Pacsin 2-dependent N-cadherin internalization regulates the migration behaviour of malignant cancer cells.

Collective cell migration is the coordinated movement of multiple cells connected by cadherin-based adherens junctions and is essential for physiological and pathological processes. Cadherins undergo dynamic intracellular trafficking, and their surface level is determined by a balance between endocytosis, recycling and degradation. However, the regulatory mechanism of cadherin turnover in collective cell migration remains elusive. In this study, we show that the Bin/amphiphysin/Rvs (BAR) domain protein pacsin 2 (protein kinase C and casein kinase substrate in neurons protein 2) plays an essential role in collective cell migration by regulating N-cadherin (also known as CDH2) endocytosis in human cancer cells. Pacsin 2-depleted cells formed cell-cell contacts enriched with N-cadherin and migrated in a directed manner. Furthermore, pacsin 2-depleted cells showed attenuated internalization of N-cadherin from the cell surface. Interestingly, GST pull-down assays demonstrated that the pacsin 2 SH3 domain binds to the cytoplasmic region of N-cadherin, and expression of an N-cadherin mutant defective in binding to pacsin 2 phenocopied pacsin 2 RNAi cells both in cell contact formation and N-cadherin endocytosis. These data support new insights into a novel endocytic route of N-cadherin in collective cell migration, highlighting pacsin 2 as a possible therapeutic target for cancer metastasis.

Blocking estrogen-induced AMH expression is crucial for normal follicle formation.

In mammals, primordial follicles assembled in fetuses or during infancy constitute the oocyte resources for life. Exposure to 17beta-estradiol and phytogenic or endocrine-disrupting chemicals during pregnancy and/or the perinatal period leads to the failure of normal follicle formation. However, the mechanisms underlying estrogen-mediated abnormal follicle formation and physiological follicle formation in the presence of endogenous natural estrogen are not well understood. Here, we reveal that estrogen receptor 1, activated by estrogen, binds to the 5' region of the anti-Mullerian hormone (Amh) gene and upregulates its transcription before follicle formation in cultured mouse fetal ovaries. Ectopic expression of AMH protein was observed in pregranulosa cells of these explants. Furthermore, the addition of AMH to the culture medium inhibited normal follicle formation. Conversely, alpha-fetoprotein (AFP) produced in the fetal liver reportedly blocks estrogen action, although its role in follicle formation is unclear. We further demonstrated that the addition of AFP to the medium inhibited ectopic AMH expression via estrogen, leading to successful follicle formation in vitro Collectively, our in vitro experiments suggest that upon estrogen exposure, the integrity of follicle assembly in vivo is ensured by AFP.

Lrp13a and Lrp13b serve as vitellogenin receptors in the ovary of zebrafish†.

In oviparous animals, egg yolk is largely derived from vitellogenin, which is taken up from the maternal circulation by the growing oocytes via the vitellogenin receptor. Recently, a novel member of the lipoprotein receptor superfamily termed low-density lipoprotein receptor-related protein 13 was identified and proposed as a candidate of vitellogenin receptor in oviparous animals. However, the roles of low-density lipoprotein receptor-related protein 13 in vitellogenesis are still poorly defined. Here, we investigated the expression, vitellogenin-binding properties, and function of low-density lipoprotein receptor-related protein 13 in zebrafish. Two different lrp13 genes termed lrp13a and lrp13b were found in zebrafish. Reverse transcription polymerase chain reaction and quantitative polymerase chain reaction revealed both lrp13s to be predominantly expressed in zebrafish ovary, and in situ hybridization detected both lrp13s transcripts in the ooplasm of early stage oocytes. Two yeast hybrid studies showed that among eight vitellogenins of zebrafish, Vtg1, 2, and 3 bind to Lrp13a, while Vtg1, 2, and 5 bind to Lrp13b. We created zebrafish lrp13a and lrp13b mutant lines using CRISPR/Cas9. Knockout of lrp13a leads to a male-biased sex ratio and decreased diameter of embryo yolk, while knockout of lrp13b and double knockout of lrp13a and lrp13b leads to the delay of vitellogenesis, followed by follicular atresia. These phenotypes of mutants can be explained by the disruption of vitellogenesis in the absence of Lrp13s. Taken together, our results indicate that both Lrp13a and Lrp13b can serve as vitellogenin receptors in zebrafish among other vitellogenin receptors that are not yet described.

