Molecular cloning, expression, and enzymatic activity of a novel endogenous cellulase from the mulberry longicorn beetle, Apriona germari

A novel endogenous beta-1,4-endoglucanase (Ag-EGase III) gene belonging to the glycoside hydrolase family (GHF) 5 was cloned from the mulberry longicorn beetle, Apriona germari. The Ag-EGase III gene spans 1061 bp and consists of a single exon coding for 325 amino acid residues. The Ag-EGase III showed 89% protein sequence identity to another beetle, Psacothea hilaris, cellulase belonging to GHF 5. The Ag-EGase III has the potential proton donor and nucleophile amino acids conserved in GHF 5 and two putative N-glycosylation sites. Northern blot and Western blot analyses showed that Ag-EGases were expressed in the gut; Ag-EGase III and Ag-EGase I were expressed in three gut regions, and no Ag-EGase II was found in hindgut, indicating that the foregut and midgut are the prime sites for cellulase synthesis in A. germari larvae. The cDNA encoding Ag-EGase III was expressed as a 47-kDa polypeptide in baculovirus-infected insect Sf9 cells and the enzyme activity of the purified recombinant Ag-EGase III was approximately 1037 U per mg of recombinant Ag-EGase III. The enzymatic property of the purified recombinant Ag-EGase III showed the highest activity at 55 degrees C and pH 6.0, and was stable at 60 degrees C at least for 10 min. In addition, the N-glycosylation of Ag-EGase III was revealed by treatment with tunicamycin of recombinant virus-infected insect Sf9 cells and with endoglycosidase F of purified recombinant Ag-EGase III, demonstrating that the carbohydrate moieties are not necessary for enzyme activity.     

Identification, cloning, and expression of a GHF9 cellulase from Tribolium castaneum (Coleoptera: Tenebrionidae)

The availability of sequenced insect genomes has allowed for discovery and functional characterization of novel genes and proteins. We report use of the Tribolium castaneum (Herbst) (red flour beetle) genome to identify, clone, express, and characterize a novel endo-β-1,4-glucanase we named TcEG1 (T. castaneum endoglucanase 1). Sequence analysis of a full-length TcEG1 cDNA clone (1356 bp) revealed sequence homology to enzymes in glycosyl hydrolase family 9 (GHF9), and verified presence of a change (Gly for Ser) in the conserved catalytic domain for GHF9 cellulases. This TcEG1 cDNA clone was predicted to encode a 49.5 kDa protein with a calculated pI of 5.39. Heterologous expression of TcEG1 in Drosophila S2 cell cultures resulted in secretion of a 51-kDa protein, as determined by Western blotting. The expressed protein was used to characterize TcEG1 enzymatic activity against two cellulose substrates to determine its specificity and stability. Our data support that TcEG1 as a novel endo-β-1,4-glucanase, the first functional characterization of a cellulase enzyme derived from an insect genome with potential applications in the biofuel industry due to its high relative activity at alkaline pH.     

Purification and cDNA cloning of a cellulase from abalone Haliotis discus hannai.

