Type Enzyme Research Articles

Biosynthesis of unique natural product scaffolds by Fe(II)/αKG-dependent oxygenases

Abstract Fe(II)/αKG-dependent oxygenases are multifunctional oxidases responsible for the formation of unique natural product skeletons. Studies of these enzymes are important because the knowledge of their catalytic functions, enzyme structures, and reaction mechanisms can be used to create non-natural enzymes through mutation and synthesize non-natural compounds. In this review, I will introduce the research we have conducted on two fungal Fe(II)/αKG-dependent oxygenases, TlxI-J and TqaL. TlxI-J is the first Fe(II)/αKG-dependent oxygenase type enzyme heterodimer that catalyzes consecutive oxidation reactions, hydroxylation followed by retro-aldol or ketal formation, to form the complex skeletons of meroterpenoids. TqaL is the first naturally occurring aziridine synthase, and I will discuss the mechanism of its unique C–N bond formation in nonproteinogenic amino acid biosynthesis. This review will advance research on the discovery of new enzymes and the analysis of their functions by reviewing the structures and functions of these extraordinary Fe(II)/αKG-dependent oxygenases, and promote their use in the synthesis of new natural medicines. Graphical Abstract

A Stereoselective Decarboxylative Aromatase/Cyclase Directs the Biosynthesis of an Axially Chiral Biphenyl Framework in Fasamycin.

Aromatic polyketides are an important class of natural products with various bioactivities, and their structural diversity arises from modifications to their aromatic frameworks. In this study, we identify a stereoselective aromatase/cyclase (ARO/CYC) FasU, which is responsible for forming the axial chiral biphenyl framework in fasamycin. FasU catalyzes sequential decarboxylation and cyclization/aromatization with strict S-stereospecificity on a previously unidentified biosynthetic intermediate. Through crystal structure analysis and site-directed mutagenesis, we reveal the enzyme's substrate binding mode, stereospecificity, and the key residues involved in catalysis. This discovery introduces a novel class of ARO/CYC enzymes in type II polyketide biosynthesis, advancing the development of biocatalysts for chiral aromatic polyketides.

Improving the Proteome-Mining of Schizophyllum commune to Enhance Medicinal Mushroom Applications

This study presents the first comprehensive proteomic profile of an Italian strain of Schizophyllum commune, a highly heterogeneous white-rot fungal species with significant potential for industrial, nutraceutical, cosmeceutical, and clinical applications. Three protein extraction methods and their impact on yield and resulting protein composition have been compared. Results revealed that the combination of Tris–Cl and urea increases the total protein yield and the variety of enzymatic species related to pivotal pathways. Notably, over 2000 proteins were identified, including enzymes involved in the growth and development of mycelium, trehalose biosynthesis, and different types of carbohydrate-active enzymes (CAZymes). These enzymes are crucial for nutraceutical and agro-industrial applications of S. commune. The multiple-step proteomic approach used could be a model for investigating other fungal species.

Differences in hair cortisol to cortisone ratio between depressed and non-depressed adolescent women

Research on stress has demonstrated that the hypothalamic-pituitary-adrenal (HPA) axis contributes to major depressive disorder in youth. Hair glucocorticoids are key biological markers of chronic stress. We assessed group differences in hair cortisol and cortisone concentrations, and the cortisol/cortisone ratio between depressed adolescent women and a non-depressed comparison group. Further, within the depression group, we explored the contribution of symptom severity and clinical correlates of depression in relation to glucocorticoid concentrations. Hair samples of three centimeters for 74 adolescent women (41 in the depression group and 33 in the comparison group), aged between 12 and 19 years old, were analyzed. Depressive and anxiety symptoms were measured using the Beck Youth Inventory II and clinical correlates of depression were measured using the Childhood Trauma Questionnaire–Short Form and the Borderline Personality Features Scale for Children. No significant differences emerged between the depression group and the comparison group on hair cortisol or hair cortisone concentrations. However, groups differed significantly on the cortisol/cortisone ratio, a proposed proxy of 11-beta-hydroxysteroid dehydrogenase activity, with a higher ratio for the depression group. Within the depression group, neither symptom severity nor clinical correlates were associated with glucocorticoid concentrations. Although cross-sectional, our findings highlight the importance of future studies to test whether the group difference found in cortisol/cortisone ratio is the result of alterations in 11-beta-hydroxysteroid dehydrogenase enzymes (type 1 or 2) activity. Further research is thus needed to clarify the role of these enzymes in major depressive disorder in youth and to develop more targeted intervention strategies.

