Differences in cellulose digestive systems among castes in two termite lineages

Abstract.  Termites (Isoptera) are eusocial insects and express polyphenism. Soldiers have specialized morphology for colony defense, but their feeding activity is dependent on other colony members. To determine differences in cellulose degradation between soldier and worker termites, enzymatic activity and cellulase gene expression, as well digestive tract histology, are examined in two phylogenetically distant species. In Hodotermopsis sjostesti (family Termopsidae) , endo-β-1,4-glucanase activity is identified in the salivary glands, whereas β-glucosidase activity is identified in salivary glands and hindgut. The relative expression levels of endo-β-1,4-glucanase genes in soldiers are significantly lower than in workers. Thin sections of salivary gland of workers and soldiers are different in H. sjostedti . In Nasutitermes takasagoensis (family Termitidae), the endo-β-1,4-glucanase activity is restricted to the midgut in four tested castes (i.e. three types of workers and soldier). Examination of activity per termite reveals the highest activity in minor workers and the lowest activity in major workers and soldiers. The β-glucosidase activity is also concentrated on the midgut in all four castes. The relative expression level of the endo-β-1,4-glucanase gene does not correspond with its activity in the midgut. In thin sections prepared from N. takasagoensis , the folds and pulvillus in the gizzards, and cuticle structure of soldiers are less developed compared with the other three worker castes. The differences in digestive system among termite castes in terms of caste development in each species are discussed.     

Coleoptile growth-inducing capacities of exo-β-(1→3)-glucanases from fungi

An exo-β-(1β3)-glucanase derived from Selerotinia libertiana induced growth of Avena sativa coleoptiles and degraded hemicelluloses and β-(1→4):(1→3) mixed linked glucan. However, neither endo-β-(1→4)- nor endo-β-(1→3)-glucanase activity could be detected in the enzyme preparation. Nojirimycin inhibited the glucan degradation caused by the enzyme but glucono-1,5-lactone did not. Another exo-β-(1→3)-glucanase derived from Basidiomycete QM 806 did not induce coleoptile growth and did not degrade the glucan. Growth-inducing properties of exo-β-(1→3)-glucanases are discussed.     

Role of a xyloglucan-specific endo-beta-1,4-glucanase inhibitor in the interactions of Nicotiana benthamiana with Colletotrichum destructivum, C. orbiculare or Pseudomonas syringae pv. tabaci

A xyloglucan-specific endo-β-1,4-glucanase inhibitor cDNA, NbXEGIP1 , was amplified from diseased leaves of Nicotiana benthamiana . The sequence was similar to the tomato xyloglucan-specific endo-β-1,4-glucanase inhibitor (XEGIP) and tobacco nectarin IV genes that have been described as binding and inactivating fungal Family 12 xyloglucan-specific endo-β-1,4-glucanases. Expression of NbXEGIP1 was not detected in healthy leaves, but the gene was induced during the later stages of infection by the fungi Colletotrichum destructivum and C. orbiculare . Induction of NbXEGIP1 also occurred during disease development by the bacterium Pseudomonas syringae pv. tabaci and during the hypersensitive response produced by P. syringae pv. tabaci expressing avrPto . A portion of NbXEGIP1 was cloned into a tobacco rattle virus vector for virus-induced gene silencing in N. benthamiana . Silencing NbXEGIP1 did not affect the interactions with either Colletotrichum species but did significantly reduce population levels of P. syringae pv. tabaci in the compatible interaction and P. syringae pv. tabaci expressing avrPto in the incompatible interaction. In the susceptible response to P. syringae pv. tabaci , silencing of NbXEGIP1 also resulted in visibly wilted leaves several hours prior to necrosis, which was not observed in control plants. This was related to a significantly higher level of electrolyte leakage and higher expression of a defensin gene from infected NbXEGIP1 -silenced leaves compared with control leaves. Silencing appeared to be specific as it did not affect expression of a related gene, NbXEGIP2 . NbXEGIP1 may act as an inhibitor of a bacterial enzyme that degrades the xyloglucan–cellulose plant cell-wall network, and degradation of the cell wall results in host membrane disruption and signalling of defence responses.     