A cellulase [endo-beta-1,4-D-glucanase (EC 3.2.1.4)] was isolated from the hepatopancreas of abalone Haliotis discus hannai by successive chromatographies on TOYOPEARL CM-650M, hydroxyapatite and Sephacryl S-200 HR. The molecular mass of the cellulase was estimated to be 66 000 Da by SDS/PAGE, thus the enzyme was named HdEG66. The hydrolytic activity of HdEG66 toward carboxymethylcellulose showed optimal temperature and pH at 38 degrees C and 6.3, respectively. cDNAs encoding HdEG66 were amplified by the polymerase chain reaction from an abalone hepatopancreas cDNA library with primers synthesized on the basis of partial amino-acid sequences of HdEG66. By overlapping the nucleotide sequences of the cDNAs, a sequence of 1898 bp in total was determined. The coding region of 1785 bp located at nucleotide position 56-1840 gave an amino-acid sequence of 594 residues including the initiation methionine. The N-terminal region of 14 residues in the deduced sequence was regarded as the signal peptide as it was absent in HdEG66 protein and showed high similarity to the consensus sequence for signal peptides of eukaryote secretory proteins. Thus, matured HdEG66 was thought to consist of 579 residues. The C-terminal region of 453 residues in HdEG66, i.e. approximately the C-terminal three quarters of the protein, showed 42-44% identity to the catalytic domains of glycoside hydrolase family 9 (GHF9)-cellulases from arthropods and Thermomonospora fusca. While the N-terminal first quarter of HdEG66 showed 27% identity to the carbohydrate-binding module (CBM) of a Cellulomonas fimi cellulase, CenA. Thus, the HdEG66 was regarded as the GHF9-cellulase possessing a family II CBM in the N-terminal region. By genomic PCR using specific primers to the 3'-terminal coding sequences of HdEG66-cDNA, a DNA of 2186 bp including three introns was amplified. This strongly suggests that the origin of HdEG66 is not from symbiotic bacteria but abalone itself.     

Cellobiohydrolase I (Cel7A) from Trichoderma reesei has chitosanase activity

Cellobiohydrolase CBH I (Cel7A) from the filamentous fungus Trichoderma reesei (TrCBHI), which is a member of glycoside hydrolase family (GHF) 7, was expressed in Aspergillus oryzae. We found that the recombinant enzyme showed significant chitosanase activity, as well as cellulase activity, and acted in an endo-type manner on soluble polymeric substrate. Furthermore, another GHF7 CBH I from Aspergillus aculeatus (AaCBHI) expressed in A. oryzae also had chitosanase activity, while endoglucanase EG I (Cel7B) from T. reesei had no activity towards chitosan. To our knowledge, this is the first report of GHF7 enzymes possessing chitosanase activity.     

pH-dependent stability of EGX, a multi-functional cellulase from mollusca, Ampullaria crossean

Cellulose and hemicellulose are the most abundant andmajor constituents of plants and the potential rawmaterials for enzyme hydrolysis related biotechnologicalprocesses. The cellulase and hemicellulase have beenconsidered as two of the most important enzymes inbiotechnology market which can hydrolyze cellulosic ma-terials to fermentable sugars for various biotechnologicalpurposes [1,2], and can also be used in textile- and paper-making industry [3]. It is important to use these enzymesunder re…     

A novel cellulase gene from the mulberry longicorn beetle, Apriona germari: gene structure, expression, and enzymatic activity

We have previously cloned a cellulase [β-1,4-endoglucanase (EGase), EC 3.2.1.4] cDNA (Ag-EGase I) belonging to glycoside hydrolase family (GHF) 45 from the mulberry longicorn beetle, Apriona germari. We report here the gene structure, expression and enzyme activity of an additional celluase (Ag-EGase II) from A. germari and also described the gene structure of Ag-EGase I. The Ag-EGase II gene spans 1033 bp and consisted of two introns and three exons coding for 239 amino acid residues. The 2713-bp-long genomic DNA of Ag-EGase I also consisted of two introns and three exons. The Ag-EGase II showed 61% protein sequence identity to Ag-EGase I and 51% to another beetle, Phaedon cochleariae, cellulase, belonging to GHF 45. The catalytic sites of GHF 45 are conserved in Ag-EGase II. The Ag-EGase II has 14 conserved cysteine residues and three putative N-glycosylation sites. Northern blot analysis confirmed midgut-specific expression of Ag-EGase II, suggesting that the midgut is the prime site for cellulase synthesis in A. germari larvae. The cDNA encoding Ag-EGase II was expressed as a 36-kDa polypeptide in baculovirus-infected insect Sf9 cells and the enzyme activity of the purified recombinant Ag-EGase II was approximately 812 U/mg of recombinant Ag-EGase II. The enzymatic properties of the purified recombinant Ag-EGase II showed the highest activity at 50 °C and pH 6.0, and were stable at 60 °C at least for 10 min.     