Dynamic Modulation of IRE1α-XBP1 Signaling by Adenovirus

The abundant production of foreign proteins and nucleic acids during viral infection elicits a variety of stress responses in host cells. Viral proteins that accumulate in the endoplasmic reticulum (ER) can trigger the unfolded protein response (UPR), a coordinated signaling program that culminates in the expression of downstream genes that collectively restore protein homeostasis. The model pathogen adenovirus serotype 5 (HAdV5) activates the UPR via the signaling axis formed by inositol-requiring enzyme type 1 (IRE1α) and the X-box binding protein 1 (XBP1), a transcription factor required for immune function. Recent studies have suggested that IRE1α-XBP1 activity supports adenovirus replication. Here, we show that HAdV5 exerted opposing effects on IRE1α and XBP1. IRE1α was activated in response to HAdV5, but the production of the XBP1 isoform, XBP1s, was post-transcriptionally blocked. The tumor suppressor p53, which is eliminated by HAdV5 after infection, inhibited IRE1α activation. The de-repression of IRE1α following the degradation of p53 conceivably reflects a novel antiviral mechanism, which HAdV5 ultimately evades by co-opting IRE1α and suppressing XBP1s. Our findings illustrate the opposing mechanisms used by adenoviruses and their host cells to exert control over the UPR, a critical determinant of cell fate.

The C-terminal structure of the N6-methyladenosine deaminase YerA and its role in deamination.

The N6-methyladenine (6mA) modification is an essential epigenetic marker and plays a crucial role in processes, such as DNA repair, replication, gene expression regulation, etc. YerA from Bacillus subtilis is considered a novel class of enzymes capable of catalyzing the deamination of 6mA to produce hypoxanthine. Despite the significance of this type of enzymes in bacterial self-defense systems and potential applications as a gene-editing tool, the substrate specificity, the catalytic mechanism and the physiological function of YerA are currently unclear due to the lack of structural information. In this study, we expressed the recombinant enzyme and conducted its reconstitution to yield the active form. Our deamination assays showed that N6-methyladenosine (N6-mAdo) served as a more favorable substrate than its base derivative 6mA. Here we report the high-resolution structure of the C-terminal region of YerA, which exhibited a compact architecture composed of two antiparallel b-sheets with no obvious close structural homologs in PDB. We also created docking models to investigate the ligand-binding pattern, and found that more favorable contacts of N6-mAdo with the enzyme binding pocket lead to its preference for 6mA. Lastly, structural comparison to the deaminase MAPDA allowed us to propose a plausible role for this C-terminal region: shielding the active site from solvent and protecting the intermediate during catalysis. Taken together, this study sheds light on the catalytic mechanism and evolutionary pathways of the promiscuous enzyme YerA, thereby contributing to our molecular understanding of epigenetic nucleoside metabolism.