Codon optimization of <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> β-1,3-1,4-glucanase gene and its expression in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

β-1,3-1,4-glucanase (EC3.2.1.73) as an important industrial enzyme has been widely used in the brewing and animal feed additive industry. To improve expression efficiency of recombinant β-1,3-1,4-glucanase from Bacillus licheniformis EGW039(CGMCC 0635) in methylotrophic yeast Pichia pastoris GS115, the DNA sequence encoding β-1,3-1,4-glucanase was designed and synthesized based on the codon bias of P. pastoris, the codons encoding 96 amino acids were optimized, in which a total of 102 nucleotides were changed, the G+C ratio was simultaneously increased from 43.6 to 45.5%. At shaking flask level, β-1,3-1,4-glucanase activity is 67.9 and 52.3 U ml⁻¹ with barley β-glucan and lichenan as substrate, respectively. At laboratory fermentor level, the secreted protein concentration is approximately 250 mg l⁻¹. The β-1,3-1,4-glucanase activity is 333.7 and 256.7 U ml⁻¹ with barley β-glucan and lichenan as substrate, respectively; however, no activity of this enzyme on cellulose is observed. Compared to the nonoptimized control, expression level of the optimized β-1,3-1,4-glucanase based on preferred codons in P. pastoris shown a 10-fold higher level. The codon-optimized enzyme was approximately 53.8% of the total secreted protein. The optimal acidity and temperature of this recombinant enzyme were pH 6.0 and 45°C, respectively.     

Characterization of an endo-1,3(4)-β-d-glucanase gene from Cellvibrio mixtus

Abstract An endo-1,3(4)-β- d -glucanase gene ( cwd2 ) of Cellvibrio mixtus encoding laminarinase activity was cloned on a 3.9-kb Pst I fragment. The Cwd2 enzyme, extracted from recombinant Escherichia coli , degraded both β-1,3 glucans and β-1,3–1,4 mixed-linkage glucans, was entohydrolytic and so conformed to the enzyme class 3.2.1.6. The pH and temperature optima of the enzyme were approximately 7 and 40°C respectively. The M _r of specifically labelled Cwd2 was approximately 34 000. This gene was quite distinct from two other C. mixtus β-1,3 glucanases previously described.     

Subcloning and functional analysis of aBacillus subtilis β-1,3-1,4-glucanase gene (bglS) inEscherichia coli

This article describes a 7.1kb EcoRI DNA fragment carrying aBacillus subtilis -1,3-1,4-glucanase gene (bglS). By subcloning, a 1.5kb EcoRI-PstI DNA fragment was inserted into the polylinker cloning sites of the plasmid pUC19 and transferred toEscherichia coli JM101. The fragment directed the synthesis inE. coli of a-1,3-1,4-glucanase that specifically degrades barley glucan and lichenan. The largest proportion (>50%) of the total enzyme activity was cellular enzyme; 25% of the-1,3-1,4-glucanse activity was present in the extracellular fractions. Through enzyme analysis, the enzymes purified fromE. coli or fromBacillus subtilis 1.88 were proved to be identical.     

Mapping of the gene for endo-β-1,3-1,4-glucanase of Bacillus subtilis

Abstract An integrating plasmid has been used to mutagenise the gene coding for endo-β-1,3-1,4-glucanase of Bacillus subtilis . The gene, named bgl , has been mapped by PBS-1 transduction to the sacA-pureA region of the B. subtilis chromosome and is closely linked to the hutP 1 locus. The order of markers in this region is sacA 321- thiC 5- bgl - hutP 1- purA 16.     