Cloning, nucleotide sequence and module structure of the gene encoding the cellulose-binding protein B (CBPB) of Eubacterium cellulosolvens 5

The nucleotide sequence of the gene encoding the cellulose-binding protein B (CBPB) of Eubacterium cellulosolvens 5 was determined. The gene consists of an open reading frame of 3,429 nucleotides. The deduced amino acid sequence of CBPB contained one module highly similar to a catalytic module of glycosyl hydrolase family 9 (GHF9), one module partially similar to a family 3 carbohydrate-binding module (CBM3), two linkers, one module similar to a CBM of cellulose-binding protein A (CBPA) from E. cellulosolvens 5, and one module almost identical to a cell wall-binding module (CWBM) of CBPA. The module similar to GHF9 showed CMCase activity, and the modules similar to CBM3 and CBM of CBPA bound to cellulose. Moreover, the module highly similar to CWBM of CBPA bound to the cell walls prepared from E. cellulosolvens 5. The amino acid sequence of CBPB had a significant homology (64.15% sequence identity) with that of CBPA. These results suggest that cbpA and cbpB genes descended from the same ancestral cellulase gene.     

Molecular cloning and characterization of two novel cellulase genes from the mollusc Ampullaria crossean

Cellulase genes have been reported not only from fungi, bacteria and plant, but also from some invertebrate animals. Here, two cellulase (endo-β-1,4-glucanase, EC 3.2.1.4) genes, eg27I and eg27II, were cloned from the freshwater snail Ampullaria crossean cDNA using degenerate primers. The nucleotide sequences of the two genes shared 94.5% identity. The open reading frames of both genes consisted of 588 bp, encoding 195 amino acids. Both EG27I and EG27II belong to the glycoside hydrolase family 45, and each lacks a carbohydrate-binding module. The presence of introns demonstrated a eukaryotic origin of the EG27 gene, and, in addition, successful cloning of EG27 cDNA supported endogenous production of EG27 cellulase by Ampullaria crossean. Investigation of the EG27 cDNA from A. crossean will provide further information on GHF45 cellulases.     

Ancient origin of glycosyl hydrolase family 9 cellulase genes

While it is widely accepted that most animals (Metazoa) do not have endogenous cellulases, relying instead on intestinal symbionts for cellulose digestion, the glycosyl hydrolase family 9 (GHF9) cellulases found in the genomes of termites, abalone, and sea squirts could be an exception. Using information from expressed sequence tags, we show that GHF9 genes (subgroup E2) are widespread in Metazoa because at least 11 classes in five phyla have expressed GHF9 cellulases. We also demonstrate that eukaryotic GHF9 gene families are ancient, forming distinct monophyletic groups in plants and animals. As several intron positions are also conserved between four metazoan phyla then, contrary to the still widespread belief that cellulases were horizontally transferred to animals relatively recently, GHF9 genes must derive from an ancient ancestor. We also found that sequences isolated from the same animal phylum tend to group together, and in some deuterostomes, GHF9 genes are characterized by substitutions in catalytically important sites. Several paralogous subfamilies of GHF9 can be identified in plants, and genes from primitive species tend to arise basally to angiosperm representatives. In contrast, GHF9 subgroup E2 genes are relatively rare in bacteria.     

Dual cellulose-digesting system of the wood-feeding termite,Coptotermes formosanus Shiraki

The distribution of endo-beta-1,4-glucanase (EG) components in the digestive system of the wood-feeding termite, Coptotermes formosanus Shiraki, was investigated by zymogram analysis using polyacrylamide gel electrophoresis, followed by N-terminal protein sequencing. EG components similar to glycoside hydrolase family (GHF) 9 members were restricted to the salivary glands, the foregut, and the midgut, whereas components similar to GHF7 members were confined to the hindgut where numerous cellulolytic flagellates were harbored. RT-PCR experiments revealed that five GHF9 EG mRNAs (1348 bp) homologous to other termite EGs were expressed in the salivary glands and the midgut. The crude extract prepared from the midgut as well as that from the hindgut produced glucose from crystalline cellulose. These data suggest that C. formosanus has two independent cellulose-digesting systems: one in the midgut where cellulose digestion is accomplished by endogenous cellulases and the other in the hindgut which makes use of other cellulases possibly from symbiotic flagellates.     

