Ultrasound-assisted enzymatic extraction of Corni Fructus alpha-glucosidase inhibitors improves insulin resistance in HepG2 cells.

Corni Fructus (CF) is a traditional medicine and beneficial food with multifaceted protective effects against diabetes and its complications. Since alpha-glucosidase inhibitors (GIs) are promising first-choice oral antihyperglycemic drugs for diabetes, we examined whether GIs from CF (GICF) are useful for diabetes treatment. Therefore, GICF was extracted by ultrasound-assisted enzymatic extraction (UAEE) that is optimized by a three-level, four-factor Box-Behnken design and determined by ultra-performance liquid chromatography. Compared to 36.31 mg g-1 without enzyme treatment, the GICF yield increased to 70.44 mg g-1via UAEE under optimum conditions (0.5% compound enzyme extracted in 23 min at 46 °C and pH 4.8). The activity (91.99%) of GICF was as predicted (93.28%). When GICF was used in an insulin-resistant HepG2 cell model, it significantly ameliorated the glucose metabolism in a dose-dependent manner. Our findings indicate that UAEE may be an innovative method for functional food extraction and a potential strategy for high-quality food ingredient (such as GI) production with high efficiency and productivity.

Bioprocess development for enhanced endoglucanase production by newly isolated bacteria, purification, characterization and in-vitro efficacy as anti-biofilm of Pseudomonas aeruginosa.

Endoglucanase producing bacteria were isolated from Egyptian soils and the most active bacterial strain was identified as Bacillus subtilis strain Fatma/1. Plackett-Burman statistical design was carried out to assess the effect of seven process variables on endoglucanase production. Carboxymethyl cellulose (CMC), yeast extract and peptone were the most significant variables that enhanced the endoglucanase production and thus were selected for further optimization using face-centered central composite design. The highest yield of endoglucanase (32.37 U/mL) was obtained in run no. 9, using 18 g/L CMC, 8 g/L peptone, 7 g/L yeast extract and 0.1 g/L FeSO4.7H2O. The optimized medium showed about eightfold increase in endoglucanase production compared to the unoptimized medium. The produced crude enzyme was further purified by ammonium sulfate precipitation, then DEAE-Sepharose CL6B column. The purified enzyme was shown to have a molecular weight of 37 kDa. The enzyme showed maximum activity at pH 8.0, temperature of 50 °C, incubation time of 60 min. The half-life time (T1/2) was 139.53 min at 50 °C, while being 82.67 min at 60 °C. Endoglucanase at concentration of 12 U/mL effectively removed 84.61% of biofilm matrix of Pseudomonas aeruginosa with marked reduction in carbohydrate content of the biofilm from 63.4 to 7.9 µg.

Screening, cloning, enzymatic properties of a novel thermostable cellulase enzyme, and its potential application on water hyacinth utilization.

Cellulose is the cheapest, natural, renewable organic substance that is used as a carbon source in various fields. Water hyacinth, an aquatic plant rich in cellulose, is often used as a raw material in fuel production. However, natural cellulase can be hardly used in industrial production on account of its low thermal stability and activity. In this study, a metagenomic library was constructed. Then, a new cellulase gene, cel1029, was screened by Congo red staining and expressed in the prokaryotic system. Enzymatic properties of Cel1029 were explored, including optimum temperature and pH, thermal and pH stability, and tolerance against organic solvents, metal ions, and salt solutions. Finally, its ability of degrading water hyacinth was identified and evaluated. Cel1029 displayed high homology with endoglucanase in the glycoside hydrolase family 5 (GH5) and had high stability across a broad temperature range. More than 86% of its enzymatic activities were retained between 4 and 60 °C after 24 h of incubation. Single-factor analysis and orthogonal design were further conducted to determine the optimal conditions for the highest reducing sugar yield of water hyacinth. Interestingly, Cel1029 efficiently transformed water hyacinth with a reducing sugar yield of 430.39 mg/g in 22 h. These findings may open the door for significant industrial applications of a novel GH5 cellulase (NCBI Reference Sequence: MK051001, Cel1029) and help identify more efficient methods to degrade cellulose-rich plants.

Expression and functional characterization in yeast of an endoglucanase from Bacillus sonorensis BD92 and its impact as feed additive in commercial broilers.

