Extracellular Cellulase Activity Research Articles

Enhanced rice plant (BRRI-28) growth at lower doses of urea caused by diazinon mineralizing endophytic bacterial consortia and explorations of relevant regulatory genes in a Klebsiella sp. strain HSTU-F2D4R

Endophytic biostimulant with pesticide bioremediation activities may reduce agrochemicals application in rice cultivation. The present study evaluates diazinon-degrading endophytic bacteria, isolated from rice plants grown in the fields with pesticide amalgamation, leading to increased productivity in high-yielding rice plants. These endophytes showed capabilities of decomposing diazinon, confirmed by FT-IR spectra analysis. Growth promoting activities of these endophytes can be attributed to their abilities to produce an increased level of IAA content and to demonstrate high level ACC-deaminase activities. Furthermore, these endophytes demonstrated enhanced level of extracellular cellulase, xylanase, amylase, protease and lignin degrading activities. Five genera including Enterobacter, Pantoea, Shigella, Acinetobacter, and Serratia, are represented only by the leaves, while four genera such as Enterobacter, Escherichia, Kosakonia, and Pseudomonas are represented only by the shoots. Five genera including, Klebsiella, Enterobacter, Pseudomonas, Burkholderia, and Bacillus are represented only by the roots of rice plants. All these strains demonstrated cell wall hydrolytic enzyme activities, except pectinase. All treatments, either individual strains or consortia of strains, enhanced rice plant growth at germination, seedling, vegetative and reproductive stages. Among four (I-IV) consortia, consortium-III generated the maximum rice yield under 70% lower doses of urea compared to that of control (treated with only fertilizer). The decoded genome of Klebsiella sp. HSTU-F2D4R revealed nif-cluster, chemotaxis, phosphates, biofilm formation, and organophosphorus insecticide-degrading genes. Sufficient insecticide-degrading proteins belonging to strain HSTU-F2D4R had interacted with diazinon, confirmed in molecular docking and formed potential catalytic triads, suggesting the strains have bioremediation potential with biofertilizer applications in rice cultivation.

The posttranscriptional regulator CsrA affects multidrug resistance and biocontrol activity in Lysobacter enzymogenes.

The posttranscriptional regulator CsrA regulates many cellular processes, including stress responses in diverse bacteria. However, the role of CsrA in multidrug resistance (MDR) and biocontrol activity in Lysobacter enzymogenes strain C3 (LeC3) remains unknown. In this study, we demonstrated that deletion of the csrA gene resulted in the initial slow growth of LeC3 and reduced its resistance to multiple antibiotics, including nalidixic acid (NAL), rifampicin (RIF), kanamycin (Km), and nitrofurantoin (NIT). Loss of the csrA gene also reduced its ability in inhibiting hypha growth of Sclerotium sclerotiorum and influenced its extracellular cellulase and protease activities. Two putative small noncoding regulatory RNAs (sRNAs), referred to as csrB and csrC, were also revealed in the genome of LeC3. Double deletion of csrB and csrC in LeC3 led to increased resistance to NAL, RIF, Km, and NIT. However, no difference was observed between LeC3 and the csrB/csrC double mutant in their suppression of S. sclerotiorum hypha growth and production of extracellular enzymes. These results suggest that CsrA in LeC3 not only conferred its intrinsic MDR, but also contributed to its biocontrol activity.

Evaluation of Non-Conventional Yeasts Isolated from Rotten Wood for Hydrolytic Activities and Xylose Fermentation