High-level expression of a specific β-1,3-1,4-glucanase from the thermophilic fungus Paecilomyces thermophila in Pichia pastoris

In this study, a novel β-1,3-1,4-glucanase gene (designated as PtLic16A) from Paecilomyces thermophila was cloned and sequenced. PtLic16A has an open reading frame of 945 bp, encoding 314 amino acids. The deduced amino acid sequence shares the highest identity (61%) with the putative endo-1,3(4)-β-glucanase from Neosartorya fischeri NRRL 181. PtLic16A was cloned into a vector pPIC9K and was expressed successfully in Pichia pastoris as active extracellular β-1,3-1,4-glucanase. The recombinant β-1,3-1,4-glucanase (PtLic16A) was secreted predominantly into the medium which comprised up to 85% of the total extracellular proteins and reached a protein concentration of 9.1 g l⁻¹ with an activity of 55,300 U ml⁻¹ in 5-l fermentor culture. The enzyme was then purified using two steps, ion exchange chromatography, and gel filtration chromatography. The purified enzyme had a molecular mass of 38.5 kDa on SDS–PAGE. It was optimally active at pH 7.0 and a temperature of 70°C. Furthermore, the enzyme exhibited strict specificity for β-1,3-1,4-d-glucans. This is the first report on the cloning and expression of a β-1,3-1,4-glucanase gene from Paecilomyces sp.     

Hemicellulase activity of antarctic microfungi

The mannanase (endo-β-1,4-mannanase; E.C. 3.2.1.78) and xylanase (endo-β-1,4-xylanase; E.C. 3.2.1.8) activity of five microfungal isolates from Antarctica were characterized at different temperatures and pH. In general, the hemicellulase activity of the antarctic strains occurred at least 10 °C and as much as 30 °C lower than that of a mesophilic reference strain. At 0 °C, two strains, a Phoma and a Penicillium , produced in excess of 40% of their measured maximum activity of mannanase. All strains had maximum hemicellulase activity in the range pH 4–5, with Penicillium , Phoma and Alternaria strains exhibiting high (in excess of 80% of maximum) mannanase activity at pH 10. Three of the antarctic isolates exhibited high levels of xylanase activity over a pH range of 3–11.     

Endogenous production of endo-β-1,4-glucanase by decapod crustaceans

The potential ability to produce cellulase enzymes endogenously was examined in decapods crustaceans including the herbivorous gecarcinid land crabs Gecarcoidea natalis and Discoplax hirtipes, the amphibious freshwater crab Austrothelphusa transversa, the terrestrial hermit crab, Coenobita variabilis the parastacid crayfish Euastacus, and the crayfish Cherax destructor. The midgut gland of both G. natalis and D. hirtipes contained substantial total cellulase activities and activities of the cellulase enzymes endo-β-1,4-glucanase and β-glucosidase. With the exception of total cellulase and β-glucosidase from D. hirtipes, the enzyme activities within the midgut gland were higher than those within the digestive juice. Hence, the enzyme activities appear to reside predominantly within midgut gland, providing indirect evidence for endogenous synthesis of cellulase enzymes by this tissue. A 900 bp cDNA fragment encoding a portion of the endo-β-1,4-glucanase amino acid sequence was amplified by RT-PCR using RNA isolated from the midgut gland of C. destructor, Euastacus, A. transversa and C. variabilis. This provided direct evidence for the endogenous production of endo-β-1,4-glucanase. The 900 bp fragment was also amplified from genomic DNA isolated from the skeletal muscle of G. natalis and D. hirtipes, clearly indicating that the gene encoding endo-β-1,4-glucanase is also present in these two species. As this group of evolutionary diverse crustacean species possesses and expresses the endo-β-1,4-glucanase gene it is likely that decapod crustaceans generally produce cellulases endogenously and are able to digest cellulose.     

Cloning of the Bacillus subtilis DLG beta-1,4-glucanase gene and its expression in Escherichia coli and B. subtilis.