Some ingredients used in poultry feed formulation contain carbohydrate polymers which are difficult to digest and thus hinder nutritional feed value. Toward overcoming this limitation, exogenous enzymes have been added to poultry feed to improve its nutritive value. The present study was designed to provide first enzymatic characterization of endoglucanase (BsEgl) from the genome of B. sonorensis BD92 expressed in Pichia pastoris. Further, we tested its impact alone and in combination with a ß-glucosidase (Bteqßgluc) on growth in commercial broilers as feed additive. The expressed enzyme displayed features of GH5 family and had optimum activity against carboxymethyl cellulose at pH 5 and 50 °C. The BsEgl was stable at a range of pH from 4 to 8 for 60 min and at 50 °C for 180 min. Supplementing broilers diet with BsEgl alone or in combination with Bteqßgluc resulted in better feed conversion ratio among treatments during a five weeks testing period. Moreover, meat percentage was also highest for this treatment, and all treatments with recombinant enzymes increased intestinal length in birds compared to treatment control group. Blood parameters and serum biochemistry profile showed non-significant difference among groups. These results support that recombinant cellulolytic enzymes supplement high fiber diets improve their nutritional performance.

Synergy between endo/exo-glucanases and expansin enhances enzyme adsorption and cellulose conversion.

Effective binding between cellulases and cellulose is essential for enzymatic hydrolysis of lignocellulose. Expansin can loosen the cellulose structure and can enhance the efficiency of cellulase. However, possible synergy between cellulases and expansin is not clear. In this work, the real-time adsorption of exoglucanases (Cel7A) or endoglucanases (Cel7B) with Bacillus subtilis expansin (BsEXLX1) and the enzymatic hydrolysis of cellulose were followed using quartz crystal microbalance with dissipation (QCM-D). Initial adsorption rate, adsorption capacity, and pseudo-steady-state rate of cellulose hydrolysis by Cel7A/Cel7B increased in the presence of BsEXLX1. When injecting Cel7A or Cel7B together with BsEXLX1 at a mass ratio of 1:1, the hydrolysis rate was almost 5 times the rate for Cel7A or Cel7B alone at 25 °C. These results increase our understanding of the real-time synergism between cellulases and expansin on cellulose, as well as the impact of their synergy on the enzymatic hydrolysis of cellulose.

Novel buffalo rumen metagenome derived acidic cellulase Cel-3.1 cloning, characterization, and its application in saccharifying rice straw and corncob biomass.

Lignocellulosic biomass (LCB) is a prominent option for second-generation biofuels production. Cellulase hydrolyses cellulose, a component of LCB by attacking the ß-1,4-glycosidic bonds, thus liberating mono, di, and oligosaccharides, which subsequently, can be converted to biofuel. In this study, a novel cellulase (Cel-3.1) of 1593 bp which encodes a 530 amino acid protein was identified from buffalo rumen metagenomic fosmid library, and functional expression was achieved through transformation into Escherichia coli. The molecular weight was estimated as 58 kDa on SDS-PAGE. Cel-3.1 belongs to glycosyl hydrolase family-5 (GH-5) and is predicted to have 14 α-helices and 15 ß-strands. The optimal temperature and pH for Cel-3.1 were experimentally determined as 5.0 and 50 °C respectively. The synergistic effect of Ca2+ with K+ ions improved Cel-3.1 activity significantly (25%) and 1% Polyethylene Glycol (PEG-400), 1% ß-mercaptoethanol enhanced the relative activity Cel-3.1 by 31.68%, 12.03% respectively. Further, the enzymatic (Cel-3.1) hydrolysis of pretreated rice straw and corncob released 13.41 ± 0.26 mg/mL and 15.04 ± 0.08 mg/mL reducing sugars respectively. High Performance Liquid Chromatography (HPLC), Scanning Electron Microscope (SEM), and Fourier Transformation Infrared spectroscopy (FTIR) analysis revealed the capability of Cel-3.1 for the breakdown and hydrolysis of both rice straw and corncob to generate various fermentable sugars.

Direct observation of endoglucanase fibrillation and rapid thickness identification of cellulose nanoplatelets using constructive interference.