Hydrolysis of lignocellulose to fermentable sugars and their subsequent conversion to ethanol remain great challenges in the biofuel industry. Rotten wood is first colonized by bacteria and molds that possess strong hydrolases. Yeasts are also an important group of microorganisms that may participate in wood hydrolysis. Decaying wood could provide a rich natural reservoir of yeasts possessing promising hydrolytic activities, including xylanases, cellulases, β-glucosidases, or abilities essential for the fermentation of pentose sugars derived from lignocellulose degradation, especially xylose. Therefore, the aim of this work was to screen yeasts isolated from rotten wood samples for the production of hydrolytic enzymes directed at lignocellulose components and the ability to ferment xylose, L-arabinose, and cellobiose. Methods. Yeast strains were isolated from 22 samples of rotten wood and identified by phenotypic characteristics according to Kurtzman et al. Hydrolytic properties and the ability of the isolated strains to ferment xylose, L-arabinose, and cellobiose were determined using conventional methods. Results. 30 strains of yeasts and yeast-like micromycetes were isolated from 22 samples of rotten wood in the Holosiivskyi Forest, Kyiv. Based on phenotypic properties, most of the isolated yeasts belonged to ascomycetous yeasts and were represented by the following genera: Candida (8 strains), Debaryomyces (5 strains), Kluyveromyces (5 strains), Pichia (5 strains), Scheffersomyces (2 strains), Lachancea, Hanseniaspora, Saccharomyces, and Geotrichum/Galactomyces. A strain of yeast-like non-photosynthetic alga Prototheca sp. was also detected. Most of the isolated microfungi (66.6% isolates) exhibited extracellular β-glucosidase activity, two Candida tropicalis strains possessed weak pectinase and xylanase activity. None of the isolates demonstrated extracellular cellulase activity. Two yeast strains preliminarily identified as Scheffersomyces stipitis were able to ferment xylose at a concentration of 20—100 g/L over a wide temperature range up to 37°C. Acetic acid at 0.25—1% (v/v) concentration resulted in the complete inhibition of xylose fermentation. Ethanol production from xylose up to 6 g/L was observed under the microaerobic fermentation conditions for 24 hr at the substrate concentration 40 g/L, but the subsequent fermentation resulted in decreasing ethanol concentration presumably due to ethanol re-assimilation. None of the isolated strains was capable of fermenting cellobiose or L-arabinose under the microaerobic conditions. Conclusions. This work provides the characterization of yeast microbiota of rotten wood that was represented predominantly by ascomycetous yeasts. The dominant extracellular hydrolytic activity of the isolates was β-glucosidase. This is the first report on the isolation of xylose-fermenting yeasts Scheffersomyces stipitis in Ukraine, which comprised 7% of all the microfungi isolated from rotten wood.

Increasing extracellular cellulase activity of the recombinant Saccharomyces cerevisiae by engineering cell wall-related proteins for improved consolidated processing of carbon neutral lignocellulosic biomass

Sustainable bioproduction usingcarbon neutral feedstocks, especially lignocellulosic biomass, has attracted increasing attention due to concern over climate change and carbon reduction. Consolidated bioprocessing (CBP) of lignocellulosic biomass using recombinantyeast of Saccharomyces cerevisiaeis a promising strategy forlignocellulosic biorefinery. However, the economic viability is restricted by low enzyme secretion levels.For more efficient CBP, MIG1spsc01isolated from the industrial yeast which encodes the glucose repression regulator derivative was overexpressed. Increased extracellular cellobiohydrolase (CBH) activity was observed with unexpectedly decreased cell wall integrity. Further studies revealed that disruption ofCWP2, YGP1, andUTH1,which are functionally related toMIG1spsc01, also enhanced CBH secretion. Subsequently, improved cellulase production was achieved by simultaneous disruption ofYGP1and overexpression ofSED5, which remarkably increased extracellular CBH activity of 2.2-fold over the control strain. These results provide a novel strategy to improve the CBP yeast for bioconversion of carbon neutral biomass.

