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.     

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.     

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.     

A Homologue of EGL1 Encoding Endo-1,4-β-glucanase in Elongating Pea Stems

A gene (EGL2) encoding an endo-1,4-β-glucanase in peas has been cloned as a homologue of EGL1. EGL2 encodes a polypeptide of 506 amino acids, including a 24-mer putative signal polypeptide. The gene product contains a domain conserved in endo-1,4-β-glucanase (family 9) showing 60% amino acid identity to EGL1. EGL2 mRNA was accumulated only in the elongating regions of pea stems, although EGL1 mRNA was abundant in both elongating and non-elongating tissues. However, the level of EGL2 mRNA was not increased by the treatment with sucrose and auxin in pea segments. These results suggest that the expression of EGL2 either requires the presence of other factors related to the auxin effect or occurs independent of auxin in the elongating pea stems.     

A thermotolerant and cold-active mannan endo-1,4-β-mannosidase from Aspergillus niger CBS 513.88: Constitutive overexpression and high-density fermentation in Pichia pastoris

The mannan endo-1,4-β-mannosidase gene man26A from Aspergillus niger CBS 513.88 was optimized according to the codon usage bias in Pichia pastoris and synthesized by splicing overlap extension PCR. It was successfully expressed in P. pastoris using constitutive expression vector pGAPzαA. The recombinant endo-beta-1,4-mannanase could work in an extremely board temperature range and over 30% relative activity were retained in the temperature range of 5-60 °C. The optimal pH value and temperature for activity were 5.0 and 45 °C, respectively. It was highly thermotolerant with a half-life time of 15 min at 90 °C. A novel fed-batch strategy was developed successfully for high cell-density fermentation and mannanase activity reached 5069 U/mL after cultivation for 56 h in 50 L fermenter. The broad working temperature range, high thermotolerance and efficient expression made this enzyme possible to be applied in food, animal feed and the production of biofuels.     

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。     

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.     

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。     

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.     

Cloning and Expression of an Endo-1,6-β-D-glucanase Gene (neg1) from Neurospora crassa

A gene (neg1) encoding an endo-1,6-β-D-glucanase from Neurospora crassa was cloned. The putative neg1 was 1443-bp long and encoded a mature endo-1,6-β-D-glucanase protein of 463 amino acids and signal peptide of 17 amino acids. The purified recombinant protein (Neg1) obtained from Escherichia coli showed 1,6-β-D-glucanase activity. No genes similar in sequence were found in yeasts and fungi.     

Expression of laccase gene lcc1 in Coprinopsis cinerea under control of various basidiomycetous promoters

Coprinopsis cinerea laccase gene lcc1 was expressed in this basidiomycete under naturally non-inductive conditions using various homologous and heterologous promoters. Laccase expression was achieved in solid and liquid media with promoter sequences from the C. cinerea tub1 gene, the Agaricus bisporus gpdII gene, the Lentinus edodes priA gene and the Schizophyllum commune Sc3 gene. As measured by enzyme activity in liquid cultures, a 277-bp gpdII promoter fragment, followed by a 423-bp priA fragment, was most efficient. A shorter priA sequence of 372 bp was inactive. tub1 promoter fragments were reasonably active, whereas the S. commune Sc3 promoter sequence was less active, in comparison. Irrespective of the promoter used, addition of copper to the medium increased enzymatic activities for highly active transformants by 10- to 50-fold and for less active transformants for 2- to 7-fold. The highest enzymatic activities (3 U/ml) were reached with the gpdII promoter in the presence of 0.1 mM CuSO₄.     

High-level production and characterization of a novel β-1,3-1,4-glucanase from Aspergillus awamori and its potential application in the brewing industry

