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Diversity and functional significance of cellulolytic microbes living in termite, pill-bug and stem-borer guts.

Bashir Z, Kondapalli VK, Adlakha N, Sharma A, Bhatnagar RK, Chandel G, Yazdani SS - Sci Rep (2013)

Bottom Line: Most of the carboxymethylcellulase positive strains also hydrolysed other amorphous substrates such as xylan, locust bean gum and β-D-glucan.Two strains, A11 and A21, demonstrated significant activity towards Avicel and p-nitrophenyl-β-D-cellobiose, indicating that they express cellobiohydrolase.These results provide insight into the co-existence of symbionts in the guts of arthropods and their possible exploitation for the production of fuels and chemicals derived from plant biomass.

View Article: PubMed Central - PubMed

Affiliation: Synthetic Biology and Biofuels Group, Aruna Asaf Ali Marg, New Delhi, India.

ABSTRACT
Arthropods living on plants are able to digest plant biomass with the help of microbial flora in their guts. This study considered three arthropods from different niches - termites, pill-bugs and yellow stem-borers - and screened their guts for cellulase producing microbes. Among 42 unique cellulase-producing strains, 50% belonged to Bacillaceae, 26% belonged to Enterobacteriaceae, 17% belonged to Microbacteriaceae, 5% belonged to Paenibacillaceae and 2% belonged to Promicromonosporaceae. The distribution of microbial families in the three arthropod guts reflected differences in their food consumption habits. Most of the carboxymethylcellulase positive strains also hydrolysed other amorphous substrates such as xylan, locust bean gum and β-D-glucan. Two strains, A11 and A21, demonstrated significant activity towards Avicel and p-nitrophenyl-β-D-cellobiose, indicating that they express cellobiohydrolase. These results provide insight into the co-existence of symbionts in the guts of arthropods and their possible exploitation for the production of fuels and chemicals derived from plant biomass.

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Qualitative assessment of hydrolytic enzymes produced by various natural isolates.Enzyme production was tested in the extracellular (A) and cell-bound fractions (B) against amorphous, crystalline and chromogenic substrates. Black and white boxes represent significant and non-significant activities, respectively, in the liquid culture assay. (C) Venn diagram for natural isolates exhibiting cellulase, hemicellulase and other glycosidase activities. Cellulase positive strains were considered to be those that hydrolysed CMC, Avicel, rice straw, pNPC or pNPG. Hemicellulase positive strains were those that hydrolysed xylan or pNPX. Strains that hydrolysed locust bean gum or β-D-glucan were considered to be positive for other glycosyl hydrolases.
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f3: Qualitative assessment of hydrolytic enzymes produced by various natural isolates.Enzyme production was tested in the extracellular (A) and cell-bound fractions (B) against amorphous, crystalline and chromogenic substrates. Black and white boxes represent significant and non-significant activities, respectively, in the liquid culture assay. (C) Venn diagram for natural isolates exhibiting cellulase, hemicellulase and other glycosidase activities. Cellulase positive strains were considered to be those that hydrolysed CMC, Avicel, rice straw, pNPC or pNPG. Hemicellulase positive strains were those that hydrolysed xylan or pNPX. Strains that hydrolysed locust bean gum or β-D-glucan were considered to be positive for other glycosyl hydrolases.

Mentions: Microbes residing in the guts of biomass feeding arthropods are likely to produce a variety of hydrolytic enzymes. In previous studies we showed that Paenibacillus ICGEB2008 isolated from the gut of the cotton bollworm produced several biomass-degrading enzymes, including cellulases and hemicellulases1819. Therefore, we tested the expression levels in the gut microbes of major glycosyl hydrolase enzymes belonging to the categories of cellulase, hemicellulase, mannanase and glucanase. The expression levels of endoglucanase, β-glucosidase, xylanase, β-xylosidase, mannanase and β-D-glucanase were determined using the substrates CMC, pNPG, Xylan, pNPX, locust bean gum and barley β-D-glucan, respectively. To test exocellulase or cellobiohydrolase expression, both Avicel and pNPC (in the presence of the β-glucosidase inhibitor glucono-δ-lactone) were used as substrates. A potential biofuel feedstock was also used, namely alkali treated rice straw, and it mainly contained crystalline cellulose as well as some amorphous forms of cellulose. These assays were used to test both extracellular and cell associated enzymes. Except for A13, A16 and T3, all natural isolates produced at least one type of glycosyl hydrolase (Figure 3A and 3B). The reason that these three strains are negative in the liquid assay despite being positive for CMC activity on the agar plates could be related to the stringency criteria set as described in the Methods section. The majority of microbes produced more than one kind of hydrolytic enzyme (Figure 3C). There were 23 microbes that produced at least one hydrolytic enzyme from each category of biomass degrading enzymes. Several strains produced only one category of enzyme, such as cellulases (5 strains) and hemicellulases (1 strain), which hydrolysed pNPG and pNPX, respectively, but did not produce significant amounts of any other type of hydrolase (Figure 3). Extracellular fractions were found to be rich in enzymes specific for amorphous polymers (Figure 3A). Only a few microbes secreted enzymes that were able to hydrolyse dimeric or oligomeric substrates, such as pNPG, pNPC and pNPX, or complex structures, such as rice straw. On the other hand, the majority of cell associated fractions hydrolysed dimeric substrates such as pNPG and pNPX (Figure 3B). However, cell associated fractions of none of the microbes were able to hydrolyse pNPC. Some strains also produced enzymes in the extracellular or cell-associated fractions for the hydrolysis of complex structures such as rice straw and Avicel.