Increasing Extracellular Cellulase Activity of the Recombinant Saccharomyces Cerevisiae by Engineering Cell Wall Related Proteins for Improved Consolidated Processing

Increasing Extracellular Cellulase Activity of the Recombinant Saccharomyces Cerevisiae by Engineering Cell Wall Related Proteins for Improved Consolidated Processing

CRISPR/Cas9-mediated genome editing directed by a 5S rRNA\u2013tRNAGly hybrid promoter in the thermophilic filamentous fungus Humicola insolens

BackgroundHumicola insolens is a filamentous fungus with high potential of producing neutral and heat- and alkali-resistant cellulase. However, the genetic engineering tools, particularly the genome-editing tool, are scarce, hindering the study of cellulase expression regulation in this organism.ResultsHerein, a CRISPR/Cas9 genome-editing system was established in H. insolens based on a hybrid 5S rRNA–tRNAGly promoter. This system is superior to the HDV (hepatitis delta virus) system in genome editing, allowing highly efficient single gene destruction in H. insolens with rates of deletion up to 84.1% (37/44). With this system, a putative pigment synthesis gene pks and the transcription factor xyr1 gene were disrupted with high efficiency. Moreover, the extracellular protein concentration and cellulase activity largely decreased when xyr1 was deleted, demonstrating for the first time that Xyr1 plays an important role in cellulase expression regulation.ConclusionsThe established CRISPR/Cas9 system is a powerful genetic operation tool for H. insolens, which will accelerate studies on the regulation mechanism of cellulase expression and engineering of H. insolens for higher cellulase production.

Deletion of the small GTPase rac1 in Trichoderma reesei provokes hyperbranching and impacts growth and cellulase production

BackgroundTrichoderma reesei is widely known for its enormous protein secretion capacity and as an industrially relevant producer of cellulases and hemicellulases. Over the last decades, rational strain engineering was applied to further enhance homologous and heterologous enzyme yields. The introduction of hyperbranching is believed to increase protein secretion, since most exocytosis is located at the hyphal apical tip. There are several genetic modifications which can cause hyperbranching, for example the deletion of the small Rho GTPase rac. Rac plays a crucial role in actin dynamics and is involved in polarisation of the cell during germination and apical extension of the hyphae.ResultsWe deleted rac1 in a T. reesei strain with an ectopically overexpressed endoglucanase, CEL12A, under Pcdna1 control. This deletion provoked a hyperbranching phenotype and strong apolar growth during germination and in mature hyphae. The strains displayed dichotomous branching and shorter total mycelium length with a larger hyphal diameter. Δrac1 strains exhibited a decreased radial growth on solid media. Biomass formation in liquid cultures was carbon source dependent; similar to the reference strain during growth on lactose, increased on d-glucose and slightly enhanced on cellulose. While extracellular cellulase activities remained at parental strain levels on d-glucose and cellulose, the specific activity on lactose cultures was increased up to three times at 72 h accompanied by an upregulation of transcription of the main cellulases. Although the morphology of the Δrac1 strains was considerably altered, the viscosity of the culture broth in fed-batch cultivations were not significantly different in comparison to the parental strain.ConclusionsDeletion of the small Rho GTPase rac1 changes the morphology of the hyphae and provokes hyperbranching without affecting viscosity, independent of the carbon source. In contrast, biomass formation and cellulase production are altered in a carbon source dependent manner in the Δrac1 strains.

Identification and characterization of an Endo-glucanase secreted from cellulolytic Escherichia coli ZH-4

BackgroundIn the previous study, the cellulolytic Escherichia coli ZH-4 isolated from bovine rumen was found to show extracellular cellulase activity and could degrade cellulose in the culture. The goal of this work was to identify and characterize the secreted cellulase of E. coli ZH-4. It will be helpful to re-understand E. coli and extend its application in industry.ResultsA secreted cellulase was confirmed to be endo-glucanase BcsZ which was encoded by bcsZ gene and located in the cellulose synthase operon bcsABZC in cellulolytic E. coli ZH-4 by western blotting. Characterization of BcsZ indicated that a broad range of pH and temperature tolerance with optima at pH 6.0 and 50 °C, respectively. The apparent Michaelis–Menten constant (Km) and maximal reaction rate (Vmax) for BcsZ were 8.86 mg/mL and 0.3 μM/min·mg, respectively. Enzyme activity of BcsZ was enhanced by Mg2+ and inhibited by Zn2+, Cu2+ and Fe3+. BcsZ could hydrolyze carboxymethylcellulose (CMC) to produce cello-oligosaccharides, cellotriose, cellobiose and glucose.ConclusionsIt is confirmed that extracellular cellulolytic capability of E. coli ZH-4 was attributed to BcsZ, which explained why E. coli ZH-4 can grow on cellulose. The endo-glucanase BcsZ from E. coli-ZH4 has some new characteristics which will extend the understanding of endo-glucanase. Analysis of the secretion characteristics of BcsZ provided a great reference for applying E. coli in multiple industrial fields.