A novel β-1,3-1,4-glucanase gene (AaBglu12A) from Aspergillus awamori was extracellularly expressed in Pichia pastoris. AaBglu12A showed amino acid identity of 96 % with a glycoside hydrolase family 12 cellulase from A. kawachii and 48 % with a β-1,3-1,4-glucanase from Magnaporthe oryzae. The highest β-1,3-1,4-glucanase activity of 159,500 ± 500 U/mL with protein concentration of 31.7 ± 0.3 g/L was achieved in a 5-L fermentor. AaBglu12A was purified until homogeneous with recovery yield of 92 %. Its maximal activity was found at 55 °C and pH 5.0. The enzyme was stable up to 60 °C and within the pH range of 2.0-9.0. It also demonstrated strict substrate specificity towards oat- and barley-glucans as well as lichenan. The K_m values for oat-, barley-glucans, and lichenan were 2.82, 3.51, and 2.53 mg/mL, respectively. The V_max values for oat-, barley-glucans, and lichenan were 12,068, 10,790, and 7236 μmol/min·mg, respectively. AaBglu12A hydrolyzed oat- and barley-β-glucans to produce tetra- and tri-saccharides. However, lichenan was hydrolyzed to yield trisaccharides as the main end product. The addition of AaBglu12A to the mashing process substantially decreased filtration time by 34.5 % and viscosity by 9.6 %. Therefore, the high-level production of AaBglu12A might be a promising strategy for the brewing industry owing to its favorable properties.     

Hydrolysis of lignocellulosic feedstock by novel cellulases originating from Pseudomonas sp. CL3 for fermentative hydrogen production

A newly isolated indigenous bacterium Pseudomonas sp. CL3 was able to produce novel cellulases consisting of endo-β-1,4-d-glucanase (80 and 100 kDa), exo-β-1,4-d-glucanase (55 kDa) and β-1,4-d-glucosidase (65 kDa) characterized by enzyme assay and zymography analysis. In addition, the CL3 strain also produced xylanase with a molecular weight of 20 kDa. The optimal temperature for enzyme activity was 50, 45, 45 and 55 °C for endo-β-1,4-d-glucanase, exo-β-1,4-d-glucanase, β-1,4-d-glucosidase and xylanase, respectively. All the enzymes displayed optimal activity at pH 6.0. The cellulases/xylanase could hydrolyze cellulosic materials very effectively and were thus used to hydrolyze natural agricultural waste (i.e., bagasse) for clean energy (H₂) production by Clostridiumpasteurianum CH4 using separate hydrolysis and fermentation process. The maximum hydrogen production rate and cumulative hydrogen production were 35 ml/L/h and 1420 ml/L, respectively, with a hydrogen yield of around 0.96 mol H₂/mol glucose.     

Cloning and functional characterization of endo-β-1,4-glucanase gene from metagenomic library of vermicompost

In the vermicomposting of paper mill sludge, the activity of earthworms is very dependent on dietetic polysaccharides including cellulose as energy sources. Most of these polymers are degraded by the host microbiota and considered potentially important source for cellulolytic enzymes. In the present study, a metagenomic library was constructed from vermicompost (VC) prepared with paper mill sludge and dairy sludge (fresh sludge, FS) and functionally screened for cellulolytic activities. Eighteen cellulase expressing clones were isolated from about 89,000 fosmid clones libraries. A short fragment library was constructed from the most active positive clone (cMGL504) and one open reading frame (ORF) of 1,092 bp encoding an endo-β-1,4-glucanase was indentified which showed 88% similarity with Cellvibrio mixtus cellulase A gene. The endo-β-1,4-glucanase cmgl504 gene was overexpressed in Escherichia coli. The purified recombinant cmgl504 cellulase displayed activities at a broad range of temperature (25–55°C) and pH (5.5–8.5). The enzyme degraded carboxymethyl cellulose (CMC) with 15.4 U, while having low activity against avicel. No detectable activity was found for xylan and laminarin. The enzyme activity was stimulated by potassium chloride. The deduced protein and three-dimensional structure of metagenome-derived cellulase cmgl504 possessed all features, including general architecture, signature motifs, and N-terminal signal peptide, followed by the catalytic domain of cellulase belonging to glycosyl hydrolase family 5 (GHF5). The cellulases cloned in this work may play important roles in the degradation of celluloses in vermicomposting process and could be exploited for industrial application in future.     

Enzymatic hydrolysis of 1,3-1,4-beta-glucosyl oligosaccharides by 1,3-1,4-beta-glucanase from Synechocystis PCC6803: a comparison with assays using polymer and chromophoric oligosaccharide substrates