Effect of Different Enzyme Treatments on Juice Yield, Physicochemical Properties, and Bioactive Compound of Several Hybrid Grape Varieties

This study investigates the effects of four enzymatic treatments on the yield, physicochemical properties, and bioactive compounds of grape juices from two red (Golubok, Regent) and two white (Muscaris, Aurora) hybrid grape varieties. A total of 20 samples were prepared using four commercial enzyme formulations (Pectinex Ultra, Safizym Clear Plus, Safizym Press, and Rapidase color) applied at a concentration of 0.02% (w/w). The juices were evaluated for yield, total phenolic content, antioxidant capacities (ABTS, DPPH, FRAP), titratable acidity, turbidity, total soluble solids, and phenolic profile. The addition of enzymes significantly improved juice yield by 10% to 20%, with the effect varying depending on the type of enzyme and the variety of grapes. Pectinex Ultra was the most effective enzyme in reducing turbidity, while enzyme treatments had minimal impact on Brix levels and sugar concentration, which were primarily determined by the characteristics of each grape variety. The enzyme addition showed a minor influence on the titratable acidity of the juices, with slight increases observed in Muscaris, but the grape variety played a major role in determining the titratable acidity levels. Color parameters revealed that white grape juices (Muscaris and Aurora) were brighter than red varieties (Golubok and Regent). Additionally, enzyme treatments influenced the color, enhancing the red hues in red grape juices. Enzyme treatments also improved the antioxidant capacity of grape juices, especially in Aurora and Muscaris, although the effect on polyphenol content was more dependent on the variety of grapes, with red varieties showing higher levels of polyphenols than white varieties. These findings highlight the significant role of both enzyme treatments and grape variety in determining the quality and health-promoting properties of grape juice.

Achillea millefolium capsule improved liver enzymes and lipid profile compared to placebo in patients with type 2 diabetes: a double-blind randomized clinical trial

BackgroundThe therapeutic properties of Achillea Millefolium (AM) in regulating blood lipids and liver enzymes have been proven in studies. Considering the abnormal lipid levels and elevated liver enzymes in diabetic patients, this study was conducted to investigate the effect of AM on the lipid profile and serum level of liver enzymes in type 2 diabetic (T2D) patients.MethodsIn this 90-day double-blind clinical trial study, 60 eligible diabetic patients were enrolled and divided into intervention and control (each 30 patients) groups. The intervention group received AM capsules (dose of 500 mg/day) for 3 months, and the control group received placebo capsules during the same period. Blood was taken from patients on day 0 and day 90. The serum levels of liver enzymes and lipid profiles of patients were measured at baseline and on day 90. The obtained values were analyzed and compared between two control and treatment groups and within each group.ResultsAM oral supplementation caused a significant decrease in alanine aminotransferases (ALT; from 28.79 ± 10.99 to 24.41 ± 6.84, p = 0.017) and aspartate aminotransferases (AST; from 24.28 ± 7.91 to 18.76 ± 6.77, p = 0.007). A significant reduction in the levels of serum lipids such as triglyceride (TG; from 161.7 ± 77.18 to 147.3 ± 66.79, p = 0.045), low-density lipoprotein (LDL; from 98.90 ± 35.67 to 80.86 ± 32.10, p = 0.001), and total cholesterol (TC; from 168.3 ± 47.46 to 150.1 ± 38.77, p = 0.006) was noted in AM group. According to these results, after the intervention, the mean of all variables (except HDL) in the two groups had a significant difference (P < 0.05).ConclusionThe study showed that the 3-month treatment of T2D patients with 500 mg/day AM capsules led to the reduction of liver enzymes and the regulation of blood lipids in them.Trial registrationIranian Registry of Clinical Trials IRCT20230612058459N1 Registered on 2023–06–24.