Cellulolytic and Xylanolytic Actinomycetes selection to degrade Lignocellulosic biomass of Robusta coffee pulp (Coffea canephora)

Actinomycetes are bacteria that have a high ability to produce hydrolytic enzymes that can be applied to degrade coffee pulp which contains lignocellulose components. Coffee pulp is residues from a coffee industry that can be used as substrates in the solid state fermentation process. In this study, cellulase and xylanase activity from actinomycetes isolates were investigated. Screening of these enzymes activity was using agar medium supplemented with 1% carboxymethylcellulose (CMC) and xylan from beechwood as substrate. Production of clear zones surrounding isolates on CMC and xylan agar medium was considered as indicative of extracellular cellulase and xylanase activity. The size of a transparent zone diameter was considered as proportional to the level of cellulase and xylanase production. Congo red 0,1% was used for staining. Selected actinomycetes isolates were continued to growth investigation on agar medium supplemented with 1% coffee pulp. The clear zone formed around isolates indicated that the isolate had the ability to degrade the lignocellulose component in the coffee pulp. The results show that of the 99 actinomycetes isolates tested on agar medium supplemented with 0.1% CMC and xylan, 16 isolates showed cellulase activity (cellulase index ≥ 2), 20 isolates showed xylanase activity (xylanase index ≥ 1), and 5 isolates showed both activities. Ten isolates have the ability to grow and produce a clear zone on the coffee pulp agar medium with cellulase and xylanase activity. Among these ten tested isolates; HJ4.5b, P2b(b).3, P2b(b).4, and P2b(b).18 are the most potential isolates that can be continued for further research on fermentation coffee cherries.

Genomic characterization of a pectinolytic isolate of Serratia oryzae isolated from lake water.

Only one isolate of Serratia oryzae, the type strain J11-6T has been characterized up to now. This strain was found in the endophytic bacterial flora of rice. As part of an ongoing investigation into pectinolytic bacteria present in lake water in France, a few Serratia strains were isolated, including S32 and J9 identified as new strains of S. oryzae. The genome of strain S32 consists of a circular chromosome of 4,810,389 bp that contains 4,584 protein-coding genes. The genome of S32, as well as those of the type strain J11-6T, contains several genes involved in pectin degradation and in the intracellular assimilation of pectin oligomers. The specific detection of enzyme activities confirmed that strain S32 secretes functional pectinases and that it also produces extracellular cellulase and protease activities. The ability to produce plant cell wall degrading enzymes shows that S. oryzae shares characteristics of plant associated bacteria, including phytopathogens.

Screening and characterization of Xanthomonas oryzae pv. oryzae strains with resistance to pheazine-1-carboxylic acid

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial leaf blight (BLB) and can be effectively controlled by phenazine-1-carboxylic acid (PCA), an antibiotic secreted by Pseudomonas spp. PCA resistance in Xoo was investigated in this research. Only four PCA-resistant strains were obtained by extensive screening, and the resistance was genetically stable in only one of them (P4). P4 was also resistant to phenazine and 1-hydroxyphezine but not to captan, bismerthiazol, or streptomycin. The following were reduced in P4 relative to the parental wild type: growth, virulence, EPS production, extracellular cellulase production and activity, biofilm formation, and swimming ability. ROS accumulation was reduced, resistance to exogenous H2O2 was increased, and expression of catalase genes and catalase activities were increased in P4, suggesting that PCA resistance in P4 results from a reduction in ROS production and/or an increased ability to metabolize ROS following PCA treatment. Given the low risk of Xoo developing PCA resistance and the reduced virulence and fitness of the resistant strain, PCA can be used in alternation with other common bactericides to control BLB in rice fields.

