Isozymes from the herbivorous gecarcinid land crab, Gecarcoidea natalis that possess both lichenase and endo-β-1,4-glucanase activity

Three isozymes with both lichenase and endo-β-1,4-glucanase activity were purified and characterised from the midgut gland of the herbivorous gecarcinid land crab, Gecarcoidea natalis. The three isozymes, termed 1a, 1b and 2, had respective molecular masses of 53 ± 0 (3), 43 ± 0 (3) and 47.4 ± 0(3) kDa. All isozymes possessed similar V(max) values and thus hydrolysed both carboxy methyl cellulose and lichenan equally. Furthermore the chromatography profiles for lichenase activities mirrored that for endo-β-1,4-glucanase activities suggesting that the same enzyme possessed both activities. Given this, the endo-β-1,4-glucanase enzymes described for other animals, may, like the isozymes described in this study, may be able to hydrolyse lichenan. However this ability needs to be confirmed. The main digestive function of these isozymes may be to hydrolyse hemicelluloses such as lichenan and mixed beta-D-glucan. All three isozymes randomly hydrolysed internal glycosidic bonds within carboxy methyl cellulose and lichenan to release short oligomers of 4-5 glucose units in length. They also hydrolysed cellotetraose to either two units of cellobiose or cellotriose and glucose. Cellotriose was hydrolysed to cellobiose and glucose. All three enzymes lacked β-1,4-glucosidase activity as they could not hydrolyse cellobiose.     

Expression ofBacillus subtilis JA18 endo-β-1,4-glucanase gene inEscherichia coli and characterization of the recombinant enzyme

Endo-β-1,4-glucanase encoded byBacillus subtilis JA18 was expressed inEscherichia coli. The recombinant enzyme was purified and characterized. The purified enzyme showed a single band of 50 kDa by SDS-PAGE. The optimum pH and temperature for this endo-β-1,4-glucanase was pH 5.8 and 60 °C. The endo-β-1,4-glucanase was highly stable in a wide pH range, from 4.0 to 12.0. Furthermore, it remained stable up to 60 °C. The endo-β-1,4-glucanase was completely inhibited by 2 mM Zn²⁺, Cu²⁺, Fe³⁺, Ag⁺, whereas it is activated in the presence of Co²⁺. In addition, the enzyme activity was inhibited by 1 mM Mn²⁺ but stimulated by 10 mM Mn²⁺. At 1% concentration, SDS completely inhibited the enzyme. The enzyme hydrolysed carboxymethylcellulose, lichenan but no activity was detected with regard to avicel, xylan, chitosan and laminarin. For carboxymethylcellulose, the enzyme had a Km of 14.7 mg/ml.     

Endoglucanase activity in <Emphasis Type="Italic">Neoteredo reynei</Emphasis> (Bivalvia,Teredinidae) digestive organs and its content

Cellulolytic enzymes have been studied in several organisms, such as insects, molluscs and other organisms, which can have enzymes endogenously produced or by symbiotic microorganisms. These enzymes are responsible for breaking down the cellulosic material upon which these organisms feed, probably with the aim of assimilating the sugars and nutrients. As Teredinidae bivalves grown in mangrove trees, this study aimed to measure endo-β-1,4-glucanase activity in different organs and its content. Endo-β-1,4-glucanase activity was detected in different organs of the Teredinidae bivalves, including gills and digestive organs tissues and its content. Moreover, organisms such as teredinids grow up inside wood and this process could perhaps be related to creating growth space. All the endoglucanase extracts, from organs tissues and contents, showed maximum activity at 40 °C. The maximum activity was observed at pH 5.5 for all the extracts, except for intestine tissue, which maximum was at pH 6. Moreover, some of the extracts showed a different profile of the activity as a pH influence, suggesting different distribution of enzymes over the digestive system of the teredinids. The results suggested that the endo-β-1,4-glucanase from Teredinidae could be applied in process that requires low temperature, such as, simultaneous saccharification and fermentation, since it presents lower optimum temperature in comparison to enzymes from terrestrial microorganisms.     

