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。     

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.     

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。     

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.     

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.     

cDNA cloning,expression, and enzymatic activity of a novel endogenous cellulase from the beetle Batocera horsfieldi

In this study, we report a novel cellulase [β-1,4-endoglucanase (EGase), EC 3.2.1.4] cDNA (Bh-EGase II) belonging to the glycoside hydrolase family (GHF) 45 from the beetle Batocera horsfieldi. The Bh-EGase II gene spans 720 bp and consists of a single exon coding for 239 amino acid residues. Bh-EGase II showed 93.72% protein sequence identity to Ag-EGase II from the beetle Apriona germari. The GHF 45 catalytic site is conserved in Bh-EGase II. Bh-EGase II has three putative N-glycosylation sites at 56–58 (N–K–S), 99–101 (N–S–T), and 237–239 (N–Y–S), respectively. The cDNA encoding Bh-EGase II was expressed in baculovirus-infected insect BmN cells and Bombyx mori larvae. Recombinant Bh-EGase II from BmN cells and larval hemolymph had an enzymatic activity of approximately 928 U/mg. The enzymatic catalysis of recombinant Bh-EGase II showed the highest activity at 50 °C and pH 6.0.     

Hydrolysis of filter-paper cellulose to glucose by two recombinant endogenous glycosyl hydrolases of Coptotermes formosanus

Abstract Genes encoding for glycosyl hydrolases (GH) in multiple families were recovered from an expression sequence tag library of Coptotermes formosanus, a xylophagous lower termite species. Functional analyses of these genes not only shed light on the mechanisms the insect employs to successfully use cellulosic materials as energy sources, which may serve as strategic targets for designing molecular-based bio-pesticides, but also enrich discoveries of new cellulolytic enzymes for conversion of biomass into biofuel. Our study demonstrated that cellulose could be converted to glucose by two recombinant endogenous glycosyl hydrolases (endo-β-1,4 glucanase in GH9 and β-glucosidase in GH1). While the former cleaved cellulose to cellobiose and cellotriose, the resulting simple cellodextrins were digested to glucose. Both of the Escherichia coli-expressed recombinant proteins showed properties that could be incorporated in a glucose-based ethanol production program.     

Molecular cloning and functional characterization of an endogenous endoglucanase belonging to GHF45 from the western corn rootworm, Diabrotica virgifera virgifera

A novel insect β-1,4-endoglucanase (DvvENGaseI) gene belonging to the glycoside hydrolase family (GHF) 45 was identified from the western corn rootworm, Diabrotica virgifera virgifera. The cDNA of the DvvENGaseI consisted of a 720 bp open reading frame encoding a 239 amino-acid protein. Analysis of the amino acid sequence revealed that DvvENGaseI exhibits 60% protein sequence identity when compared with an endoglucanase belonging to GHF45 from another beetle, Leptinotarsa decemlineata. Western blot analyses using a polyclonal antiserum developed from a partial peptide sequence revealed that DvvENGaseI expression coincided with body regions corresponding to the fore-, mid- and hindgut, although regions corresponding to the midgut and hindgut were the primary sites for DvvENGaseI expression. Functional analysis of the DvvENGaseI by RNA interference (RNAi) indicated that nearly complete knock-down of gene expression could be obtained by injection of dsRNA based on qRT-PCR and western blot analysis. However, suppression only resulted in slight developmental delays suggesting that this gene may be part of a larger system of cellulose degrading enzymes.     

内生枯草芽孢杆菌SWB8 菌株β-1，3-1，4-葡聚糖酶的抗菌作用及细胞毒性

【目的】本文研究从药用植物黄姜中分离的内生枯草芽孢杆菌菌株SWB8分泌的β-1,3-1,4-葡聚糖酶的抗菌活性和细胞毒性。【方法】利用液体发酵、凝胶渗透色谱(GPC)、十二烷基-聚丙烯酰胺凝胶电泳(SDS-PAGE)和液相层析串联质谱(LC-MS/MS)等方法纯化和鉴定枯草芽孢杆菌株SWB8合成的β-1,3-1,4-葡聚糖酶;利用纸片扩散法,检测葡聚糖酶抑制临床致病性细菌和真菌生长的活性;应用MTT法和流式细胞术(FCM)评估此葡聚糖酶对人肺腺癌细胞(A549)和骨髓间质干细胞(MSCs)的细胞毒性。【结果】细菌性β-1,3-1,4-葡聚糖酶显示了广谱的抗菌活性;抗肿瘤活性主要以细胞凋亡的方式选择性的抑制人肺腺癌细胞系A549细胞的增殖,而对人骨髓间质干细胞系MSC细胞无明显影响。【结论】首次报道β-1,3-1,4-葡聚糖酶的抗菌和抗肿瘤细胞的活性。内生枯草芽孢杆菌SWB8菌株有可能成为抗菌和高效低毒的抗肿瘤药物的潜在来源。     

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.     