The specificity of 1,3-1,4-β-glucanase from Synechocystis PCC6803 (SsGlc) was investigated using novel substrates 1,3-1,4-β-glucosyl oligosaccharides, in which 1,3- and 1,4-linkages are located in various arrangements. After the enzymatic reaction, the reaction products were separated and determined by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). As a result, SsGlc was found to hydrolyze the pentasaccharides, which possess three contiguous 1,4-β-glycosidic linkages (cellotetraose sequence) adjacent to 1,3-β-linkage, but none of the other oligosaccharides were hydrolyzed. To further analyze the specificity, kinetic measurements were performed using polymeric substrates and 4-methylumbelliferyl derivatives of laminaribiose and cellobiose (1,3-β-(Glc)₂-MU and 1,4-β-(Glc)₂-MU). The k_cat/K_m value obtained for barley β-glucan was considerably larger than that for lichenan, indicating that SsGlc prefers 1,3-1,4-β-glucan possessing a larger amount of cellotetraose sequence. This is consistent with the data obtained for 1,3-1,4-β-glucosyl oligosaccharides. However, the k_cat/K_m value obtained for 1,4-β-(Glc)₂-MU was considerably lower than that for 1,3-β-(Glc)₂-MU, suggesting inconsistency with the data obtained from the other natural substrates. It is likely that the kinetic data obtained from such chromophoric substrates do not always reflect the true enzymatic properties.     

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.     

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.     

Structural and catalytic roles of residues located in β13 strand and the following β-turn loop in Fibrobacter succinogenes 1,3-1,4-β-d-glucanase

Background

Fibrobacter succinogenes 1,3-1,4-β-d-glucanase (Fsβ-glucanase) is the only naturally occurring circularly permuted β-glucanase among bacterial glucanases with reverse protein domains. We characterized the functional and structural significance of residues 200–209 located in the domain B of Fsβ-glucanase, corresponding to the major surface loop in the domain A region of Bacillus licheniformis glucanase.

Methods

Rational design approaches including site-directed mutagenesis, initial-rate kinetics, and structural modeling analysis were used in this study.

Results

Our kinetic data showed that D202N and D206N exhibited a 1.8- and 1.5-fold increase but G207N, G207−, F205L, N208G and T204F showed a 7.0- to 2.2-fold decrease, in catalytic efficiency (k_cat/K_M) compared to the wild-type enzyme. The comparative energy ΔΔG_b value in individual mutant enzymes was well correlated to their catalytic efficiency. D206R mutant enzyme exhibited the highest relative activity at 50 °C over 10 min, whereas K200F was the most heat-sensitive enzyme.

Conclusions

This study demonstrates that Phe205, Gly207, and Asn208 in the Type II turn of the connecting loop may play a role in the catalytic function of Fsβ-glucanase.

General significance

Residues 200–209 in Fsβ-glucanase resided at the similar structural topology to that of Bacillus enzyme were found to play some similar catalytic function in glucanase.     

NMR characterization of a 264-residue hyperthermostable endo-β-1,3-glucanase

Insight into the hyperthermostable endo-β-1,3-glucanase pfLamA from Pyrococcus furiosus is obtained by using NMR spectroscopy. pfLamA functions optimally at 104 °C and recently the X-ray structure of pfLamA has been obtained at 20 °C, a temperature at which the enzyme is inactive. In this study, near-complete (>99%) NMR assignments are presented of chemical shifts of pfLamA in presence and absence of calcium at 62 °C, a temperature at which the enzyme is biologically active. The protein contains calcium and the effects of calcium on the protein are assessed. Calcium binding results in relatively small chemical shift changes in a region distant from the active site of pfLamA and thus causes only minor conformational modifications. Removal of calcium does not significantly alter the denaturation temperature of pfLamA, implying that calcium does not stabilize the enzyme against global unfolding. The data obtained form the basis for elucidation of the molecular origins involved in conformational stability and biological activity of hyperthermophilic endo-β-1,3-glucanases at extreme temperatures.     

Cloning and Characterization of the Gene Encoding Endo-β-1,3-glucanase from Arthrobacter sp. NHB-10

The gluA gene, encoding an endo-β-1,3-glucanase from Arthrobacter sp. (strain NHB-10), was cloned and analyzed. The deduced endo-β-1,3-glucanase amino acid sequence was 750 amino acids long and contained a 42 amino acid signal peptide with a mature protein of 708 amino acids. There was no similarity to known endo-β-1,3-glucanases, but GluA was partially similar to two fungal exo-β-1,3-glucanases in glycoside hydrolase (GH) family 55. Of five possible residues for catalysis and two motifs in two β-helix heads of GH family 55, three residues and one motif were conserved in GluA, suggesting that GluA is the first bacterial endo-β-1,3-glucanase in GH family 55. Significant similarity was also found to two proteins of unknown function from Streptomyces coelicolor A3(2) and S. avermitilis.