Properties of the endogenous cellulase from Panesthia cribrata Saussure and purification of major endo-β-1, 4-glucanase components
The cellulase of Panesthia cribrata consists of at least six endo-β-1,4-glucanase (EC 126.96.36.199) and two β-glucosidase (EC 188.8.131.52) components. The two major endo-β-1,4-glucanase components, named EG1 and EG2, comprise 13% of the soluble protein in the foregut and midgut contents. They were purified by a combination of gel chromatography and ion-exchange FPLC and had Mr of 53,600 and 48,800, respectively. With carboxymethylcellulose (CMC) the Km and Vmax values for EG1 and 9.4 mg/ml and 22.2 mg reducing sugar/min/mg protein; the corresponding values for EG2 were 6.8 and 88.3l. Values for Km, Vmax and kcat were also calculated for activity against cellotetraose and cellopentaose. Neither component hydrolysed cellobiose or cellotriose. The relative activities of EG1 and EG2 against CMC and microcrystalline cellulose Sigmacell Type 20 were 5200:1 and 3500:1. It is proposed that the inefficiency of EG1 and EG2 is compensated for by their secretion in large amounts into the gut. Four minor endo-β-1,4-glucanase components, comprising between 4 and 10% of the endo-β-1,4-glucanase activity were also present but could not be separated from the β-glucosidase components and therefore were not characterised. There were two β-glucosidase components, named GD1 and GD2, which were partially purified and characterised. The Km for GD1, the major component, was 10.6 mM with . Km values were also determined for both components with cellodextrins ranging from cellobiose to cellopentaose. GD1 was competitively inhibited by gluconon-δ-1, 5-lactone (Ki = 0.33 mM) but was unaffected by glucose at physiological concentrations. Neither component was active against crystalline cellulose or CMC.
Available from: Shing Yip Lee
- "β-glucosidase releases glucose from the non-reducing end of cellobiose or oligomers (Watanabe and Tokuda, 2010). Endo-β-1,4-glucanases from some arthropods, however, have both endocellulase and exocellulase activities (Scrivener and Slaytor, 1994; Allardyce and Linton, 2008). Different from herbivorous vertebrates, which rely on cellulase enzymes produced by symbiotic gut microflora for cellulose digestion, many invertebrates are capable of producing their own enzymes for Comparative Biochemistry and Physiology, Part B 179 (2015) 27–36 cellulose hydrolysis. "
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ABSTRACT: The sesarmid crab Parasesarma erythodactyla consumes large amounts of mangrove leaf litter but its biochemical capacity for cellulose digestion is poorly known. We demonstrate the presence of endo-β-1,4-glucanase, β-glucosidase and total cellulase activities in the digestive juice of this crab. The highest total cellulase activity was observed at mildly acidic pH (5 to 6) and temperature between 30 and 50 °C. A 1752 bp cDNA containing an open reading frame of 1386 bp encoding a putative endo-β-1,4-glucanase (EG) of 461 amino acids was identified in the crab’s hepatopancreas using polymerase chain reaction (PCR), cloning and sequencing techniques. P. erythodactyla endo-β-1,4-glucanase (PeEG) contains a glycosyl hydrolase family 9 (GHF9) catalytic domain with all catalytically important residues conserved, and shows high sequence identity to GHF9 EGs reported from other arthropods. The endogenous origin of PeEG was confirmed by PCR amplification of a ~ 1.5 kb DNA fragment, containing a phase 1 intron flanked by two exon sequences identical to the cDNA, from genomic DNA isolated from the crab’s muscle tissue. PeEG encoding cDNA is the first endogenous EG sequence reported from the brachyuran crabs. Using degenerate primers, we also isolated 204 bp cDNA fragments with sequences affiliated to EG from the hepatopancreas of eight other mangrove crabs of the Sesarmidae (Neosarmatium trispinosum and Sesarmoides borneensis), Macrophthalmidae (Ilyograpsus daviei, Australoplax tridentata, and Macrophthalmus setosus), Varunidae (Pseudohelice subquadrata), Heloeciidae (Heloecius cordiformis), and Ocypodidae (Uca perplexa) families, suggesting that endogenous cellulase production may be a common characteristic among the detritivorous mangrove crabs.