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.     

Structural and mutagenetic analyses of a 1,3–1,4-β-glucanase from Paecilomyces thermophila

The thermostable 1,3–1,4-β-glucanase PtLic16A from the fungus Paecilomyces thermophila catalyzes stringent hydrolysis of barley β-glucan and lichenan with an outstanding efficiency and has great potential for broad industrial applications. Here, we report the crystal structures of PtLic16A and an inactive mutant E113A in ligand-free form and in complex with the ligands cellobiose, cellotetraose and glucotriose at 1.80 Å to 2.25 Å resolution. PtLic16A adopts a typical β-jellyroll fold with a curved surface and the concave face forms an extended ligand binding cleft. These structures suggest that PtLic16A might carry out the hydrolysis via retaining mechanism with E113 and E118 serving as the nucleophile and general acid/base, respectively. Interestingly, in the structure of E113A/1,3–1,4-β-glucotriose complex, the sugar bound to the − 1 subsite adopts an intermediate-like (α-anomeric) configuration. By combining all crystal structures solved here, a comprehensive binding mode for a substrate is proposed. These findings not only help understand the 1,3–1,4-β-glucanase catalytic mechanism but also provide a basis for further enzymatic engineering.     

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.     

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.     

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.     

A cryoprotective and cold-adapted 1,3-β-endoglucanase from cherimoya (Annona cherimola) fruit

A 1,3-β-glucanase with potent cryoprotective activity was purified to homogeneity from the mesocarp of CO₂-treated cherimoya fruit (Annona cherimola Mill.) stored at low temperature using anion exchange and chromatofocusing chromatography. This protein was characterized as a glycosylated endo-1,3-β-glucanase with a M_r of 22.07 kDa and a pI of 5.25. The hydrolase was active and stable in a broad acidic pH range and it exhibited maximum activity at pH 5.0. It had a low optimum temperature of 35 °C and it retained 40% maximum activity at 5 °C. The purified 1,3-β-glucanase was relatively heat unstable and its activity declined progressively at temperatures above 50 °C. Kinetic studies revealed low k_cat (3.10 ± 0.04 s⁻¹) and K_m (0.32 ± 0.03 mg ml⁻¹) values, reflecting the intermediate efficiency of the protein in hydrolyzing laminarin. Moreover, a thermodynamic characterization revealed that the purified enzyme displayed a high k_cat at both 37 and 5 °C, and a low E_a (6.99 kJ mol⁻¹) within this range of temperatures. In vitro functional studies indicated that the purified 1,3-β-glucanase had no inhibitory effects on Botrytis cinerea hyphal growth and no antifreeze activity, as determined by thermal hysteresis analysis using differential scanning calorimetry. However, a strong cryoprotective activity was observed against freeze-thaw inactivation of lactate dehydrogenase. Indeed, the PD₅₀ was 8.7 μg ml⁻¹ (394 nM), 9.2-fold higher (3.1 on a molar basis) than that of the cryoprotective protein BSA. Together with the observed accumulation of glycine-betaine in CO₂-treated cherimoya tissues, these results suggest that 1,3-β-glucanase could be functionally implicated in low temperature-defense mechanism activated by CO₂.     