Structure and function of a cellulase gene in redclaw crayfish, Cherax quadricarinatus

The most abundant organic compound produced by plants is cellulose; however, it has long been accepted that most animals do not produce endogenous enzymes required for its degradation, but rely instead on symbiotic relationships with microbes that produce the necessary enzymes. Here, we present the genomic organisation of an endogenous glycosyl hydrolase family (GHF) 9 gene in redclaw crayfish (Cherax quadricarinatus), consolidated from a cDNA sequence determined by Byrne et al. [Gene 239 (1999) 317–324.]. Comparison with several other invertebrate GHF9 genes reveals the conservation of both intron position/phase and splice sequence, which adds support to an argument for an ancestral animal cellulase gene. Furthermore, two introns in plant GHF9 genes are also identical in position, implying a more ancient origin for this class of animal cellulase.

Protein purification from redclaw gastric fluid via fast performance liquid chromatography (FPLC) indicated the presence of two endoglucanase enzymes. The molecular weights of these components were determined by matrix-assisted laser desorption/ionisation—time-of-flight (MALDI-TOF) to be 47,887 Da (Cel1) and 50,295 Da (Cel2). Cel1 is possibly the functional product of the described cellulase gene, with N-terminal amino acid residues identical to the translated amino acid sequence from the corresponding gene region. Cel2 was identical to Cel1 for 7 of 11 N-terminal residues and likely to be the product of a paralogous endoglucanase gene. These results suggest that redclaw crayfish possess at least one and possibly two functional, endoglucanase enzymes, although further work is required to confirm their origin and attributes.     

A GHF7 CELLULASE FROM THE PROTIST SYMBIONT COMMUNITY OF Reticulitermes flavipes ENABLES MORE EFFICIENT LIGNOCELLULOSE PROCESSING BY HOST ENZYMES

Termites and their gut microbial symbionts efficiently degrade lignocellulose into fermentable monosaccharides. This study examined three glycosyl hydrolase family 7 (GHF7) cellulases from protist symbionts of the termite Reticulitermes flavipes. We tested the hypotheses that three GHF7 cellulases (GHF7‐3, GHF7‐5, and GHF7‐6) can function synergistically with three host digestive enzymes and a fungal cellulase preparation. Full‐length cDNA sequences of the three GHF7s were assembled and their protist origins confirmed through a combination of quantitative PCR and cellobiohydrolase (CBH) activity assays. Recombinant versions of the three GHF7s were generated using a baculovirus‐insect expression system and their activity toward several model substrates compared with and without metallic cofactors. GHF7‐3 was the most active of the three cellulases; it exhibited a combination of CBH, endoglucanase (EGase), and β‐glucosidase activities that were optimal around pH 7 and 30°C, and enhanced by calcium chloride and zinc sulfate. Lignocellulose saccharification assays were then done using various combinations of the three GHF7s along with a host EGase (Cell‐1), beta‐glucosidase (β‐glu), and laccase (LacA). GHF7‐3 was the only GHF7 to enhance glucose release by Cell‐1 and β‐glu. Finally, GHF7‐3, Cell‐1, and β‐glu were individually tested with a commercial fungal cellulase preparation in lignocellulose saccharification assays, but only β‐glu appreciably enhanced glucose release. Our hypothesis that protist GHF7 cellulases are capable of synergistic interactions with host termite digestive enzymes is supported only in the case of GHF7‐3. These findings suggest that not all protist cellulases will enhance saccharification by cocktails of other termite or fungal lignocellulases.     