DNA Methylation: Its Role and Interaction with Epigenetic Modifications in Cancer

DNA methylation is an epigenetic mark involving the addition of a methyl group to DNA, particularly at cytosine residues. This methylation plays an important role in regulating gene expression through direct gene repression or through the control of other epigenetic modifications such as histone modification or chromatin remodeling. DNA methylation is catalyzed by de novo and maintenance DNMT methyltransferase type enzymes and requires the transfer of a methyl group to cytosine to transform it into 5-methyl-cytosine. Currently, several investigations have highlighted the involvement of aberrant DNA methylation with certain tumors. Indeed, the methylation of antioncogenes and/or the demethylation of oncogenes are the major alterations that are strongly linked to human cancers. DNMTs play a central role in the epigenetic regulation of the human genome, both in normal and pathological processes. Recent discoveries on the differentiated roles of DNMTs in DNA methylation, and their implication in various cancers, open the way to new therapeutic approaches targeting these enzymes to treat epigenetic diseases. It is essential to continue exploring the roles of DNMTs to better understand their implication in tumorigenesis mechanisms and to develop more effective treatment strategies.

The development and application of an engineered direct electron transfer enzyme for continuous levodopa monitoring

Levodopa, the primary treatment for Parkinson’s Disease, has a narrow therapeutic window further complicated by the lack of real-time feedback, primarily due to the absence of an enzyme specific to levodopa. We addressed this by developing a novel direct electron transfer type (DET) enzyme, copper dehydrogenase (CoDH), engineered from an extremophile derived multicopper oxidase (MCO), for use in a continuous levodopa sensor. By introducing mutations into the type 2 and type 3 copper ligand histidine residues, the enzyme drastically decreased its oxidase activity while enhancing DET activity with the electrode. Using this developed CoDH, a chronoamperometric levodopa sensor was constructed, which was minimally affected by environmental changes, or by interferents, including levodopa metabolites, adjunct medications, and common plasma and interstitial fluid components. A miniaturized levodopa sensor was constructed and was able to detect levodopa as low as 138 nM, suggesting its future application for in vivo subcutaneous measurement.

CUL4-Based Ubiquitin Ligases in Chromatin Regulation: An Evolutionary Perspective.

Ubiquitylation is a post-translational modification that modulates protein function and stability. It is orchestrated by the concerted action of three types of enzymes, with substrate specificity governed by ubiquitin ligases (E3s), which may exist as single proteins or as part of multi-protein complexes. Although Cullin (CUL) proteins lack intrinsic enzymatic activity, they participate in the formation of active ubiquitin ligase complexes, known as Cullin-Ring ubiquitin Ligases (CRLs), through their association with ROC1 or ROC2, along with substrate adaptor and receptor proteins. Mammalian genomes encode several CUL proteins (CUL1-9), each contributing to distinct CRLs. Among these CUL proteins, CUL1, CUL3, and CUL4 are believed to be the most ancient and evolutionarily conserved from yeast to mammals, with CUL4 uniquely duplicated in vertebrates. Genetic evidence strongly implicates CUL4-based ubiquitin ligases (CRL4s) in chromatin regulation across various species and suggests that, in vertebrates, CRL4s have also acquired a cytosolic role, which is facilitated by a cytosol-localizing paralog of CUL4. Substrates identified through biochemical studies have elucidated the molecular mechanisms by which CRL4s regulate chromatin and cytosolic processes. The substantial body of knowledge on CUL4 biology amassed over the past two decades provides a unique opportunity to explore the functional evolution of CRL4. In this review, we synthesize the available structural, genetic, and biochemical data on CRL4 from various model organisms and discuss the conserved and novel functions of CRL4s.

Recovery of Polysaccharides from Red Grape Marc and White Grape Pomace by Degradation of Cell Walls by Enzymes with Different Activities.

The recovery of polysaccharides (PS) from red grape marc and white grape pomace by enzymatic degradation of their cell walls is an interesting green extraction technique that preserves the structure and bioactivity of PS. The type and dose of enzyme, and the liquid/solid (L/S) ratio in PS extraction were studied using four commercial enzymes. Four different doses per enzyme were used, with tartaric acid as solvent and L/S ratios of 1.3/1 and 4/1 for 24 h at 20 °C, compared with a control. The highest dose of enzyme E1, polygalacturonase + pectin lyase + pectin-methyl-esterase (with the highest activity) was the most effective in the degradation of high and medium molecular weight PS. At the lower L/S ratio, the fact that the highest dose of E1 degraded a higher percentage of high and medium molecular weight PS in the marc was explained by the difference in cell wall deconstruction between pomace and marc. The highest total PS purity was achieved in pomace with E1 at the maximum dose in both ratios, and in marc at the 1.3/1 ratio. The extraction efficiency of total PS was low for all enzymes. In the future, extraction with E1 combined with other green extraction techniques will be studied.

