising method for 2-αGG manufacturing. However, there are only a few SPases available for 2-αGG production. Here, we report on the characterization and application of SPase from Lactobacillus reuteri (LrSPase). The results of transglycosylation properties assay showed that LrSPase was a potential glycerol glycosylating tool with high activity at pH 8.0 and 45 °C. And the transglycosylation activity of LrSPase was seriously inhibited by Fe3+, Zn2+ and Cu2+. Moreover, the result of substrate specificity assay showed LrSPase was able to catalyze the transglycosylation of 13 phenolic compounds. To produce commercially relevant concentrations of 2-αGG, we have developed a practical, efficient and scalable process for 2-αGG production using sucrose batch-feeding strategy by whole-cell catalyst. The maximum titer of 2-αGG was 237.68 g L-1 with a productivity of 23.39 mM h-1 and the molar conversion rate of glycerol reached 62.38%. To the best of our knowledge, this is the highest 2-αGG production level by using only SPase to synthesize 2-αGG until now. This study provides an effective way for industrial production of 2-αGG.

Evaluation of chiral triticonazole in three kinds of fruits: enantioseparation, degradation, and dietary risk assessment.

The enantioselective behaviors of chiral pesticides would affect the accuracy of risk assessment. This study evaluated the enantioselectivity of chiral triticonazole (a widely used fungicide) in three kinds of fruits. Firstly, the enantioseparation of triticonazole enantiomers was carried out within 1.2 min utilizing CHIRALPAK OJ-3 column with a mixture of CO2 and methanol (93:7, v/v) using SFC-MS/MS. Secondly, field trials were conducted to clarify the enantioselective degradation and residue of S-( +)-triticonazole and R-(-)-triticonazole in fruits. The initial concentrations of rac-triticonazole were 25.1-93.1 ng/g, and enantioselective degradation was observed in pear, peach, and jujube after 2 h, 10 days, and 3 days, respectively. The degradation of S-( +)-triticonazole was fastest in pear (T1/2, 2.01 days), while the T1/2 of R-(-)-triticonazole was 5.02 days. The residue concentrations of rac-triticonazole were less than the MRL set by EU (10 ng/g) on the 3rd and 21st day in pear and peach, respectively, which were lower than 10 ng/g in jujube on the 30th day (no MRL). Finally, we found that the dietary intake risks of rac-triticonazole in fruits were low for 2-7 age, 20-50 age/female, and 20-50 age/male. The current study could provide complimentary references for the rational usage, MRL formulation, and risk assessment of chiral triticonazole.

Maleimide structure: a promising scaffold for the development of antimicrobial agents.

Natural compounds bearing maleimide rings are a series of secondary metabolites derived from fungi/marine microorganisms, which are characterized by a general structure -CO-N(R)-CO-, and the R group is normally substituted with alkyl or aryl groups. Maleimide compounds show various biological activities such as antibacterial, antifungal, and anticancer activity. In this review, the broad-spectrum antimicrobial activities of 15 maleimide compounds from natural sources and 32 artificially synthesized maleimides were summarized, especially against Candida albicans, Sclerotinia sclerotiorum, and Staphylococcus aureus. It highlights that maleimide scaffold has tremendous potential to be utilized in the development of novel antimicrobial agents.

Efficient production of valinomycin by the soil bacterium, Streptomyces sp. ZJUT-IFE-354.

A novel strain with antifungal activity against Sclerotinia sclerotiorum was isolated from soil, and identified as Streptomyces sp. ZJUT-IFE-354 using morphological and 16S rDNA sequence analysis. The bioactive metabolite produced by strain ZJUT-IFE-354 was identified and characterized as valinomycin by spectroscopic and chemical methods. The yield of valinomycin was 191.26 mg/L from the culture of Streptomyces sp. ZJUT-IFE-354, which was the highest yield to our knowledge. The in vitro antifungal activity of valinomycin against S. sclerotiorum was investigated as 0.056 ± 0.012 (EC50) and 0.121 ± 0.023 µg/mL (EC95), respectively, which was approximately 10.696- and 30.960-fold more active than that of carbendazim. The results from scanning electron microscopy, cell membrane permeability, and D-sorbitol and ergosterol assay indicated that valinomycin exerted the antifungal activity probably by increasing permeability of fungal cell membrane, leading to mycelial electrolyte leakage, and eventually resulting in the death of S. sclerotiorum. Thus, valinomycin may be a promising antifungal agent to control S. sclerotiorum. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-03055-5.