Better understanding through direct observation of the mechanisms involved in chemical and enzymatic hydrolysis of biomass is of great importance, to implement a substitute for the common cellulose standards. We report the hydrolysis of biomass, using exclusively the parenchyma, to isolate cellulose nanoplatelets using a less harsh pretreatment. Then, we show direct evidence of the effect of endoglucanase on the structure of cellulose nanoplatelets, finding that amorphous cellulose is exclusively digested, loosening the cellulose nanofibrils in the process. The analysis of micrographs demonstrates that when cellulose nanoplatelets are deposited on a silicon wafer, its thickness can be qualitatively measured by the interference color detected using an optical microscope. This finding facilitates further studies of mechanisms involved in lignin removal and cellulose nanofibrils production by specific enzymatic digestion.

Improving the thermostability of a thermostable endoglucanase from Chaetomium thermophilum by engineering the conserved noncatalytic residue and N-glycosylation site.

Endoglucanases provide an attractive avenue for the bioconversion of lignocellulosic materials into fermentable sugars to supply cellulosic feedstock for biofuels and other value-added chemicals. Thermostable endoglucanases with high catalytic activity are preferred in practical processes. To improve the thermostability and activity of the thermostable ß-1,4-endoglucanase CTendo45 isolated from the thermophilic fungus Chaetomium thermophilum, structure-based rational design was performed by using site-directed mutagenesis. When inactivated mutation of the unique N-glycosylation sequon (N88-E89-T90) was implemented and the conserved Y173 residue was substituted with phenylalanine, a double mutant T90A/Y173F demonstrated enzymatic activity that dramatically increased 2.12- and 1.82-fold towards CMC-Na and ß-D-glucan, respectively. Additionally, T90A/Y173F exhibited extraordinary heat endurance after 300 min of incubation at elevated temperatures. This study provides a valid approach to the improvement of enzyme redesign protocols and the properties of this endoglucanase mutant distinguish it as an excellent candidate enzyme for industrial biomass conversion.

Characterization and efficient production of a thermostable, halostable and organic solvent-stable cellulase from an oil reservoir.

The manufacture of biofuels from cellulose is regarded as one of practicable strategies to meet increasing energy demand and alleviate environmental issues. Cellulases, which play an important role in the production of second-generation biofuels, are expected to be highly thermostable, halostable and organic solvent-stable to adapt to the harsh conditions in practical application. Here we cloned and characterized a novel cellulase (MaCel) from Mahella australiensis 50-1 BON, an anaerobic thermophile isolated from an oil reservoir. MaCel exhibited excellent thermostability, halostability as well as organic solvent stability, and could be efficiently produced in a yield of 1.7 × 106 U/L in 15 h with inexpensive culture medium. These results indicate that MaCel may be a suitable candidate for industrial applications, illustrating the potential benefits of enzymes from oil reservoir extremophiles in the manufacture of biofuels.

Non-chromatographic purification of thermostable endoglucanase from Thermotoga maritima by fusion with a hydrophobic elastin-like polypeptide.

Endoglucanase EG12B from Thermotoga maritima is a thermophilic cellulase that has great potential for industrial applications. Here, to enable the selective purification of EG12B in a simple and efficient manner, an elastin-like polypeptide (ELP), which acts as a thermally responsive polypeptide, was fused with EG12B to enable its inverse phase transition cycling (ITC). A small gene library comprising ELPs from ELP5 to ELP50 was constructed using recursive directional ligation by plasmid reconstruction. ELP50 was added to the C-terminus of EG12B as a fusion tag to obtain the expression vector pET28-EG12B-ELP50, which was transformed into Escherichia coli BL21 (DE3) to enable the expression of fusion protein via IPTG induction. Gray scanning analysis revealed that the EG12B-ELP50 expression level was up to about 35% of the total cellular proteins. After three rounds of ITC, 8.14 mg of EG12B-ELP50 was obtained from 500-mL lysogeny broth culture medium. The recovery rate and purification fold of EG12B-ELP50 purified by ITC reached 78.1% and 11.8, respectively. The cellulase activity assay showed that EG12B-ELP50 had a better thermostability, higher optimal temperature, and longer half-life than those of free EG12B. Overall, our results suggested that ELP50 could be used as a favorable fusion tag, providing a rapid, simple, and inexpensive strategy for non-chromatographic target-protein purification.

Overexpression and characterization of TnCel12B, a hyperthermophilic GH12 endo-1,4-ß-glucanase cloned from Thermotoga naphthophila RKU-10T.