Glucose metabolism is required for oocyte maturation of zebrafish.

Glucose is an essential source of energy production for animal cells. The importance of glucose metabolism in oocyte maturation has been studied extensively in mammals. However, such roles in non-mammalian species are still largely unknown. Here, we used zebrafish as a model, which is phylogenetically distant from mammals, and analyzed the role of glucose metabolism in oocyte maturation. Major glucose transporters (GLUT/Slc2A) were analyzed in zebrafish, two Slc2a1 (Slc2a1a and Slc2a1b), one Slc2a2, and two Slc2a3 (Slc2a3a and Slc2a3b) were identified. Among these five Slc2a genes, slc2a1b exhibited the highest expression level in fully grown follicles. The expression of slc2a1b gradually increased during folliculogenesis, and also significantly increases during the oocyte maturation process. Consistently, the glucose concentration increases during natural oocyte maturation. By using a fluorescent glucose derivative (6-NBDG) to trace glucose transport, the uptake of glucose by ovarian follicles in a time-dependent manner could be observed. Intriguingly, by treatment of glucose in vitro, oocyte maturation could be induced in a time-, dose- and stage-dependent manner. Glucose can be metabolized by glycolysis, the pentose phosphate pathway (PPP), the hexosamine biosynthesis pathway (HBP), and the polyol pathway. Using the inhibitors for these pathways, we found only PPP but not glycolysis, HBP or polyol pathway is essential for oocyte maturation. All these results clearly demonstrate for the first time that the glucose metabolism is required for oocyte maturation of zebrafish, suggesting the highly conserved role of glucose metabolism in control of oocyte maturation between fish and mammals.

Dynamin 2 and BAR domain protein pacsin 2 cooperatively regulate formation and maturation of podosomes.

Podosomes are actin-rich adhesion structures formed in a variety of cell types, such as monocytic cells or cancer cells, to facilitate attachment to and degradation of the extracellular matrix (ECM). Previous studies showed that dynamin 2, a large GTPase involved in membrane remodeling and actin organization, is required for podosome function. However, precise roles of dynamin 2 at the podosomes remain to be elucidated. In this study, we identified a BAR (Bin-Amphiphysin-Rvs167) domain protein pacsin 2 as a functional partner of dynamin 2 at podosomes. Dynamin 2 and pacsin 2 interact and co-localize to podosomes in Src-transformed NIH 3T3 (NIH-Src) cells. RNAi of either dynamin 2 or pacsin 2 in NIH-Src cells inhibited podosome formation and maturation, suggesting essential and related roles at podosomes. Consistently, RNAi of pacsin 2 prevented dynamin 2 localization to podosomes, and reciprocal RNAi of dynamin 2 prevented pacsin 2 localization to podosomes. Taking these results together, we conclude that dynamin 2 and pacsin 2 co-operatively regulate organization of podosomes in NIH-Src cells.

Igf3: a novel player in fish reproduction†.

As in other vertebrates, fish reproduction is tightly controlled by gonadotropin signaling. One of the most perplexing aspects of gonadotropin action on germ cell biology is the restricted expression of gonadotropin receptors in somatic cells of the gonads. Therefore, the identification of factors conveying the action of gonadotropins on germ cells is particularly important for understanding the mechanism of reproduction. Insulin-like growth factors (Igfs) are recognized as key factors in regulating reproduction by triggering a series of physiological processes in vertebrates. Recently, a novel member of Igfs called Igf3 has been identified in teleost. Different from the conventional Igf1 and Igf2 that are ubiquitously expressed in a majority of tissues, Igf3 is solely or highly expressed in the fish gonads. The role of Igf3 in mediating the action of gonadotropin through Igf type 1 receptor on several aspects of oogenesis and spermatogenesis have been demonstrated in several fish species. In this review, we will summarize existing data on Igf3. This new information obtained from Igf3 provides insight into elucidating the molecular mechanism of fish reproduction, and also highlights the importance of Igf system in mediating the action of gonadotropin signaling on animal reproduction.

Igf3 is essential for ovary differentiation in zebrafish†.