Synthesis of a series of validoxylamine A esters and their biological activities.

BACKGROUND: The worldwide reduction in food production due to pests and diseases is still an important challenge facing today. Validoxylamine A (VAA) is a natural polyhydroxyl compound derived from validamycin, acting as an efficient trehalase inhibitor with insecticidal and antifungal activities. To extend the application and discover green pesticide, a series of ester derivatives were prepared based on VAA as a lead compound. Their biological activities were investigated against three typically agricultural disease, Rhizoctonia solani, Sclerotinia sclerotiorum and Aphis craccivora. RESULTS: This study involved 30 novel validoxylamine A fatty acid esters (VAFAEs) synthesized by Novozym 435 and they were characterized with high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) and proton nuclear magnetic resonance (1 H-NMR). Of these 30 derivatives, most compounds showed improved antifungal activity, and 12 novel compounds showed improved insecticidal activity. When reacted with pentadecanoic acid, compound 14 showed the highest inhibitory activity against R. solani [median effective concentration (EC50 ) 0.01 µmol L-1 ], while the EC50 value of VAA was 34.99 µmol L-1 . Furthermore, 21 novel VAFAEs showed higher inhibitory activity against S. sclerotiorum. Validoxylamine A oleic acid ester, compound 21, exhibited the highest insecticidal activity against A. craccivora [median lethal concentration (LC50 ) 39.63 µmol L-1 ], while the LC50 value of Pymetrozine was 50.45 µmol L-1 , a commercialized pesticide against A. craccivora. CONCLUSION: Combining our results, esterification of VAA by introducing different acyl donors was beneficial for the development of new eco-friendly drugs in the field of pesticides.

Efficient production of l-menthyl α-glucopyranoside from l-menthol via whole-cell biotransformation using recombinant Escherichia coli.

l-Menthyl α-D-glucopyranoside (α-MenG) is a glycoside derivative of l-menthol with improved water-solubility and new flavor property as a food additive. α-MenG can be synthesized through biotransformation, but its scale-up production was rarely reported. In this study, the properties of an α-glucosidase from Xanthomonas campestris pv. campestris 8004 (Agl-2) in catalyzing the glucosylation of menthol was investigated. Agl-2 can almost completely glycosylate l-menthol (> 99%) when using 1.2 M maltose as glycosyl donor. Accumulated glucose resulted from maltose hydrolysis and transglycosylation caused the inhibition of the glucosylation rate (40% reduction of the glucosylation rate in the presence of 1.2 M glucose) which can be avoided through whole-cell catalysis with recombinant E. coli. Interestingly, in spite of the poor solubility of menthol, the productivity of α-MenG reached 24.7 g/(L·h) in a 2 L catalyzing system, indicating industrialization of the reported approach.

[Application of sucrose phosphorylase in glycosylation].

Water solubility, stability, and bioavailability, can be substantially improved after glycosylation. Glycosylation of bioactive compounds catalyzed by glycoside hydrolases (GHs) and glycosyltransferases (GTs) has become a research hotspot. Thanks to their rich sources and use of cheap glycosyl donors, GHs are advantageous in terms of scaled catalysis compared to GTs. Among GHs, sucrose phosphorylase has attracted extensive attentions in chemical engineering due to its prominent glycosylation activity as well as its acceptor promiscuity. This paper reviews the structure, catalytic characteristics, and directional redesign of sucrose phosphorylase. Meanwhile, glycosylation of diverse chemicals with sucrose phosphorylase and its coupling applications with other biocatalysts are summarized. Future research directions were also discussed based on the current research progress combined with our working experience.

[Application of immobilized glycosidase in the synthesis of glycoside compounds].

Glycoside compounds are widely used in medicine, food, surfactant, and cosmetics. The glycosidase-catalyzed synthesis of glycoside can be operated at mild reaction conditions with low material cost. The glycosidase-catalyzed processes include reverse hydrolysis and transglycosylation, appropriately reducing the water activity in both processes may effectively improve the catalytic efficiency of glucosidase. However, glucosidase is prone to be deactivated at low water activity. Thus, glucosidase was immobilized to maintain its activity in the low water activity environment, and even in neat organic solvent system. This article summarizes the advances in glycosidase immobilization in the past 30 years, including single or comprehensive immobilization techniques, and immobilization techniques combined with genetic engineering, with the aim to provide a reference for the synthesis of glycosides using immobilized glycosidases.