A putative cellulolytic gene (825 bp) from Thermotoga naphthophila RKU-10T was overexpressed as an active soluble endo-1,4-ß-glucanase (TnCel12B), belongs to glycoside hydrolase family 12 (GH12), in a mesophilic expression host. Heterologous expression and engineered bacterial cell mass was improved through specific strategies (induction and cultivation). Hence, intracellular activity of TnCel12B was enhanced in ZYBM9 modified medium (pH 7.0) by 8.38 and 6.25 fold with lactose (200 mM) and IPTG (0.5 mM) induction, respectively; and 6.95 fold was increased in ZYP-5052 auto-inducing medium after 8 h incubation at 26 °C (200 rev min-1). Purified TnCel12B with a molecular weight of ~32 kDa, was optimally active at 90 °C and pH 6.0; and exhibited prodigious stability over a wide range of temperature (50-85 °C) and pH (5.0-9.0) for 8 h TnCel12B displayed great resistance towards different chemical modulators, though activity was improved by Mg2+, Zn2+, Pb2+ and Ca2+. Purified TnCel12B had affinity with various substrates but peak activity was observed toward barley ß-glucan (1664 U mg-1) and carboxymethyl cellulose (736 U mg-1). The values of Km, Vmax, kcat, and kcatKm-1 were found to be 4.63 mg mL-1, 916 µmol mg-1min-1, 1326.7 s-1 and 286.54 mL mg-1 s-1, respectively using CMC substrate. All noteworthy features of TnCel12B make it an appropriate industrial candidate for bioethanol production and various other potential applications.

Purification and Biochemical Characterization of Alkalophilic Cellulase from the Symbiotic Bacillus subtilis BC1 of the Leopard Moth, Zeuzera pyrina (L.) (Lepidoptera: Cossidae).

In the current study, an extracellular cellulase belonging to symbiotic Bacillus subtilis Bc1 of the leopard moth is purified and characterized. The molecular mass of enzyme was 47.8 kDa using SDS-PAGE. The purified enzyme had optimum activity in temperature and pH around 60 °C and 8, respectively. The purified cellulase was introduced as a stable enzyme in a wide variety of temperature (20-80 °C) and pH (4-10) and remained active to more than 74% at 80 °C for 1 h. Moreover, the cellulase extremely was stabled in the presence of metal ions and organic solvents and its activity was increased by acetone (20% v/v), CaCl2 and CoCl2 and inhibited by MnCl2 and NiCl2. The values of enzyme's Km and Vmax were found to be 1.243 mg/mL and 271.3 µg/mL/min, respectively. The purified cellulase hydrolyzed cellulose, avicel and carboxymethyl cellulose (CMC) and the final product of CMC hydrolysis was cellobiose using thin-layer chromatography analysis. Consequently, owing to exo/endoglucanase activity and organic solvent, temperature and pH stability of the purified cellulase belong to B. subtilis BC1, it can be properly employed for various industrial purposes.

Efficient enzymatic saccharification of macroalgal biomass using a specific thermostable GH 12 endoglucanase from Aspergillus terreus JL1.

Stranded green macroalgae represents an important and renewable biomass that remains under valorized despite the numerous environmental problems generated by their accumulation in coastal regions. This work describes the isolation of a filamentous thermophile fungus identified as Aspergillus terreus JL1 that produces an efficient cellulolytic activity for green macroalgae saccharification. The characterization of the endoglucanase activity obtained after submerged fermentation showed a differential induction depending on the carbon source used with a unique isoform released when Ulva lactuca was used as inducer. The crude extract obtained hydrolyzed efficiently the untreated algal biomass (70.5%) compared to other cellulolytic extracts. The unique endoglucanase released was then purified to homogeneity (Yield: 49.6%; Specific activity: 30.1 U/mg; Purification fold: 4.36) and characterized biochemically. Its peptidic sequence was then determined and showed its belonging to the GH12. The described enzyme represents a promising biotechnological tool for algal biomass conversion.

Preparation and characterization of reusable magnetic combi-CLEA of cellulase and hemicellulase.