A comparison of seasonal flexibility in pectoralis muscle fiber type and enzyme activity in migratory and resident sparrow species.

The pectoralis muscle in birds is important for flight and thermogenesis. In migratory songbirds this muscle exhibits seasonal flexibility in size, but whether this flexibility reflects changes in muscle fiber type has not been well documented. We investigated how seasonal changes in photoperiod affected pectoralis muscle fiber type and metabolic enzymes, comparing among three closely related sparrow species: two seasonal migrants and one year-round, temperate climate resident. We quantified fast oxidative glycolytic (FOG) and fast glycolytic (FG) fibers histologically, and measured activities of citrate synthase (CS) and lactate dehydrogenase (LDH) in the pectoralis muscle of the three species that were acclimated to long or short periods of daylight. In all species, FOG was the predominant fiber type, but song sparrows had FG fibers regardless of daylight conditions. By contrast, Lincoln's sparrows incorporated FG fibers only under short-daylight conditions, and house sparrows did not significantly express FG fibers, regardless of daylight length. Both migratory species increased LDH activity in short-daylight conditions but did not alter CS activity. In contrast, resident house sparrows did not alter CS or LDH activity with changes in daylight length. Our findings suggest that the presence of FG fibers is important for seasonal flexibility in LDH activity. Additionally, migratory species exhibited seasonal flexibility in muscle fiber type and enzyme activity, presumably to support migratory flight, while the resident species did not exhibit such seasonal flexibility, suggesting that this consistent phenotype is important year-round, despite changing thermogenic requirements.

Hollow mesoporous silica nanoparticles for drug formulation and delivery: Opportunities for cancer therapy.

The advantages of large surface area, high volume ratio, good biocompatibility, and controllable surface functionalization make hollow mesoporous silica nanoparticles (HMSNs) an ideal drug carrier. HMSNs can achieve high efficiency, targeting, and controlled release by adjusting the microstructure and surface modification of its particles, which makes it broad application prospects in the field of medical therapy, especially in cancer therapy. Numerous studies have shown that preparation method, shape, particle size, hollow inner diameter, aperture and wall thickness of the HMSNs, the characteristics of the drugs, the interaction between the drugs and the carriers, and the external environment all closely affect the drug delivery, release, and efficacy. The external environment includes temperature, pH value, light intensity, magnetic field intensity, enzyme type and concentration, etc. This review summarizes the research progress of HMSNs as carrier materials in the past five years, analyzes the existing problems in the application process and presents the development prospects of HMSNs.

Proteoglycan-degrading enzymes engineered for enhanced tumor microenvironment interaction in renal cell carcinoma.