Enzymatic properties,crystallization, and deduced amino acid sequence of an alkaline endoglucanase from Bacillus circulans

A high-isoelectric-point (pI), alkaline endo-1,4-β-glucanase (Egl-257) of Bacillus circulans KSM-N257 was purified to homogeneity and crystallized. The purified enzyme hydrolyzed carboxymethyl cellulose (CMC) with optima of pH 8.5 and 55 °C. The molecular mass was 43 kDa, and the pI was pH 9.3. The structural gene contained a single open reading frame of 1221 bp, corresponding to 407 amino acids (aa), including a 30-aa signal peptide (377 aa and 41,680 Da for the mature enzyme). Egl-257 hydrolyzed lichenan and showed 76.3% aa identity to a lichenase from B. circulans WL-12 belonging to glycosyl hydrolase family 8 but did not hydrolyze laminarin, curdran, and xylan at all. This indicates that Egl-257 is a true endo-1,4-β-glucanase. However, this enzyme was not active on p-nitrophenyl β-d-cellotrioside and p-nitrophenyl β-d-cellotetraoside. It was crystallized by the hanging-drop vapor-diffusion method with phosphate plus CdCl₂ as precipitant. Pyramid-like crystals were formed, and they diffracted X-rays beyond 2.2 Å resolution. It belongs to the space group P2₁2₁2₁ with unit cell parameters of a=62.5 Å, b=71.7 Å, and c=88.6 Å.     

Distribution and Properties of Endo-β-1,4-glucanase from a Lower Termite,Coptotermes formosanus (Shiraki)

A multi-enzyme distribution of endo-β-1,4-glucanase activity was found in the digestive system of a worker caste of the lower termite Coptotermes formosanus (Shiraki) by zymogram analysis. Its distribution analysis demonstrated that about 80% of this activity was localized in salivary glands from where only one component (EG-E) was secreted into the digestive tract.

EG-E was isolated by a combination of chromatographic and electrophoretic techniques. Its molecular mass, optimal pH and temperature, isoelectric point, and K _m were 48 kDa, 6.0, 50°C, 4.2, and 3.8 (mg/ml on carboxymethylcellulose), respectively. EG-E hydrolyzed cellooligosaccharides with a degree of polymerization of 4 and larger, and had low activity on crystalline cellulose. Main reaction products from low molecular weight cellulose were cellobiose and cellotriose. The N-terminal amino acid sequence of EG-E has similarity with fungal endo-β-1,4-glucanases and cellobiohydrolases of the glycosyl hydrolase family 7 rather than the other insect endo-β-1,4-glucanases of family 9.     

Symbiotic adaptation of bacteria in the gut of Reticulitermes speratus: Low endo-β-1,4-glucanase activity

The termite is a good model of symbiosis between microbes and hosts and possesses an effective cellulose digestive system. Oxygen-tolerant bacteria, such as Dyella sp., Chryseobacterium sp., and Bacillus sp., were isolated from Reticulitermes speratus gut. Notably, the endo-β-1,4-glucanase (EG) activity of all 16 strains of isolated bacteria was low. Due to the combined activity of EG from the termites and their symbiotic protozoa, the bacteria might not be compelled to express EG. This observation demonstrates how well intestinal bacteria have assimilated themselves into the efficient cellulose digestive systems of termites.     

特异腐质霉内切葡聚糖酶Ⅱ基因在毕赤酵母中的表达及酶学性质

【目的】在毕赤酵母中表达特异腐质霉Humicola insolens的中性内切葡聚糖酶Ⅱ,并对其性质加以研究。【方法】利用RT-PCR的方法,以特异腐质霉(Humicola insolens)NC3总RNA为模板,克隆到中性内切葡聚糖酶Ⅱ基因(egⅡ)的cDNA。将其插入表达载体pPIC9K,重组质粒经线性化后电击转化毕赤酵母(Pichia pastoris)菌株GS115。【结果】SDS-PAGE和酶活的检测结果均表明:egⅡ基因在毕赤酵母中成功表达。重组酶的部分酶学性质研究表明,该酶的最适反应温度为70°C,且在65°C以下具有较好的热稳定性。最适反应pH为6.5,在pH 6.0?7.0之间有较好的稳定性。【结论】用重组毕赤酵母可高效表达外源中性内切葡聚糖酶,为其今后在工业应用奠定了基础。     

Molecular cloning and expression of a Bacillus subtilis β-glucanase gene in Escherichia coli

A Bacillus subtilis gene coding for an endo-β-1,3-1,4-glucanase has been transferred to Escherichia coli by molecular cloning using bacteriophage λ and plasmid vectors. The gene is contained within a 1.6-kb EcoRI-PvuI DNA fragment and directs the synthesis in E. coli of a β-glucanase which specifically degrades barley glucan and lichenan. A novel dye-staining method has been developed to detect β-glucanase activity in colonies on agar plates.     