Cross-linked enzyme aggregate (CLEA) is a technology to overcome the limitation of enzymes for its application in chemical industries. The inability of repeated use of enzymes, stability and ease of separation from reaction mixture limits its applications. Here, magnetic combi-CLEA has been synthesised by adding amino-functionalized magnetic nanoparticles into pectinase ultra-clear (containing pectinases, xylanases and cellulases). Enzymes were precipitated on the surface of amino-functionalized magnetic nanoparticles with ethanol and cross-linked using glutaraldehyde. The structural characterization of magnetic combi-CLEA was studied by Scanning Electron Microscopy. Thermal stability was performed at 70 °C for pectinase and 80 °C for xylanase and cellulase respectively. Half-life (t1/2) of the xylanase, cellulase and pectinase in free form remarkably enhance from 84.51, 29.36, and 25.29 min respectively to 533.07, 187.29 and 147.44 min in magnetic-combi CLEA respectively. Magnetic combi-CLEA can be efficiently reused till 12th cycle after which pectinase, xylanase and cellulase retain 86.45%, 90.3% and 88.62% activity respectively. Using this CLEA preparation bioethanol concentration increases to 1.82-fold as compared to free enzyme, when simultaneous saccharification and fermentation was performed using wheat straw as the substrate. Magnetic combi-CLEA can be used for a variety of industrial applications like food processing, textile industry and bioethanol production.

A highly thermostable crude endoglucanase produced by a newly isolated Thermobifida fusca strain UPMC 901.

A thermophilic Thermobifida fusca strain UPMC 901, harboring highly thermostable cellulolytic activity, was successfully isolated from oil palm empty fruit bunch compost. Its endoglucanase had the highest activity at 24 hours of incubation in carboxymethyl-cellulose (CMC) and filter paper. A maximum endoglucanase activity of 0.9 U/mL was achieved at pH 5 and 60 °C using CMC as a carbon source. The endoglucanase properties were further characterized using crude enzyme preparations from the culture supernatant. Thermal stability indicated that the endoglucanase activity was highly stable at 70 °C for 24 hours. Furthermore, the activity was found to be completely maintained without any loss at 50 °C and 60 °C for 144 hours, making it the most stable than other endoglucanases reported in the literature. The high stability of the endoglucanase at an elevated temperature for a prolonged period of time makes it a suitable candidate for the biorefinery application.

Purification and characterization of a glycoside hydrolase family 5 endoglucanase from Tricholoma matsutake grown on barley based solid-state medium.

An endoglucanase was isolated from solid-state culture of the ectomycorrhizal fungus Tricholoma matsutake (TmEgl5A) grown on rolled barley and vermiculite. The enzyme was purified by ammonium sulfate fractionation, ion-exchange, hydrophobic, and gel filtration. TmEgl5A showed a molecular mass of approximately 40 kDa as determined by SDS-PAGE. The single band of the protein was analyzed by peptide-mass-finger-printing using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and the trypsin-digested peptide sequences were matched to a putative endoglucanase sequence (protein ID1465229) in the JGI T. matsutake 945 v3.0 genome database. Based on the sequence information, the gene encoding TmEgl was cloned and expressed in Pichia pastoris KM71H. The deduced amino acid sequence was similar to GH5 family endoglucanases from Basidiomycetes. The enzyme acts on barley ß-glucan, lichenan, and CMC-Na. The hydrolyzation products from these substrates were detected by thin-layer chromatography as oligosaccharides with minimal disaccharides. These results suggested that T. matsutake produces a typical endoglucanase in solid-state culture, and the fungus has the potential to degrade ß-linkage polysaccharides.

Purification and Characterization of Cellulase from Obligate Halophilic Aspergillus flavus (TISTR 3637) and Its Prospects for Bioethanol Production.

A cellulase from the extreme obligate halophilic fungus, Aspergillus flavus, isolated from a man-made solar saltern in Phetchaburi, Thailand, was purified by ammonium sulfate precipitation and using Sephadex G-100 gel filtration column chromatography. The cellulase was found to be approximately 55 kDa by SDS-PAGE. Using CMC as a substrate, the specific activity of the cellulase was 62.9 units (U) mg-1 with Vmax and Km values of 37.87 mol min-1 mg-1 and 3.02 mg mL-1, respectively. Characterization of the enzyme revealed it to be an extremozyme, having an optimum activity at pH 10, 60 °C, and 200 g L-1 of NaCl. The enzyme activity was not significantly altered by the addition of divalent metal cations at 2 mM and neither did ß-mercaptoethanol, while EDTA was found strongly inhibiting the cellulase. Compared with commercial cellulase, the purified cellulase from A. flavus was more active in the extremity of conditions, especially at pH 10, 60 °C, and 150 g L-1 NaCl, whereas the commercial cellulase had a very low activity.