Zebrafish gonadal sexual differentiation is an important but poorly understood subject. Previously, we have identified a novel insulin-like growth factor (Igf) named insulin-like growth factor 3 (Igf3) in teleosts. The importance of Igf3 in oocyte maturation and ovulation has been recently demonstrated by us in zebrafish. In this study, we have further found the essential role of Igf3 in gonadal sexual differentiation of zebrafish. A differential expression pattern of igf3 between ovary and testis during sex differentiation (higher level in ovary than in testis) was found in zebrafish. An igf3 knockout zebrafish line was established using TALENs-mediated gene knockout technique. Intriguingly, all igf3 homozygous mutants were males due to the female-to-male sex reversal occurred during sex differentiation. Further analysis showed that Igf3 did not seem to affect the formation of so-called juvenile ovary and oocyte-like germ cells. Oocyte development was arrested at primary growth stage, and the ovary was gradually sex-reversed to testis before 60 day post fertilization (dpf). Such sex reversal was likely due to decreased germ cell proliferation by suppressing PI3K/Akt pathway in early ovaries of igf3 mutants. Estrogen is considered as a master regulator in fish sex differentiation. Here, we found that igf3 expression could be upregulated by estrogen in early stages of ovarian follicles as evidenced in in vitro treatment assays and cyp19a1a mutant zebrafish, and E2 failed to rescue the defects of igf3 mutants in ovarian development, suggesting that Igf3 may serve as a downstream factor of estrogen signaling in sex differentiation. Taken together, we demonstrated that Igf3 is essential for ovary differentiation in zebrafish.

Dual roles of PDE9a in meiotic maturation of zebrafish oocytes.

The essential role of cyclic guanosine monophosphate (cGMP) signaling in regulating the oocyte meiotic cell cycle has been established. However, control of the level of cGMP in ovarian follicles is unclear. The cGMP-hydrolyzing phosphodiesterases (PDEs) are important in regulating the cellular cGMP level. We used zebrafish as a model to study the role of a cGMP-hydrolyzing phosphodiesterase-9a (PDE9a) in meiotic maturation of oocytes. Three PDE9a coding genes (PDE9aa, PDE9ab, and PDE9ac) were identified in zebrafish. Both pde9aa and pde9ac are expressed in most adult tissues including the ovary, but pde9ab is only expressed in the ovary, kidney, pituitary, and brain. All three pde9as mRNA exhibited different expression profiles during folliculogenesis. All of them are highly expressed in the oocyte but not in the follicular cell. The expression of both pde9aa and pde9ab, but not pde9ac, in ovarian follicles increases during oocyte maturation either in natural ovulatory cycle or induced by administration of hCG in vivo. We overexpressed pde9aa by injection of capped pde9aa mRNA into the oocytes. The cGMP level was decreased, and oocyte maturation was stimulated. When the activity of PDE9a was blocked by a specific inhibitor, Bay736691, the oocyte maturation was also stimulated. The stimulatory effect could be blocked by a gap junction blocker. However, the spontaneous oocyte maturation of denuded oocytes was not largely affected after treatment with Bay736691. All of the mature oocytes obtained by either treatment of Bay736691 or injection of pde9aa mRNA, could be fertilized in vitro. These results demonstrate the dual roles of PDE9a in oocyte maturation. The basal level of PDE9a is responsible for maintaining the meiotic arrest, and the increased level of PDE9a induced by LH signaling is helpful for stimulating meiotic maturation by hydrolyzing cGMP in oocytes.

Efficacy of Bacillus methylotrophicus SY200 strain as feed additive against experimental Salmonella typhimurium infection in mice.

To investigate the effects of Bacillus methylotrophicus SY200 on Salmonella typhimurium (STM) infection in mice, a total of 36 three-week-old male mice were selected and randomly divided into 3 equal groups (N = 12). Group A and group B were fed with basal diet while group C was fed the basal diet supplemented with 0.1% (w/w) B. methylotrophicus SY200 during the 21 days experimental period. On the 14th day of the experiment, mice of group A were intragastrically administered with 0.5 ml of normal saline, group B and C were orally administered with 0.5 ml of STM suspension. On the first day and seventh day after STM challenge, the number of total white blood cells (WBCs) and neutrophils, relative weight of visceral organs, the number of Salmonella spp., Escherichia coli, Lactobacillus spp. and Bifidobacterium spp. in ileum and cecum, and diversity of cecal microflora were measured. The results showed that: on the first day and seventh day after STM challenge, the number of WBCs and neutrophils in the blood of the mice was the highest in group B, then followed by group C, and group A. On the first day after STM challenge, the relative weight of spleen in group C was significantly higher than that in group B (p < 0.05), moreover, compared with group B, B. methylotrophicus SY200 significantly reduced the number of Salmonella spp. and E. coli (p < 0.05), and increased the number of Lactobacillus spp. and Bifidobacterium spp. (p < 0.05) in the intestines of mice, and improved the Shannon-Wiener diversity (H), Simpson (E) and richness (S) indices of cecal flora of mice (p < 0.05). The results indicated that B. methylotrophicus SY200 could alleviate the inflammatory reaction after STM infection and resist the adverse effects of STM infection on mice intestinal flora.