Available from: Jonathan D. Willis
- "Purification procedures usually consist of multiple steps, including size exclusion, anion exchange and/or hydrophobic interaction chromatography (Marana et al., 1995, 2000; Ferreira et al., 2001; Yapi et al., 2009). Even though proteins displaying CBH activity have not been purified from insect systems, EG enzymes have been purified and characterized from cockroach species (Scrivener & Slaytor, 1994; Genta et al., 2003) or termite symbiotic flagellates (Li et al., 2003) using also liquid chromatographic procedures. Alternative reported purification methods for glycosidases include isoelectric focusing (Ferreira & Terra, 1983) and preparative electrophoresis (Chipoulet & Chararas, 1985a; Sugimura et al., 2003; Sami & Shakoori, 2008). "
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ABSTRACT: Abstract Cellulosic ethanol has been identified as a crucial biofuel resource due to its sustainability and abundance of cellulose feedstocks. However, current methods to obtain glucose from lignocellulosic biomass are ineffective due to recalcitrance of plant biomass. Insects have evolved endogenous and symbiotic enzymes to efficiently use lignocellulosic material as a source of metabolic glucose. Even though traditional biochemical methods have been used to identify and characterize these enzymes, the advancement of genomic and proteomic research tools are expected to allow new insights into insect digestion of cellulose. This information is highly relevant to the design of improved industrial processes of biofuel production and to identify potential new targets for development of insecticides. This review describes the diverse methodologies used to detect, quantify, purify, clone and express cellulolytic enzymes from insects, as well as their advantages and limitations.
Available from: John Bland
- "These differential enzymatic patterns could be due to the structural difference of the substrates. The recombinant cellulase in this study resembles other endo-b-1,4-glucanases, extracted and/or purified from the higher termite (Nasutitermes walkeri; Schulz et al., 1986), the lower termite (R. speratus; Watanabe et al., 1997) and the cockroach (Panesthia cribrata; Scrivener and Slaytor, 1994), in which the hydrolytic products were mainly cellobiose and a small amount of glucose (R.s.) or cellobiose, cellotriose and a small amount of glucose (N.w.) or equimolar amount of cellobiose and cellotriose and no glucose (P.c.) when using crystalline cellulose as the substrate. Structurally, both crystalline cellulose and filter paper are made from cotton linters and composed of more than 98% acellulose with the later being an amorphous form and water soluble. "
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ABSTRACT: An endogenous cellulase gene (CfEG3a) of Coptotermes formosanus, an economically important pest termite, was cloned and overexpressed in both native form (nCfEG) and C-terminal His-tagged form (tCfEG) in Escherichia coli. Both forms of recombinant cellulases showed hydrolytic activity on cellulosic substrates. The nCfEG was more active and stable than tCfEG even though the latter could be purified to near homogeneity with a simple procedure. The differential activities of nCfEG and tCfEG were also evidenced by hydrolytic products they produced on different substrates. On CMC, both acted as an endoglucanase, randomly hydrolyzing internal β-1,4-glycosidic bonds and resulting in a smear of polymers with different lengths, although cellobiose, cellotriose, and cellotetraose equivalents were noticeable. The hydrolytic products of tCfEG were one unit sugar less than those produced by nCfEG. Using filter paper as substrate, however, the major hydrolytic products of nCfEG were cellobiose, cellotriose and trace of glucose; those of tCfEG were cellobiose, cellotriose and trace of cellotetraose, indicating a property similar to that of cellobiohydrolase, an exoglucanase. The results presented in this report uncovered the biochemical properties of the recombinant cellulase derived from the intact gene of Formosan subterranean termites. The recombinant cellulase would be useful in designing cellulase-inhibiting termiticides and incorporating into a sugar-based biofuel production program.
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