This work optimized proteoglycan-degrading enzymes through targeted mutagenesis to enhance their interaction with the tumor microenvironment in Renal Cell Carcinoma (RCC). A comprehensive mutagenesis approach identified 60 key mutations significantly improving enzymatic activity, stability, and structural integrity. When compared to Wild Type (WT) enzyme, a remarkable increase in specific activity by 35 % (p < 0.001) and a considerable decrease in the Km values for hyaluronidases from 2.5 mM to 1.5 mM (p < 0.05), as a result of these modifications. Computational methods are then employed to analyze the active site of the enzymes to detect potential residues that may alter. These computational techniques include molecular docking and protein structure prediction. The structural models of the enzymes are created by utilizing homology modeling and crystallography. These models demonstrate the spatial arrangement of the amino acid enzymes. It also illustrated the specific mutations to improve the potential of enzymes to relate to the Extracellular Matrix (ECM) of tumors. The computational screening methods effectively predicted how the modifications impact enzyme catalytic efficiency and stability. The modified enzymes retained 85 % of the enzyme activity, while the WT retained 60 %. Thus, the modified enzymes demonstrated better thermal stability than WT. Vitro test analyses show that the proteoglycan breakdown was significantly reduced by 70 % (p < 0.001), and for effective proteoglycan breakdown, hyaluronidase concentration is needed. This work proposed a novel therapeutic approach called proteoglycan-degrading enzymes for the treatment of RCC. These proteoglycan-degrading enzymes are more stable and effective for treating RCC, as demonstrated in the outcomes. Customized proteoglycan-degrading enzymes make the therapy more effective. The effective breakdown of the tumor's ECM in RCC models establishes this customized proteoglycan-degrading enzyme. These enzymes are effective for this customized cancer treatment as they improve stability, activity, and interaction with the TME.

Classification of polyphenol oxidases shows ancient gene duplication leading to two distinct enzyme types

Classification of polyphenol oxidases shows ancient gene duplication leading to two distinct enzyme types

The effect of hydrolase enzyme on antibiofilm activity in oral cavity microbes

Background : Every surface of the human body that comes into contact with the outside environment is covered with a layer of microorganisms called the human microbiome, characterized by species diversity and a high number of cells. Biofilms are surface-related, three-dimensional multicellular structures whose integrity depends on the extracellular matrix produced by their constituent bacteria. Hydrolase enzymes are a class of enzymes belonging to the anti-biofilm enzymes and are categorized as polysaccharide-degrading enzymes, proteolytic enzymes, and antiquorum sensing enzymes. Purpose : To know the effect of hydrolase enzyme towards antibiofilm activity of oral cavity microbe. Method : The method used is literature study by searching databases from various national and international sources. Results : There is an anti-biofilm effect of several types of hydrolase enzymes on oral microbes such as P. aeruginosa, S. aureus, E. faecalis, and E. coli. The greatest effect was found in the enzymes PelAh &amp; PslGh with the mechanism of action of polysaccharide-degrading enzymes and their microbial target, P. aeruginosa, can reduce biofilm biomass by 58-94%. Conclusion : Of the several existing mechanisms can provide different effects depending on the microbial target.

Efficacy and Safety of Aerosol Inhalation of Colistin Sulfate for the Treatment of Carbapenem-Resistant Klebsiella pneumoniae Infection in the Peri-Operative Period of Liver Transplantation: A Single-Center Retrospective Study.

Objective: This study intended to evaluate the clinical efficacy and safety of colistin sulfate aerosol inhalation in combination with ceftazidime-avibactam for the treatment of pulmonary carbapenem-resistant Klebsiella pneumoniae (CRKP) infection during the peri-operative period of liver transplantation. Materials and Methods: A retrospective analysis was designed to investigate 52 patients who developed pulmonary CRKP infection after liver transplantation between December 1, 2019, and November 30, 2022. On the basis of whether they received colistin sulfate aerosol inhalation, the patients were divided into the treatment group (n = 29) and the control group (n = 23). The baseline information, infection status, CRKP enzyme type, inflammatory markers, liver and kidney function, and prognosis were compared and analyzed. Results: There were no significant differences in patient characteristics, infection status, and drug resistance enzyme type between the treatment group (treated with colistin sulfate aerosol inhalation and ceftazidime and avibactam sodium for injection) and the control group (treated with ceftazidime and avibactam sodium for injection alone). Colistin sulfate aerosol inhalation treatment reduced concentrations of inflammatory markers, with post-treatment white blood cell count, procalcitonin, and C-reactive protein significantly lower than pre-treatment levels (p < 0.05). Except for C-reactive protein at 14 days (p = 0.032), the two groups had no significant differences in other indicators. There were no significant differences in alanine aminotransferase, aspartate aminotransferase, total bilirubin, and glomerular filtration rate after treatment, indicating no discernible alteration in liver and kidney function. In addition, the treatment group took a significantly shorter time to normalize body temperature compared with the control group (p = 0.025), but there were no significant differences in the cure with no colonization rate and all-cause mortality rate between the two groups. Conclusions: The combination of colistin sulfate aerosol inhalation and ceftazidime and avibactam sodium for injection is effective in treating pulmonary CRKP infection during the peri-operative period of liver transplantation. It does not impose an additional burden on liver and kidney function, providing a new treatment option for this type of infection.