Co-expression of two fibrolytic enzyme genes in CHO cells and transgenic mice

Cellulose is the main non-starch polysaccharides (NSP) in plant cell walls and acts as anti-nutritional factor in animal feed. However, monogastric animals do not synthesize enzymes that cleave such plant structural polysaccharides and thus waste of resources and pollute the environment. We described the vectors construction and co-expressions of a multi-functional cellulase EGX (with the activities of exo-β-1,4-glucanase, endo-β-1,4-glucanase, and endo-β-1,4-xylanase activities) from mollusca, Ampullaria crossean and a β-glucosidase BGL1 from Asperjillus niger in CHO cells and the transgenic mice. The recombinant enzymes were synthesised, secreted by the direction of pig PSP signal peptide and functionally active in the eukaryote systems including both of CHO cells and transgenic mice by RT-PCR analysis, western blot analysis and cellulolytic enzymes activities assays. Expressions were salivary glands-specific dependent under the control of pig PSP promoter in transgenic mice. 2A peptide was used as the self-cleaving sequence to mediate co-expression of the fusion genes and the cleavage efficiency was very high both in vitro and in vivo according to the western blot analysis. In summary, we have demonstrated that the single ORF containing EGX and BGL1 were co-expressed by 2A peptide in CHO cells and transgenic mice. It presents a viable technology for efficient disruption of plant cell wall and liberation of nutrients. To our knowledge, this is the first report using 2A sequence to produce multiple cellulases in mammalian cells and transgenic animals.     

Expression of multi-functional cellulase gene mfc in Coprinus cinereus under control of different basidiomycete promoters

Multi-functional cellulase gene mfc was expressed in Coprinus cinereus under naturally non-inductive conditions using three heterologous promoters. Endo-β-1,4-glucanase expression was achieved in solid and liquid media with promoter sequences from the Lentinula edodesgpd gene, the Flammulina velutipes gpd gene and the Volvariella volvaceagpd gene. As measured by enzyme activity in liquid cultures, a 613-bp gpd promoter fragment from L. edodes was most efficient, followed by a 752-bp gpd fragment from F. velutipes. The V. volvacea gpd promoter sequence was less active, in comparison. Irrespective of the promoter used, enzymatic activities increase 34-fold for highly active transformants and 29-fold for less active one by using cellulase-inducing medium. The highest activities of endo-β-1,4-glucanase (34.234 U/ml) and endo-β-1,4-xylanase (263.695 U/ml) were reached by using the L. edodesgpd promoter.     

Cellulolytic Activity of Three Fusarium culmorum (W. G. Sm.) Sacc. Isolates Pathogenic towards Wheat Seedlings

Fusarium culmorum isolates pathogenic to wheat (severely, medium and weakly pathogenic) proved to be producers of endo-1,4-β-glucanase and exo-1,4-β-glucanase. The activity of the latter enzyme was low. There was no correlation between the activities in vitro of the two enzymes and pathogenicity of the isolates.     

白蚁内切-β—1，4-葡聚糖酶基因的克隆、表达、纯化及酶学性质的研究

提取了台湾家白蚁总RNA并反转录获得eDNA,PCR扩增出白蚁内切葡聚糖酶的基因,并将目的基因分别克隆到大肠杆菌和酿酒酵母载体中,构建了产内切-β-1,4-葡聚糖酶的基因工程菌。由于大肠杆菌会有少量的泄漏表达,而所用的酿酒酵母表达载体是本实验室构建带有INU信号肽的表达载体,故都可采用刚果红平板染色法筛选具有羧甲基纤维素酶（CMCase）活性的重组转化子。利用金属镍亲和层析对大肠杆菌表达的内切-β-1,4-葡聚糖酶进行纯化,CMC酶活检测显示纯化酶的最适温度和最适pH值分别为42℃、6．5;内切-β-1,4-葡聚糖酶的Vmax为0．071mg／mL·min,Km值为80．2712mg／mL。     

Cellulose digestion in termites and cockroaches: What role do symbionts play?