The gene encoding beta-1,4-glucanase in Bacillus subtilis DLG was cloned into both Escherichia coli C600SF8 and B. subtilis PSL1, which does not naturally produce beta-1,4-glucanase, with the shuttle vector pPL1202. This enzyme is capable of degrading both carboxymethyl cellulose and trinitrophenyl carboxymethyl cellulose, but not more crystalline cellulosic substrates (L. M. Robson and G. H. Chambliss, Appl. Environ. Microbiol. 47:1039-1046, 1984). The beta-1,4-glucanase gene was localized to a 2-kilobase (kb) EcoRI-HindIII fragment contained within a 3-kb EcoRI chromosomal DNA fragment of B. subtilis DLG. Recombinant plasmids pLG4000, pLG4001a, pLG4001b, and pLG4002, carrying this 2-kb DNA fragment, were stably maintained in both hosts, and the beta-1,4-glucanase gene was expressed in both. The 3-kb EcoRI fragment apparently contained the beta-1,4-glucanase gene promoter, since transformed strains of B. subtilis PSL1 produced the enzyme in the same temporal fashion as the natural host B. subtilis DLG. B. subtilis DLG produced a 35,200-dalton exocellular beta-1,4-glucanase; intracellular beta-1,4-glucanase was undetectable. E. coli C600SF8 transformants carrying any of the four recombinant plasmids produced two active forms of beta-1,4-glucanase, an intracellular form (51,000 +/- 900 daltons) and a cell-associated form (39,000 +/- 400 daltons). Free exocellular enzyme was negligible. In contrast, B. subtilis PSL1 transformed with recombinant plasmid pLG4001b produced three distinct sizes of active exocellular beta-1,4-glucanase: approximately 36,000, approximately 35,200, and approximately 33,500 daltons. Additionally, B. subtilis PSL1(pLG4001b) transformants contained a small amount (5% or less) of active intracellular beta-1,4-glucanase of three distinct sizes: approximately 50,500, approximately 38,500 and approximately 36,000 daltons. The largest form of beta-1,4-glucanase seen in both transformants may be the primary, unprocessed translation product of the gene.     

The Schizosaccharomyces pombe endo-1,3-beta-glucanase Eng1 contains a novel carbohydrate binding module required for septum localization

Cell separation in Schizosaccharomyces pombe is achieved through the concerted action of the Eng1 endo-β-1,3-glucanase and the Agn1 endo-α-1,3-glucanase, which are transported to the septum and localize to a ring-like structure that surrounds the septum. Correct localization of these hydrolases requires the presence of both the septins and the exocyst. In this work, we show that the glucanase Eng1 contains a region at the C-terminus that acts as a carbohydrate-binding module (CBM) and that it is not present in other members of glycoside hydrolases family 81 (GH81). In vitro , the purified CBM has affinity for β-1,3-glucan chains with a minimum degree of polymerization of 30 glucose units. Deletion of the CBM results in a protein that is largely defective in complementing the separation defect of eng1 Δ mutants. This defect is due to a reduction in the catalytic activity against insoluble substrates and to a defect in targeting of Eng1 to the septum, as the truncated protein localizes to the lateral cell wall of the cell. Thus, the targeting of Eng1 to the primary septum requires not only trans -factors (septins and the exocyst complex) but also a cis -element localized to the C-terminus of the protein.     

β(1,3)-Glucanases from Geotrichum lactis: activity on its own nascent and preformed β(1,3)-glucan

Abstract Actively growing mycelium of Geotrichum lactis contains at least three β(1,3)-glucanase activities. Two of the activities have been characterized as exo- and the third as endo-hydrolytic. The action of the activities on β(1,3)-glucan synthesized in vitro by the β(1,3)-glucan synthase system from G. lactis has been studied. One of the exo-β-glucanases and the endo-β-glucanase were active on this β(1,3)-glucan and the degradation rates were higher on nascent than on preformed β(1,3)-glucan.     

Construction and characterization of a fusion β-1,3-1,4-glucanase to improve hydrolytic activity and thermostability

A new fusion gene (Bgl-licMB), encoding β-1,3-1,4-glucanase both from Bacillus amyloliquefaciens (Bgl) and Clostridium thermocellum (licMB), was constructed via end-to-end fusion and expressed in Escherichia coli to improve hydrolytic activity and thermostability of β-1,3-1,4-glucanase. The results of enzymatic properties showed that the catalytic efficiency (K_cat/K_m) of the fusion enzyme for oat β-glucan was 2.7 and 20-fold higher than that of the parental Bgl and licMB, respectively, and that the fusion enzyme can retain more than 50% of activity following incubation at 80°C for 30 min, whereas the residual activities of Bgl and licMB were both less than 30%. These properties make this particular β-1,3-1,4-glucanase a good candidate for application in brewing and animal-feed industries.     

