Lichenase and Cellobiohydrolase Activities of a Novel Bi-Functional β-Glucanase from the Marine Bacterium Streptomyces sp. J103
Youngdeuk LeeEunyoung JoYeon‐Ju LeeMin Jin KimNavindu Dinara GajanayakaMahanama De ZoysaHo‐Jin ParkChulhong Oh
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Abstract:
In this study, we report the molecular and enzymatic characterisation of Spg103, a novel bifunctional β-glucanase from the marine bacterium Streptomyces sp. J103. Recombinant Spg103 (rSpg103) functioned optimally at 60 °C and pH 6. Notably, Spg103 exhibited distinct stability properties, with increased activity in the presence of Na+ and EDTA. Spg103 displays both lichenase and cellobiohydrolase activity. Despite possessing a GH5 cellulase domain, FN3 and CBM3 domains characteristic of cellulases and CBHs, biochemical assays showed that rSpg103 exhibited higher activity towards mixed β-1,3-1,4-glucan such as barley β-glucan and lichenan than towards beta-1,4-linkages. The endolytic activity of the enzyme was confirmed by TLC and UPLC-MS analyses, which identified cellotriose as the main hydrolysis product. In addition, Spg103 exhibited an exo-type activity, selectively releasing cellobiose units from cellooligosaccharides, which is characteristic of cellobiohydrolases. These results demonstrate the potential of Spg103 for a variety of biotechnological applications, particularly those requiring tailor-made enzymatic degradation of mixed-linked β-glucans. This study provides a basis for further structural and functional investigations of the bifunctional enzyme and highlights Spg103 as a promising candidate for industrial applications.Keywords:
Glucanase
Hydrolase
Beta-glucosidase
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Cellulolytic fungi have evolved a sophisticated genetic regulatory network of cellulase synthesis to adapt to the natural environment. Even in the absence of lignocellulose, it still secretes low levels of "constitutive" cellulase for standby application. However, the mechanisms of this constitutive expression remain incompletely understood. Here we identified a cellobiose synthetase (CBS) from Rhizopus stolonifer, which has the capacity to catalyse the synthesis of cellobiose from uridine diphosphate glucose (UDPG). Through the construction of R. stolonifer Δcbs strain, we found that CBS plays a key role in the synthesis of cellulase. Further analysis of cellulase synthesis under glucose culture reveals that the cellobiose-responsive regulator CLR1 was activated by CBS-synthesized cellobiose, thereby promoting the expression of CLR2 and finally opening the transcription of cellulase genes. Our results suggest that R. stolonifer can be induced by self-synthesized cellobiose to produce cellulase, which can be used to reconstruct the expression regulation network to achieve rapid production of cellulase using simple carbon source. Based on our data, the "constitutive expression" of cellulase actually derives from the induction of cellobiose that synthesized by CBS from carbohydrate metabolites, which updates our knowledge of cellulase, and provides a novel insight into the regulation of cellulase synthesis.
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The cellulases of Streptomyces thermodiastaticus (strain 2Sts) and thermomonospora fusca (strain 190Th) were produced with carboxymethyl-cellulose (CMC) serving as the carbon source during growth. Both cellulases act by random internal hydrolysis of the CMC chain, producing cellobiose, glucose, and intermediate length oligosaccharides. Cellobiase was not detected in culture filtrates produced under these conditions.
Carboxymethyl cellulose
Streptomycetaceae
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Bioconversion
Carbon source
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Cellobiase (β-glucosidase) production was compared for two streptomycetes: Streptomyces flavogriseus , a known producer of cellulase complex, and Streptomyces sp. strain CB-12, a strain isolated for its rapid growth on cellobiose. The optimal conditions for enzyme activity were established in relation to pH, temperature, enzyme stability, and substrate affinity. The production of β-glucosidase by the two strains depended on the carbon substrate in the medium. Cellobiose was found to repress the biosynthesis of the enzyme in S. flavogriseus and to stimulate its production in strain CB-12. The biosynthesis of the enzyme correlated well with the accumulation of glucose in the culture filtrates. The combined action of the β-glucosidases produced by the two Streptomyces strains might allow a better utilization of the reaction products which arise during the biodegradation of cellulose.