The role of PDE5a in oocyte maturation of zebrafish.

The importance of cyclic guanosine monophosphate (cGMP) signaling pathway in oocyte maturation has recently attracted much attention in vertebrates. Previously, using zebrafish as a model, we have revealed the role of cGMP and the action of cGMP protein kinase (PKG) in oocyte maturation. In the present study, the function of a cGMP specific phosphodiesterase (PDE5a) is further analyzed in oocyte maturation in zebrafish. Two distinct PDE5a coding genes (named PDE5aa and PDE5ab) were identified in zebrafish, and expressed in most adult tissues including ovary. Both pde5aa and pde5ab mRNA are predominantly expressed in the oocyte but not in follicular cells. Two commercial antibodies targeted to mammalian PDE5a and phosphorylated PDE5a were validated in zebrafish, and we found both antibodies can be used to detect PDE5ab and phosphorylated PDE5ab of zebrafish, respectively. Using both antibodies, we found PDE5ab is only expressed in the oocyte and the phosphorylation of PDE5ab in oocyte could be activated during oocyte maturation induced by human chronic gonadotropin. Intriguingly, we found that the oocyte maturation could be stimulated by treatment of either two different PDE5a inhibitors, sildenafil or tadalafil, and such effects could be completely blocked by a PKG inhibitor KT5823 and two gap junction blockers, respectively. All of these results clearly demonstrate the importance of PDE5a in maintaining the oocyte maturation of zebrafish. When compared with mammals, the functional model of PDE5a is different in zebrafish, suggesting the function of PDE5a might shift from the oocyte in fish to the granulosa cell in mammals during evolution.

Zinc mediates the action of androgen in acting as a downstream effector of luteinizing hormone on oocyte maturation in zebrafish†.

The role of androgenic steroids on ovarian development has attracted much attention in recent years, but the molecular mechanism is still largely unknown. In this study, using zebrafish as a model, we found that the trace metal zinc mediates the action of androgen on oocyte maturation. The ovarian and serum testosterone is transiently stimulated by LH during oocyte maturation. Testosterone could mimic the action of LH on oocyte maturation, and its action appears to be independent of the classical nuclear androgen receptor. Consistent with a recent finding that a zinc transporter (Zip9) has been suggested as a novel androgen receptor, we found the labile zinc concentration could be induced by testosterone in the ovarian follicular cells, and zinc could mimic the action of testosterone on oocyte maturation and signaling. Moreover, the action of testosterone on oocyte maturation could be abolished by the chelation of zinc. Thus, the evidence supports the notion that zinc could mediate the action of androgen on oocyte maturation in zebrafish. This finding would shed light on understanding the role of androgen in ovary development and the molecular mechanism of oocyte maturation in fish.

Insulin-like growth factor binding proteins inhibit oocyte maturation of zebrafish.

The function of insulin-like growth factor (Igf) system in ovary has attracted much attention, but the role of Igf binding proteins (Igfbps) in ovary is still largely unknown. In this study, the role of Igfbps in oocyte maturation was investigated in zebrafish. The expression of all eight identified Igfbps except Igfbp6b could be detected in the adult ovary and exhibited differential expression profiles during folliculogenesis. The expression of several Igfbps is dynamically changed during oocyte maturation induced by human chorionic gonadotropin (hCG). By treatment of an Igfbps inhibitor NBI-31772 in vitro, the oocyte maturation could be stimulated in a clear dose-, time- and stage-dependent manner. Such effects were also observed by administration of NBI-31772 in vivo. Igfbps are differentially expressed in both follicular cells and oocytes, but the effect of NBI-31772 could only be found in intact follicles and not in the denuded oocytes. Previous studies have demonstrated that Igf3 is the major Igf member in regulating oocyte maturation of zebrafish. Interestingly, NBI-31772 could increase the effect of Igf3 on oocyte maturation. Furthermore, we found the effect of NBI-31772 on oocyte maturation could be blocked by an Igf type 1 receptor inhibitor BMS-536924 in vitro, suggesting the Igfbps can inhibit the oocyte maturation via Igf/Igf1r pathway. Together, we provided the first evidence in fish that Igfbps inhibit oocyte maturation of zebrafish.