Effect of 1,3;1,6-β-D-glucan on Infection of Detached Tobacco Leaves with Tobacco Mosaic Virus

Glucan preparations were obtained by transformation of laminaran from the alga Laminaria cichorioides with endo-β-1,3-glucanase from marine mollusks. These preparations, like those of other sources, have an inhibitory effect on tobacco mosaic virus (TMV) infection of detached leaves of local and systemic host tobacco plants.     

Codon optimization,expression and characterization of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> MA139 <Emphasis Type="Italic">β</Emphasis>-1,3-1,4-glucanase in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

β-1,3-1,4-Glucanase has been broadly used in feed and brewing industries. According to the codon bias of Pichia pastoris, the Bacillus subtilis MA139 β-1,3-1,4-glucanase gene was de novo synthesized and expressed in P. pastoris X-33 strain under the control of the alcohol oxidase 1 promoter. In a 10-L fermentor, the β-1,3-1,4-glucanase was overexpressed with a yield of 15,000 U/mL by methanol induction for 96 h. The recombinant β-1,3-1,4-glucanase exhibited optimal activity at 40°C and pH 6.4. The activity of the recombinant β-1,3-1,4-glucanase was not significantly affected by various metal ions and chemical reagents. To our knowledge, the expression of this β-1,3-1,4-glucanase from Bacillus sp. in P. pastoris is in relatively high level compared to previous reports. These biochemical characteristics suggest that the recombinant β-1,3-1,4-glucanase has a prospective application in feed and brewing industries.     

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.     

Characterization of production and enzyme properties of an endo-β-1,4-glucanase fromBacillus subtilis CK-2 isolated from compost soil

Bacillus subtilis CK-2, isolated from garden organic waste compost, was found to have high hydrolytic activity against carboxymethylcellulose (CMC) due to the secretion of an endo--1,4-glucanase. Enzyme production was related to the sporulation process, and was regulated by the concentration of readily metabolizable carbohydrate in growth medium. Enzyme production did not require CMC or other cellulose containing materials. The endo--1,4-glucanase activity was optimal at pH 5.6–5.8 and at 65 MoC, and achieved thermal stability up to 55 MoC. The activity was inhibited by Hg²⁺. The purified enzyme gave a single band corresponding to a MW of 35.5 kDa on SDS-PAGE, while the Sephadex G-75 chromatography revealed a molecular weight of the active enzyme around 70 kDa, indicating a dimeric form of the active enzyme. The enzyme activity was irreversibly inhibited by SDS. Native PAGE and IEF revealed three different isoelectric forms of the enzyme, all with an identical N-terminal amino-acid sequence.Abbreviations CMC carboxymethylcellulose - DNS dinitrosalicylic - SDS sodium dodecyl sulfate     

Subcellular targeting is a key condition for high-level accumulation of cellulase protein in transgenic maize seed

Ethanol from lignocellulosic biomass is being pursued as an alternative to petroleum-based transportation fuels. To succeed in this endeavour, efficient digestion of cellulose into monomeric sugar streams is a key step. Current production systems for cellulase enzymes, i.e. fungi and bacteria, cannot meet the cost and huge volume requirements of this commodity-based industry. Transgenic maize ( Zea mays L.) seed containing cellulase protein in embryo tissue, with protein localized to the endoplasmic reticulum, cell wall or vacuole, allows the recovery of commercial amounts of enzyme. E1 cellulase, an endo-β-1,4-glucanase from Acidothermus cellulolyticus , was recovered at levels greater than 16% total soluble protein (TSP) in single seed. More significantly, cellobiohydrolase I (CBH I), an exocellulase from Trichoderma reesei , also accumulated to levels greater than 16% TSP in single seed, nearly 1000-fold higher than the expression in any other plant reported in the literature. The catalytic domain was the dominant form of E1 that was detected in the endoplasmic reticulum and vacuole, whereas CBH I holoenzyme was present in the cell wall. With one exception, individual transgenic events contained single inserts. Recovery of high levels of enzyme in T₂ ears demonstrated that expression is likely to be stable over multiple generations. The enzymes were active in cleaving soluble substrate.