Cold-tolerant endoglucanase producing ability of Mrakia robertii A2-3 isolated from cryoconites, Hamtha glacier, Himalaya.

A psychrotolerant yeast strain Mrakia robertii A2-3 isolated from cryoconites of Hamtah glacier, Himalaya, India was investigated for the production of cold-tolerant endoglucanase. Optimum endoglucanase production was found at 15°C with an initial pH of 5.5, and potent inducers were 1% wt/vol of xylose and KNO3 and 0.1% wt/vol of NaCl. Under optimum conditions, the enzyme production was 1.81-fold higher than the unoptimized conditions. Crude enzyme was partially purified by ammonium sulfate precipitation followed by dialysis. The enzyme was purified to 2.53-fold and the yield was 6.03% with specific activity of 17.38 U/mg and molecular weight ~57 kDa. The Km and Vmax values of the partially purified enzyme were found to be 1.57 mg/ml and 142.85 U/mg, respectively. The characterization study revealed that the best temperature was 15°C for activity and stability. Furthermore, the enzyme showed the highest activity at pH 11.0 and was stable at pH 6.0. Fe2+ , Mn2+ , Na2+ , Cu2+ , Co2+ , Ca2+ proved to be activators of endoglucanase. Ethylenediamine tetraacetic acid showed very low effect on the enzyme activity whereas it was active with Tween-80 and sodium deoxycholate. The present study successfully produced a cold-active endoglucanase with novel properties making it promising as a biocatalyst for industrial processes.

Haloferax sulfurifontis GUMFAZ2 producing xylanase-free cellulase retrieved from Haliclona sp. inhabiting rocky shore of Anjuna, Goa-India.

Salt stable cellulases are implicated in detritic food webs of marine invertebrates for their role in the degradation of cellulosic material. A haloarchaeon, Haloferax sulfurifontis GUMFAZ2 producing cellulase was successfully isolated from marine Haliclona sp., a sponge inhabiting the rocky intertidal region of Anjuna, Goa. The culture produced extracellular xylanase-free cellulase with a maximum activity of 11.7 U/ml, using carboxymethylcellulose-Na (CMC-Na), as a sole source of carbon in 3.5 M NaCl containing medium, pH 7 at 40°C and produced cellobiose and glucose, detectable by thin-layer chromatography. Nondenaturing polyacrylamide gel electrophoresis of the crude enzyme, revealed a single protein band of 19.6 kDa which on zymographic analysis exhibited cellulase activity while corresponding sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a molecular weight of 46 kDa. Unlike conventional cellulases, this enzyme is active in presence of 5 M NaCl and does not have accompanying xylanase activity, hence can be considered as xylanase-free cellulase. Such enzymes from haloarchaea offer great potential for biotechnological application because of their stability at high salinity and is therefore worth pursuing.

Characterization of the gut microbiota of invasive Agrilus mali Matsumara (Coleoptera: Buprestidae) using high-throughput sequencing: uncovering plant cell-wall degrading bacteria.

The genus Agrilus comprises diverse exotic and agriculturally important wood-boring insects that have evolved efficient digestive systems. Agrilus mali Matsumara, an invasive insect, is causing extensive mortality to endangered wild apple trees in Tianshan. In this study, we present an in-depth characterization of the gut microbiota of A. mali based on high-throughput sequencing of the 16S rRNA gene and report the presence of lignocellulose-degrading bacteria. Thirty-nine operational taxonomic units (OTUs) were characterized from the larval gut. OTUs represented 6 phyla, 10 classes, 16 orders, 20 families, and 20 genera. The majority of bacterial OTUs belonged to the order Enterobacteriales which was the most abundant taxa in the larval gut. Cultivable bacteria revealed 9 OTUs that all belonged to Gammaproteobacteria. Subsequently, we examined the breakdown of plant cell-wall compounds by bacterial isolates. Among the isolates, the highest efficiency was observed in Pantoea sp., which was able to synthesize four out of the six enzymes (cellulase, cellobiase, ß-xylanase, and ß-gluconase) responsible for plant-cell wall degradation. One isolate identified as Pseudomonas orientalis exhibited lignin peroxidase activity. Our study provides the first characterization of the gut microbial diversity of A. mali larvae and shows that some cultivable bacteria play a significant role in the digestive tracts of larvae by providing nutritional needs.