Endogenous cellulase enzymes in the stick insect (Phasmatodea) gut

High cellulase (endo-beta-1,4-glucanase) activity was detected in the anterior midgut of the walking stick (Phasmatodea) Eurycantha calcarata. The enzyme was isolated and analyzed via mass spectrometry. RT-PCR revealed two endoglucanase genes, EcEG1 and EcEG2. Mascot analysis of the purified enzyme confirms it to be the product of gene EcEG1. Homologous cDNAs were also isolated from a distantly related species, Entoria okinawaensis, suggesting a general distribution of cellulase genes in phasmids. Phasmid cellulases showed high homology to endogenously-produced glycoside hydrolase family 9 (GH9) endoglucanases from insects, especially to those of termites, cockroaches, and crickets. The purified E. calcarata enzyme showed clear antigency against an anti-serum for termite GH9 cellulase, which, together with the sequence homology, further suggests an endogenous origin of the enzyme. This discovery suggests a possible nutritive value for cellulose in the leaf-feeding phasmids, unlike in herbivorous Lepidoptera.     

Identification and characterization of a cellulase-encoding gene from the buffalo rumen metagenomic library

Microorganisms residing in the rumens of cattle represent a rich source of lignocellulose-degrading enzymes, since their diet consists of plant-based materials that are high in cellulose and hemicellulose. In this study, a metagenomic library was constructed from buffalo rumen contents using pCC1FOS fosmid vector. Ninety-three clones from the pooled library of approximately 10,000 clones showed degrading activity against AZCL-HE-Cellulose, whereas four other clones showed activity against AZCL-Xylan. Contig analysis of pyrosequencing data derived from the selected strongly positive clones revealed 15 ORFs that were closely related to lignocellulose-degrading enzymes belonging to several glycosyl hydrolase families. Glycosyl hydrolase family 5 (GHF5) was the most abundant glycosyl hydrolase found, and a majority of the GHF5s in our metagenomes were closely related to several ruminal bacteria, especially ones from other buffalo rumen metagenomes. Characterization of BT-01, a selected clone with highest cellulase activity from the primary plate screening assay, revealed a cellulase encoding gene with optimal working conditions at pH 5.5 at 50 °C. Along with its stability over acidic pH, the capability efficiently to hydrolyze cellulose in feed for broiler chickens, as exhibited in an in vitro digestibility test, suggests that BT-01 has potential application as a feed supplement.     

里氏木霉不同糖苷水解酶基因转录水平比较分析

以前期里氏木霉RNA-seq中发现的7个糖苷水解酶基因为对象,分析其不同条件下的表达特性,以期为寻找新的纤维素降解功能酶提供证据。运用生物信息学方法,分析了7个基因可能的编码产物和结构特征。以不同的产纤维素酶菌株（QM 9414、RUT C30）为材料,采用实时荧光定量PCR,对7个糖苷水解酶基因（编号4–10）在各种碳源条件下转录情况与主要的3个纤维素酶基因cbh1,cbh2,egl1（编号1–3）进行了比较分析。信息学分析表明,7个基因编码蛋白分属于GH47（4号、5号）,GH92（6–8号）,GH16（9号）,GH31（10号）糖苷水解酶家族,具有典型的信号肽序列。cbh1,cbh2,egl1基因在纤维素酶诱导条件下,转录水平均表现显著的增加,上调倍数以QM 9414菌株表现的最高。QM 9414菌株中,cbh1,cbh2,egl1基因在纤维素条件下的上调倍数显著高于乳糖,3个基因在RUT C30菌株中的转录水平则显示乳糖条件下上调幅度更大。7个糖苷水解酶基因也存在类似的情况,而且编码α-甘露糖苷酶和内切β-葡聚糖酶的8号、9号基因上调倍数在纤维素酶诱导条件下仅次于纤维素酶基因,而以甘油为碳源条件下,8号、9号基因上调倍数高于纤维素酶基因。4号基因在上述碳源条件下,转录水平变化不大。结果表明：4号基因可能是组成型表达。基因5、6、7、8、9、10的表达呈现明显的菌株和碳源依赖性,且在纤维素酶诱导条件下基本上是和3个纤维素酶基因共转录的。     

A monovalent anion affected multi-functional cellulase EGX from the mollusca,Ampullaria crossean