Diversity and functional significance of cellulolytic microbes living in termite, pill-bug and stem-borer guts.

Bashir Z, Kondapalli VK, Adlakha N, Sharma A, Bhatnagar RK, Chandel G, Yazdani SS - Sci Rep (2013)

Qualitative assessment of hydrolytic enzymes produced by various natural isolates.Enzyme production was tested in the extracellular (A) and cell-bound fractions (B) against amorphous, crystalline and chromogenic substrates. Black and white boxes represent significant and non-significant activities, respectively, in the liquid culture assay. (C) Venn diagram for natural isolates exhibiting cellulase, hemicellulase and other glycosidase activities. Cellulase positive strains were considered to be those that hydrolysed CMC, Avicel, rice straw, pNPC or pNPG. Hemicellulase positive strains were those that hydrolysed xylan or pNPX. Strains that hydrolysed locust bean gum or β-D-glucan were considered to be positive for other glycosyl hydrolases.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3757366&req=5

f3: Qualitative assessment of hydrolytic enzymes produced by various natural isolates.Enzyme production was tested in the extracellular (A) and cell-bound fractions (B) against amorphous, crystalline and chromogenic substrates. Black and white boxes represent significant and non-significant activities, respectively, in the liquid culture assay. (C) Venn diagram for natural isolates exhibiting cellulase, hemicellulase and other glycosidase activities. Cellulase positive strains were considered to be those that hydrolysed CMC, Avicel, rice straw, pNPC or pNPG. Hemicellulase positive strains were those that hydrolysed xylan or pNPX. Strains that hydrolysed locust bean gum or β-D-glucan were considered to be positive for other glycosyl hydrolases.
Mentions: Microbes residing in the guts of biomass feeding arthropods are likely to produce a variety of hydrolytic enzymes. In previous studies we showed that Paenibacillus ICGEB2008 isolated from the gut of the cotton bollworm produced several biomass-degrading enzymes, including cellulases and hemicellulases1819. Therefore, we tested the expression levels in the gut microbes of major glycosyl hydrolase enzymes belonging to the categories of cellulase, hemicellulase, mannanase and glucanase. The expression levels of endoglucanase, β-glucosidase, xylanase, β-xylosidase, mannanase and β-D-glucanase were determined using the substrates CMC, pNPG, Xylan, pNPX, locust bean gum and barley β-D-glucan, respectively. To test exocellulase or cellobiohydrolase expression, both Avicel and pNPC (in the presence of the β-glucosidase inhibitor glucono-δ-lactone) were used as substrates. A potential biofuel feedstock was also used, namely alkali treated rice straw, and it mainly contained crystalline cellulose as well as some amorphous forms of cellulose. These assays were used to test both extracellular and cell associated enzymes. Except for A13, A16 and T3, all natural isolates produced at least one type of glycosyl hydrolase (Figure 3A and 3B). The reason that these three strains are negative in the liquid assay despite being positive for CMC activity on the agar plates could be related to the stringency criteria set as described in the Methods section. The majority of microbes produced more than one kind of hydrolytic enzyme (Figure 3C). There were 23 microbes that produced at least one hydrolytic enzyme from each category of biomass degrading enzymes. Several strains produced only one category of enzyme, such as cellulases (5 strains) and hemicellulases (1 strain), which hydrolysed pNPG and pNPX, respectively, but did not produce significant amounts of any other type of hydrolase (Figure 3). Extracellular fractions were found to be rich in enzymes specific for amorphous polymers (Figure 3A). Only a few microbes secreted enzymes that were able to hydrolyse dimeric or oligomeric substrates, such as pNPG, pNPC and pNPX, or complex structures, such as rice straw. On the other hand, the majority of cell associated fractions hydrolysed dimeric substrates such as pNPG and pNPX (Figure 3B). However, cell associated fractions of none of the microbes were able to hydrolyse pNPC. Some strains also produced enzymes in the extracellular or cell-associated fractions for the hydrolysis of complex structures such as rice straw and Avicel.

Bottom Line: Most of the carboxymethylcellulase positive strains also hydrolysed other amorphous substrates such as xylan, locust bean gum and β-D-glucan.Two strains, A11 and A21, demonstrated significant activity towards Avicel and p-nitrophenyl-β-D-cellobiose, indicating that they express cellobiohydrolase.These results provide insight into the co-existence of symbionts in the guts of arthropods and their possible exploitation for the production of fuels and chemicals derived from plant biomass.

View Article: PubMed Central - PubMed

Affiliation: Synthetic Biology and Biofuels Group, Aruna Asaf Ali Marg, New Delhi, India.

ABSTRACT
Arthropods living on plants are able to digest plant biomass with the help of microbial flora in their guts. This study considered three arthropods from different niches - termites, pill-bugs and yellow stem-borers - and screened their guts for cellulase producing microbes. Among 42 unique cellulase-producing strains, 50% belonged to Bacillaceae, 26% belonged to Enterobacteriaceae, 17% belonged to Microbacteriaceae, 5% belonged to Paenibacillaceae and 2% belonged to Promicromonosporaceae. The distribution of microbial families in the three arthropod guts reflected differences in their food consumption habits. Most of the carboxymethylcellulase positive strains also hydrolysed other amorphous substrates such as xylan, locust bean gum and β-D-glucan. Two strains, A11 and A21, demonstrated significant activity towards Avicel and p-nitrophenyl-β-D-cellobiose, indicating that they express cellobiohydrolase. These results provide insight into the co-existence of symbionts in the guts of arthropods and their possible exploitation for the production of fuels and chemicals derived from plant biomass.

Show MeSH