Surface Display of Antigen Protein VP8* of Porcine Rotavirus on Bacillus Subtilis Spores Using CotB as a Fusion Partner.

Porcine rotavirus is a major cause of acute viral gastroenteritis in suckling piglets, and vaccination is considered to be an effective measure to control these infections. The development of a live mucosal vaccine using Bacillus subtilis spores as an antigen delivery vehicle is a convenient and attractive vaccination strategy against porcine rotavirus. In this study, a shuttle vector was constructed for the spore surface display of the spike protein VP8* from porcine rotavirus (the genotype was G5P[7]). A successful display of the CotB-VP8* fusion protein on the spore surface was confirmed by Western blot and immunofluorescence microscopy analysis. The capacity for immune response generated after immunization with the recombinant strain was evaluated in a mouse model. The intestinal fecal IgA and serum IgG were detected by enzyme-linked-immunosorbent serologic assay (ELISA). Importantly, recombinant strain spores could elicit strong specific mucosal and humoral immune responses. These encouraging results suggest that recombinant B. subtilis BV could provide a strategy for a potential novel application approach to the development of a new and safe mucosal subunit vaccine against porcine rotavirus.

The role of PKG in oocyte maturation of zebrafish.

Recently the importance of cyclic guanosine monophosphate (cGMP) signaling pathway in oocyte maturation has been well demonstrated in several species. However, as the primary downstream effector of the cGMP signaling pathway, little is known on the role of cGMP-dependent protein kinase (PKG) in oocyte maturation. In the present study, the expression, regulation and function of PKG in oocyte maturation was investigated in zebrafish. We identified four distinct PKG coding genes (named Prkg1a, Prkg1b, Prkg2, and Prkg3) in zebrafish. All prkgs are expressed in the ovary, and both prkg1a and prkg1b could be regulated by human chronic gonadotropin in follicular cells during oocyte maturation. We found that a cGMP analogue, 8-Br-cGMP, could stimulate oocyte maturation in a dose- and time-dependent manner. Such stimulatory effects of cGMP could be totally blocked by a PKG specific inhibitor, KT-5823. Intriguingly, we further found KT5823 could significantly attenuate spontaneous oocyte maturation in intact follicles but not in the denuded oocytes, suggesting that the activity of PKG in follicular cells is important for oocyte maturation. All of these results clearly demonstrate that PKG is involved in oocyte maturation in zebrafish.

The dual role of cGMP in oocyte maturation of zebrafish.

The roles of cyclic guanosine monophosphate (cGMP) signaling in oocyte maturation attracts much attention in mammals, but its roles in fish are still largely unknown. Using zebrafish as a model, we demonstrated for the first time in fish that cGMP is involved in oocyte maturation, and its functional model in oocyte maturation is different from that of mammals. The intracellular cGMP could be regulated by nitric oxide (NO), we found that all three NO synthase enzymes and four soluble guanylyl cyclases (sGC) are expressed in the zebrafish ovary. Intriguingly, either the activation or inhibition of the NO/sGC/cGMP pathway in fully grown follicles could lead to oocyte maturation. During oocyte maturation, cGMP levels increased in the follicular cell layer but decreased in oocytes, while NO levels increased in follicular cells but remained constant in oocytes. Based on these findings in zebrafish, we propose a hypothetical model on the dual role of cGMP in oocyte maturation: in follicular cells the LH signal could increase the level of NO and cGMP which induces oocyte maturation, while in the oocyte the decreased cGMP level can also induce oocyte maturation. These findings help us to understand the molecular mechanism of fish oocyte maturation.

Evolution of gonadotropin signaling on gonad development: insights from gene knockout studies in zebrafish.

Gonadal development is precisely regulated by the two gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Much progress on understanding the functions of LH and FSH signaling on gonad development has been achieved in the past decades, mostly from studies in mammals, especially genetic studies in both mouse and human. The functions of both LH and FSH signaling in nonmammalian species are still largely unknown. In recent years, using zebrafish, a teleost phylogenetically distant from mammals, we and others have genetically analyzed the functions of gonadotropins and their receptors through gene knockout studies. In this review, we will summarize the pertinent findings and discuss how the actions of gonadotropin signaling on gonad development have evolved during evolution from fish to mammals.