A cellulose hydrolytic enzyme was isolated from the stomach juice of Ampullaria crossean, a kind of herbivorous mollusca. The enzyme was purified 45.3-fold to homogenety by ammonium sulfate precipitation, DEAE-Sephadex A-50 column, Bio-gel P-100 gel filtration column, and phenyl-Sepharose CL-4B column chromatography. The enzyme was designated as cellulase EGX. The purified enzyme is a multi-functional enzyme with the activities of exo-beta-1,4-glucanase (14.84 U/mg for p-nitrophenyl beta-D-cellobioside), endo-beta-1,4-glucanase (40.3 U/mg for carboxymethyl cellulose), and endo-beta-1,4-xylanase (196 U/mg for soluble xylan from birchwood). The monovalent anions such as F(-), Cl(-), Br(-), I(-), and NO(3)(-) are essential for its exo-beta-1,4-glucanase activity but have no effect on the activity for xylan, while I(-) higher than 5mM would inhibit the exo-beta-1,4-glucanase activity. The monovalent anions Cl(-) and Br(-) activate its endo-beta-1,4-glucanase activity. Binding of Cl(-) enhances the thermostability of EGX, but does not affect its fluorescence emission spectrum. The molecular mass of EGX is 41.5 kDa, as determined by SDS-PAGE. The pI value is about pH 7.35. The xylan hydrolytic activity of EGX reaches to the maximum between pH 4.8 and 6.0 and the pNPC hydrolytic activity reaches the maximum between pH 4.8 and 5.6, while that for CMC hydrolytic activity is between pH 4.4 and 4.8. Preliminary results showed that the enzyme was secreted by the mollusca itself.     

Isolation and cloning of an endo-beta-1,4-mannanase from Pacific abalone Haliotis discus hannai

An endo-beta-1,4-mannanase was isolated from digestive fluid of Pacific abalone, Haliotis discus hannai, by successive chromatographies on TOYPEARL CM-650M, hydroxyapatite, and TOYOPEARL HW50F. The abalone mannanase, named HdMan in the present paper, showed a molecular mass of approximately 39,000 Da on SDS-PAGE, and exhibited high hydrolyic activity on both galactomannan from locust bean gum and glucomannan from konjac at an optimal pH and temperature of 7.5 and 45 degrees C, respectively. HdMan could degrade either beta-1,4-mannan or beta-1,4-mannooligosaccharides to mannotriose and mannobiose similarly to beta-1,4-mannanases from Pomacea, Littorina, and Mytilus. In addition, HdMan could disperse the fronds of a red alga Porphyra yezoensis into cell masses consisting of 10-20 cells that are available for cell engineering of this alga. cDNAs encoding HdMan were amplified by polymerase chain reaction from an abalone-hepatopancreas cDNA library. From the nucleotide sequences of the cDNAs, the sequence of 1232 bp in total was determined and the amino-acid sequence of 377 residues was deduced from the translational region of 1134 bp locating at nucleotide positions 15-1148. The N-terminal region of 17 residues except for the initiation Met, was regarded as the signal peptide of HdMan because it was absent in the HdMan protein and showed high similarity to the consensus sequence for signal peptides of eukaryote secretory proteins. Accordingly, mature HdMan was considered to consist of 359 residues with the calculated molecular mass of 39,627.2 Da. HdMan is classified into glycoside hydrolase family 5 (GHF5) on the basis of sequence homology to GHF5 enzymes.     

The celA gene, encoding a glycosyl hydrolase family 3 beta-glucosidase in Azospirillum irakense, is required for optimal growth on cellobiosides

The CelA beta-glucosidase of Azospirillum irakense, belonging to glycosyl hydrolase family 3 (GHF3), preferentially hydrolyzes cellobiose and releases glucose units from the C(3), C(4), and C(5) oligosaccharides. The growth of a DeltacelA mutant on these cellobiosides was affected. In A. irakense, the GHF3 beta-glucosidases appear to be functional alternatives for the GHF1 beta-glucosidases in the assimilation of beta-glucosides by other bacteria.