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Two highly purified cellulases [EC 3.2.1.4], II-A, and II-B, were obtained from the cellulase system of Trichoderma viride. Both cellulases split cellopentaose, retaining the β-configuration of the anomeric carbon atoms in the hydrolysis products at both pH 3.5 and 5.0. The Km values of cellulases II-A and II-B for cellotetraose were different, but their Vmax values were similar and those for cellooligosaccharides increased in parallel with chain length. Both cellulases produced predominantly cellobiose and glucose from various cellulosic substrates as well as from higher cellooligosaccharides. Cellulase II-A preferentially attacked the holoside linkage of p-nitrophenyl β-D-cellobioside, whereas cellulase II-B attacked mainly the aglycone linkage of this cellobioside. Both cellulases were found to catalyze the synthesis of cellotriose from p-nitrophenyl β-D-cellobioside by transfer of a glucosyl residue, possibly to cellobiose produced in the reaction mixture. They were also found to catalyze the rapid synthesis of cellotetraose from cellobiose, with accompanying formation of cellotriose and glucose, which seemed to be produced by secondary random hydrolysis of the cellotetraose produced. The capacity to synthesize cellotetraose from cellobiose appeared to be greater with cellulase II-B than with cellulase II-A.
Trichoderma viride
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Carboxymethyl cellulose
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The location and formation of cellulases and β‐glucosidase in the fungus Trichoderma viride were studied on different substrates. The cellulases were found to be cell‐bound during active growth on cellulose, CMC, and cellobiose. On CMC, much CM‐cellulase was found cell‐free but sonication released cellulase from the washed mycelium. Analysis of the carbohydrates of the mycelial cell wall after hydrolysis revealed glucose, mannose, and galactose—the same carbohydrates as reported to be present in purified cellulase from the same organism. Glucose repressed the formation of both CM‐cellulase and Avicelase and cellobiose apparently the formation of Avicelase. Relatively little CM‐cellulase was formed on cellobiose but a straight line was obtained when a differential plot of CM‐cellulase versus protein was made.
Trichoderma viride
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Abstract Apricot gummosis pathogens — the fungi Hendersonula toruloidea , a Cytosporina sp. and a Cytospora sp., produce cellulase (endo‐1,4‐β‐glucanase, carboxymethylcellulase, CMCase) in culture. Cytospora sp. produced significantly lower amounts of cellulase than H. toruloidea and Cytosporina sp. The tendency of cellulase accumulation inside apricot stems infected with either H. toruloidea or Cytosporina sp. is similar: most of the cellulase was found in the necrotic area, whereas comparatively small amounts accumulated at the advancing edge of the disease symptom. H. toruloidea produces cellulase with a molecular mass of 76 kDa.
Glucanase
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Abstract Cellulase can hydrolyze cellulose to produce reducing sugars such as cellobiose and cellotriose. β-glucosidas can further hydrolyze cellobiose and cellobiose produced by cellulase to produce glucose. According to the hydrolysis mechanisms of cellulase and β-glucosidas, we selected two enzyems, cellulase and β-glucosidase to study optimization of co-immobilization of cellulase and β-glucosidas. Meanwhile, we selected two materials, activated carbon and sodium alginate to co-immobilize cellulase and β-glucosidas by the immobilization method of embedding-adsorption. The immobilization conditions, alginate, CaCl 2 and activated carbon was optimized. The results showed that the optimum concentrations of sodium alginate and CaCl 2 were 2% and 2%, respectively, and the quality of activated carbon is 0.15 g. The optimal ratio of cellulase to β - glucosidase was 1:1.5. The results indicated that cellulase and β-glucosidase had a synergistic effect and that their compound degradation of cellulose was better than the separate effects of the two enzymes acting independently.
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