Igf3 serves as a mediator of luteinizing hormone in zebrafish ovulation.

Both oocyte maturation and ovulation is triggered by the luteinizing hormone (LH) surge in vertebrates, but exactly how these processes are regulated by LH remains to be fully elucidated. Previously, we found that Igf3, a fish-specific member of the igf family predominantly expressed in the gonads, could mediate the action of LH on oocyte maturation in zebrafish. Here, we further reveal the importance of Igf3 in mediating the action of LH on ovulation in zebrafish. All the four igf gene family members are expressed in the zebrafish ovary but only the igf3 transcript level is increased in hCG-induced ovulation in vivo. The expression of Igf3 protein in the follicles is also increased during ovulation. The actions of hCG on the expression of ovulatory enzymes and on ovulation itself could be largely mimicked by the recombinant zebrafish Igf3 protein. Intriguingly, the phosphorylation of Igf1r, the receptor for Igf3, could be activated by hCG in the follicular cells during ovulation. And inhibition of Igf3 signaling by Igf1r inhibitors and Igf3 antiserum could significantly attenuate the hCG-induced ovulation. Collectively, all these data support the notion that Igf3 serves as a mediator of LH action in zebrafish ovulation.

Germline-specific dgcr8 knockout in zebrafish using a BACK approach.

Zebrafish is an important model to study developmental biology and human diseases. However, an effective approach to achieve spatial and temporal gene knockout in zebrafish has not been well established. In this study, we have developed a new approach, namely bacterial artificial chromosome-rescue-based knockout (BACK), to achieve conditional gene knockout in zebrafish using the Cre/loxP system. We have successfully deleted the DiGeorge syndrome critical region gene 8 (dgcr8) in zebrafish germ line and demonstrated that the maternal-zygotic dgcr8 (MZdgcr8) embryos exhibit MZdicer-like phenotypes with morphological defects which could be rescued by miR-430, indicating that canonical microRNAs play critical role in early development. Our findings establish that Cre/loxP-mediated tissue-specific gene knockout could be achieved using this BACK strategy and that canonical microRNAs play important roles in early embryonic development in zebrafish.

Knockdown of zebrafish Nanog increases primordial germ cells during early embryonic development.

Nanog is a homeodomain transcription factor that plays a prominent role in maintaining the pluripotency and self-renewal capacity of embryonic stem cells (ESCs) in mammals. Medaka Nanog is necessary for S-phase transition and proliferation during embryonic development. However, whether Nanog regulates the proliferation of primordial germ cells (PGCs) during embryonic development has not yet been investigated. In this study, we identified the homologue of the mammalian Nanog gene in zebrafish (zNanog). The expression of both zNanog mRNA and protein was demonstrated in the spermatogonia (male germ stem cells) of the testis and the early oocytes of the ovary. During the embryonic development, zNanog mRNA is expressed in the cytoplasm of PGCs, and its protein is localized to the PGC nuclei. We also found that zNanog depletion using morpholinos resulted in the increases and aberrant localization of PGCs in the zebrafish embryos from the sphere stage to the 50% epiboly stage. These data indicated that zNanog inhibits the PGCs proliferation in early embryonic development of zebrafish.

Co-immobilized multi-enzyme biocatalytic system on reversible and soluble carrier for saccharification of corn straw cellulose.

Herein, three enzymes (cellulase, ß-glucosidase, and pectinase) with synergistic effects were co-immobilized on the Eudragit L-100, and the recovery of co-immobilized enzymes from solid substrates were achieved through the reversible and soluble property of the carrier. The optimization of enzyme ratio overcomed the problem of inappropriate enzyme activity ratio caused by different immobilization efficiencies among enzymes during the preparation process of co-immobilized enzymes. The co-immobilized enzymes were utilized to catalytically hydrolyze cellulose from corn straw into glucose, achieving a cellulose conversion rate of 74.45% under conditions optimized for their enzymatic characteristics and hydrolytic reaction conditions. As a result of the reversibility and solubility of the carrier, the co-immobilized enzymes were recovered from the solid substrate after five cycles, retaining 54.67% of the enzyme activity. The aim of this study is to investigate the potential of co-immobilizing multiple enzymes onto the Eudragit L-100 carrier for the synergistic degradation of straw cellulose.