• 1.1. Termites and cockroaches are excellent models for studying the role of symbionts in cellulose digestion in insects: they eat cellulose in a variety of forms and may or may not have symbionts.
• 2.2. The wood-eating cockroach, Panesthia cribrata, can be maintained indefinitely, free of microorganisms, on a diet of crystalline cellulose. Under these conditions the RQ is 1, indicating that the cockroach is surviving on glucose produced by endogenous cellulase.
• 3.3. The in vitro rate at which glucose is produced from crystalline cellulose by gut extracts from P. cribrata and Nasutitermes walkeri is comparable to the in vivo production of CO₂ in these insects, clearly indicating that the rate of glucose production from crystalline cellulose is sufficient for their needs.
• 4.4. In all termites and cockroaches examined, cellulase activity was found in the salivary glands and predominantly in the foregut and midgut. These regions are the normal sites of secretion of digestive enzymes and are either devoid of microorganisms (salivary glands) or have very low numbers.
• 5.5. Endogeneous cellulases from termites and cockroaches consist of multiple endo-β-1,4-glucanase (EC 3.2.1.4) and β-1,4-glucosidase (EC 3.2.1.21) components. There is no evidence that an exo-β-1,4-glucanase (cellobiohydrolase) (EC 3.2.1.91) is involved in, or needed for, the production of glucose from crystalline cellulose in termites or cockroaches as the endo-β-1,4-glucanase components are active against both crystalline cellulose and carboxymethylcellulose.
• 6.6. There is no evidence that bacteria are involved in cellulose digestion in termites and cockroaches. The cellulase associated with the fungus garden of M. michaelseni is distinct from that in the midgut; there is little indication that the fungal enzymes are acquired or needed. Lower termites such as Coptotermes lacteus have Protozoa in their hindgut which produce a cellulase(s) quite distinct from that in the foregut and midgut.

     

Functional morphology of the gastric mills of carnivorous,omnivorous, and herbivorous land crabs

Terrestrial decapods consume a wide variety of plant and animal material. The potential adaptations of carnivorous, omnivorous, and herbivorous terrestrial crustaceans were studied by examining the functional morphology of the gastric mill. Two closely related species from each feeding preference group were examined to identify which features of the mill were due to phylogeny and which were due to adaptation. The morphology of the gastric mill matched the diet well; the gastric mills of the carnivorous species (Geograpsus grayi and Geograpsus crinipes) possessed a blunt, rounded medial tooth and flattened lateral teeth with a longitudinal grinding groove. These features make them well suited to a carnivorous diet of soft animal tissue as well as hard material, such as arthropod exoskeleton. In contrast, the mill of the herbivorous gecarcinids (Gecarcoidea natalis and Discoplax hirtipes) consisted of a medial tooth with sharp transverse ridges and lateral teeth with sharp interlocking cusps and ridges and no grinding surface. These features would efficiently shred fibrous plant material. The morphology of the mill of the omnivorous coenobitids (Coenobita perlatus and Birgus latro) was more generalized toward a mixed diet. However, the mill of B. latro was more adapted to deal with highly nutritious food items, such as nuts and heavily calcified decapods. Its mill possessed lateral teeth with extended ridges, which sat close to the calcified cardiopyloric valve to form a flattened floor. Hard items trapped in the mill would be crushed against this surface by the medial tooth. J. Morphol. 2010. © 2009 Wiley‐Liss, Inc.     

A membrane-associated creatine kinase (EC 2.7.3.2) identified as an acidic species of the non-receptor,peripheral v-proteins in Torpedo acetylcholine receptor membranes

Human liver mRNA isolated from subjects phenotyped as homozygous PiMM or PiZZ α₁-antitrypsin, was translated in a reticulocyte cell-free system, and α₁-antitrypsin identified by immunoprecipitation. In the presence of dog pancreas membranes the translated α₁-antitrypsin appeared as a larger product. Treatment with endo-β-N-glucosaminidase yielded a protein smaller than the reticulocyte translated product, presumably due to removal of the N-terminal signal sequence by membranes and sugar residues by endo-β-N-glucosaminidase. Quantitation of α₁-antitrypsin translated from PiMM and PiZZ livers suggests that both mRNA species were present at the same cellular concentration, and that processing to the core glycosylation stage proceeded at identical rates.     

Cellulase formation by Aspergillus nidulans

The optimum pH, temperature and concentration of the substrate, carboxymethyl-cellulose (CMC), for the production of cellulases by Aspergillus nidulans were found to be 3.05, 37°C and 1%, respectively. When grown on CMC under optimum conditions, it produced the three components of the cellulase complex, exo-β-1,4-glucanase, endo-β-1,4-glucanase and β-1,4-glucosidase, both in cell free as well as cell-associated states. The enzyme yields in shake cultures were lower than those obtained during stationary cultivation. Among the defined substrates, lactose emerged as the best inducer for exo-glucanase and endo-glucanase, while β-glucosidase was best induced by pectin. Endo-glucanase production increased significantly when A. nidulans was grown on insoluble delignified lognocellulosic substrates, with the maximum being on paddy straw.It appears that the synthesis of individual components of the cellulase system of A. nidulans may not be regulated in a strictly coordinated manner.     