Digestive enzymes of two brachyuran and two anomuran land crabs from Christmas Island,Indian Ocean

The digestive ability of four sympatric land crabs species (the gecarcinids, Gecarcoidea natalis and Discoplax celeste and the anomurans, Birgus latro and Coenobita perlatus) was examined by determining the activity of their digestive enzymes. The gecarcinids are detritivores that consume mainly leaf litter; the robber crab, B. latro, is an omnivore that preferentially consumes items high in lipid, carbohydrate and/or protein; C. perlatus is also an omnivore/detritivore. All species possess protease, lipase and amylase activity for hydrolysing ubiquitous protein, lipid and storage polysaccharides (glycogen and starch). Similarly all species possess enzymes such as N-acetyl-β-d-glucosaminidase, the cellulases, endo-β-1,4-glucanase and β-glucohydrolase and hemicellulases, lichenase and laminarinase for the respective hydrolysis of structural substrates chitin, cellulose and hemicelluloses, lichenan and laminarin. Except for the enzyme activities of C. perlatus, enzyme activity could not be correlated to dietary preference. Perhaps others factors such as olfactory and locomotor ability and metabolic status may determine the observed dietary preferences. The digestive fluid of C. perlatus possessed higher endo-β-1,4-glucanase, lichenase and laminarinase activities compared to that of the other species. Thus, C. perlatus may be efficient at digestion of cellulose and hemicellulose within plant material. Zymography indicated that the majority of protease, lipase, phosphatase, amylase, endo-β-1,4-glucanase, β-glucohydrolase and N-acetyl-β-d-glucosaminidase isozymes were common to all species, and hence were inherited from a common aquatic ancestor. Differences were observed for the phosphatase, lipase and endo-β-1,4-glucanase isozymes. These differences are discussed in relation to phylogeny and possible evolution to cope with the adoption of a terrestrial diet.     

Expression of an endoglucanase from Tribolium castaneum (TcEG1) in Saccharomyces cerevisiae

Insects are a largely unexploited resource in prospecting for novel cellulolytic enzymes to improve the production of ethanol fuel from lignocellulosic biomass. The cost of lignocellulosic ethanol production is expected to decrease by the combination of cellulose degradation (saccharification) and fermentation of the resulting glucose to ethanol in a single process, catalyzed by the yeast Saccharomyces cerevisiae transformed to express efficient cellulases. While S. cerevisiae is an established heterologous expression system, there are no available data on the functional expression of insect cellulolytic enzymes for this species. To address this knowledge gap, S. cerevisiae was transformed to express the full‐length cDNA encoding an endoglucanase from the red flour beetle, Tribolium castaneum (TcEG1), and evaluated the activity of the transgenic product (rTcEG1). Expression of the TcEG1 cDNA in S. cerevisiae was under control of the strong glyceraldehyde‐3 phosphate dehydrogenase promoter. Cultured transformed yeast secreted rTcEG1 protein as a functional β‐1,4‐endoglucanase, which allowed transformants to survive on selective media containing cellulose as the only available carbon source. Evaluation of substrate specificity for secreted rTcEG1 demonstrated endoglucanase activity, although some activity was also detected against complex cellulose substrates. Potentially relevant to uses in biofuel production rTcEG1 activity increased with pH conditions, with the highest activity detected at pH 12. Our results demonstrate the potential for functional production of an insect cellulase in S. cerevisiae and confirm the stability of rTcEG1 activity in strong alkaline environments.     

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.     

Molecular and biochemical characterizations of a novel arthropod endo-β-1,3-glucanase from the Antarctic springtail,Cryptopygus antarcticus,horizontally acquired from bacteria

Collembolan species have been known to have β-1,3-glucanase activity and yet the genes coding such enzymes have not been demonstrated. We report here a novel arthropod endo-β-1,3-glucanase gene CaLam from the Antarctic springtail, Cryptopygus antarcticus. The open reading frame consists of 813 bp encoding 270 amino acids with a putative signal peptide and a typical motif of glycosyl hydrolase family 16 (GHF16), E–I–D–I–T–E. The recombinant protein expressed in E. coli shows the hydrolytic activity toward laminarin (K_m ～ 9.98 mg/mL) with an optimal temperature 50 °C and an optimal pH 6.0. CaLam digests laminarin and laminarioligosaccharides except laminaribiose as an endo-β-1,3-glucanase, releasing glucose, laminaribiose and laminaritriose as the major products. Analyses of molecular phylogeny of CaLam and its protein structure reveal that CaLam is closely related with bacterial β-1,3-glucanases more than with the eukaryotic homologues. Even so, the genomic structure of the CaLam gene consisting of six exons interspersed with approximately 57 to 63 bp introns confirms that it is endogenous in the genome of the Antarctic springtail. These results suggest that CaLam should have been transferred from bacteria to the lineage of the Collembolan species by horizontal gene transfer.