A Refined Nomenclature System to Better Discriminate Endo- and Exo-Type Fructanases and Glucanases.

Distinguishing between endo- and exo-type enzymes within the glycoside hydrolase (GH) classification presents significant challenges. Traditional methods, often based on endpoint activity measurements, do not capture the full range of products generated, leading to inconsistencies in classification. Not all exo-acting fructanases and glucanases produce monosaccharides (like fructose or glucose), while endo-acting enzymes do not solely produce higher-degree polymerization oligosaccharides. In practice, both enzyme types can yield a variety of products throughout the reaction, complicating classification efforts. To address these challenges, we propose a refined nomenclature system for GH enzymes, including fructanases and glucanases, based on good practices and initial product formation. This system classifies enzymes into four categories for each type: Fr, Fn, Fn,n+1 and F1 for fructanases, and Gr, Gn, Gn,n+1 and G1 for glucanases, based on their mode of action (endo- or exo-based) and initial product profiles. Our refined nomenclature system will advance enzyme structure-function research and support the production and application of fructan and glucan oligosaccharides as prebiotics, priming agents, and potential signaling molecules in microbe-microbe and plant-microbe interactions. Ultimately, this system could benefit agronomy and the food industry, contributing to health improvements.

Clarification of Bio-Degumming Enzymes Based on a Visual Analysis of the Hemp Roving Structure.

Hemp fibers, recognized for their breathability, specific strength, and ultraviolet resistance, are widely utilized in textile manufacturing and composite materials. Bio-degumming is a promising alternative technology to traditional chemical degumming that can be used to produce hemp fibers due to its eco-friendly nature. However, its lower efficiency has hindered its widespread adoption. The unclear and complex structure of the gums leads to a poor understanding on the enzyme types required for bio-degumming, thereby restricting improvements in its efficiency. In this study, the morphological characteristics, polysaccharide composition, and branched structure of hemp stem, roving fibers, and refined fibers were investigated using scanning electron microscopy and laser scanning confocal microscopy in combination with immunofluorescence techniques, with a view to identify the enzymes necessary for the efficient bio-degumming of hemp. The results revealed that the gums were primarily located in the middle lamella, phloem parenchyma, and certain xylem tissues. These tissues showed chunk-like, fence-like, and plate-like shapes, respectively, and tightly wrapped around the fiber bundles. In these tissues, pectin comprised low-esterified homogalacturonan, along with rhamnogalacturonan carrying galactan and arabinan branches. Xylan exhibited acetyl, arabinose, and glucuronic acid branches, while mannan displayed acetyl and galactose branches. Partial xylan and mannan were masked by pectin, and the branching structures impeded their enzymatic removal. As a consequence, the necessary enzymes and their synergistic effects for effective hemp roving degumming were elucidated. Pectin degradation was facilitated by pectate lyase and rhamnogalacturonan-degrading enzymes. Xylan and mannan were effectively removed by endo-xylanase and endo-mannanase, a process necessitating the synergistic action of branched-chain-degrading enzymes, including the esterase, α-L-arabinofuranosidase, α-galactosidase, and α-glucuronidase. This study provided practical strategies to enhance the efficiency of hemp bio-degumming.