A phylogenetic study of endo-beta-1,4-glucanase in higher termites

Cellulose is the most abundant polymer in the world and termites are the most important metazoan cellulose processors. Termites are divided into lower and higher termites, with the latter being the most derived and most specious. Although termites are known for their ability to digest wood, members of the family Termitidae (higher termites) are nutritionally diverse in their use of cellulose. This study investigated the evolution of endogenous cellulases in 25 species of higher termites, using phylogenetic inferences from mitochondrial (16S) and nuclear (28S) ribosomal RNA and endo-β-1,4-glucanase sequences. The translated endo-β-1,4-glucanase amino acid order in all 41 sequences obtained showed high similarity to endo-β-1,4-glucanases in the glycosyl hydrolase family 9. The inferred endo-β-1,4-glucanase phylogenetic tree showed congruency with the mitochondrial/nuclear tree, with the fungus-growers being the most basal group and the soil/litter- and wood/lichen/grass/litter-feeders being the most distal diphyletic feeding groups. The bacterial comb-grower formed a separate clade from the fungus-growers and is sister groups with the soil/litter- and wood/lichen/grass/litter-feeders. There was also a strong diphyletic relationship between endo-β-1,4-glucanases of upper layer soil-feeders and the other soil-feeders. Within the monophyletic wood/lichen/grass/litter-feeding termites’ subclade, the nasutitermitines were polyphyletic and a strong diphyletic relationship was observed in the most distal lichen- and the grass/litter-feeders groups.     

Family 28 carbohydrate-binding module of the thermostable endo-1,4-β-glucanase CelD from <Emphasis Type="Italic">Caldicellulosiruptor bescii</Emphasis> maximizes enzyme activity and irreversibly binds to amorphous cellulose

The nucleotide sequence of a chromosome fragment of the thermophilic anaerobic bacterium Caldicellulosiruptor bescii (syn. Anaerocellum thermophilum) has been determined. The fragment contains four open reading frames with the second encoding a 749 aa multimodular endo-1,4-β-glucanase CelD (85019 Da). The N-terminal region of the protein includes a signal peptide and a catalytic module of glycoside hydrolase family 5 (GH5), followed by a carbohydrate-binding module of family 28 (CBM28). The C-terminal region bears three SLH modules. The recombinant endoglucanase and its two separate modules, the catalytic module and CBM28, were produced in E. coli cells and purified to homogeneity. An analysis of the catalytic properties showed CelD to be an endo-1,4-β-glucanase with maximum activity on barley β-glucan at pH 6.2 and 70°C. The enzyme was stable at 50°C for 30 days. Upon removal of the C-terminal CBM28, the activity of GH5 was decreased on cellulose substrates, and its thermostability has dropped. Binding of CBM28 to amorphous cellulose has been almost irreversible as it could not be removed from this substrate in a range of pH of 4–11, temperatures of 0–75°C, and NaCl concentrations of 0–5 M. Only 100% formamide or 1% SDS have been able to remove the protein.     

High expression of a neutral endo-β-glucanase gene from Humicola insolens in Trichoderma reesei

The neutral endo-β-glucanase gene cel5A from Humicola insolens was cloned and connected with the cellobiohydrolase 1 promoter from Trichoderma reesei to construct a recombinant plasmid pCB-hEG with the hygromycin B resistance marker. The plasmid was introduced into conidia of T. reesei using the Agrobacterium tumefaciens mediated transformation method. Eight transformants were obtained on screening plates with sodium carboxymethyl cellulose as the sole carbon source. Stable integration of the cel5A gene into the chromosomal DNA of T. reesei was confirmed by PCR. An obvious protein band (approximately 52 kDa) was detected by SDS-PAGE from fermentation broth, which showed that the cel5A gene in recombinant T. reesei successfully fulfilled efficient expression and extracellular secretion. After 96 h shaking-flask fermentation, the endo-β-glucanase activity at pH 6.5 from recombinant T. reesei reached 3,068 U/ml, which was 11 times higher than that of the host strain. In a 2 m³ fermenter, the endo-β-glucanase activity could be further increased to 8,012 U/ml after 96 h fermentation. The results showed a good prospect for application of neutral endo-β-glucanase in the textile industry.