Diversity Analysis and Bioresource Characterization of Halophilic Bacteria Isolated from a South African Saltpan.

Though intensive research has been channeled towards the biotechnological applications of halophiles and other extremophilic microbes, these studies have not been, by any means, exhaustive. Saline environments still offer a vast diversity of microbes with potential to produce an array of natural products which can only be unlocked by concerted research efforts. In this study, a combination of culture and molecular approaches were employed to characterize halophilic bacteria from saltpan water samples and profile their potential biotechnological applications. Physicochemical analysis of the water samples showed that pH was alkaline (pH 8.8), with a salinity of 12.8%. 16S rRNA gene targeted amplicon analysis produced 10 bacterial phyla constituting of Bacteroidetes (30.57%), Proteobacteria (15.27%), Actinobacteria (9.05%), Planctomycetes (5.52%) and Cyanobacteria (3.18%). Eighteen strains were identified using sequencing analysis of the culturable bacterial strains. From these, the strains SP7 and SP9 were positive for cellulase production while the strains SP4, SP8 and SP22 were positive for lipase production. Quantitative enzyme assays showed moderate extracellular cellulase activity (1.95 U/mL) and lipase activity (3.71 U/mL) by the isolate SP9 and SP4 respectively. Further, of the six isolates, the isolate SP9 exhibited exploitable potential in the bioremediation of hydrocarbon pollution as demonstrated by its fairly high activity against benzanthracene (70% DCPIP reduction). Elucidation of the isolates secondary metabolites showed the production of the molecules 2,3-butanediol, hexahydro-3-(2-methylpropyl)pyrrole[1,2a]pyrazine-1,4-dione, aziridine, dimethylamine and ethyl acetate (GC-MS) and oxypurinol and 5-hydroxydecanoic acid (LC-MS), particularly by the isolate Salinivibrio sp. SP9. Overall, the study showed that the isolated halophiles can produce secondary metabolites with potential industrial and pharmaceutical application.

Disruption of comA homolog in Ralstonia solanacearum does not impair its twitching motility.

Ralstonia solanacearum is an important phyto-pathogenic bacterium. The bacterium exhibits type IV pili meditated twitching motility that has been implicated in the process of natural transformation in it. A comA gene homolog, alike in several other naturally competent bacteria, has been already reported in this bacterium. However, there are no report of direct link between comA and twitching motility during the natural transformation process in this pathogen. In order to figure out any connection between comA and twitching motility, we created an insertion mutation in comA gene homolog of R. solanacearum F1C1 strain. As anticipated, the insertion mutant (CBRS01 strain) was inefficient for natural transformation. CBRS01 strain was found to be proficient for twitching motility alike the wild-type F1C1. This is interesting since recent findings of Salzer et al. (2016;Environ Microbiol;18:65-74) showed deficiency of twitching motility due to comEC gene (comA homolog) mutation in another naturally competent Gram-negative bacterium Thermus thermophilus. Additionally, we also found CBRS01 strain to be proficient for extracellular cellulase activity and virulence on tomato seedlings. Our findings in this work indicate that an R. solanacearum strain inefficient in undergoing natural transformation can, however, be proficient in exhibiting twitching motility.

Relationship Between Extracellular Cellulase, Pectinase and Xylanase Activity of Isolated Aspergillus oryzae Strains Grown on Koji and the Umami-Tasting Amino Acid Content of Soy Sauce

ABSTRACTWe compared the koji-based extracellular carboxymethylcellulases (CM-cellulase), pectinases and xylanases of four Aspergillus oryzae isolates from soy sauce koji, A1, PRB-1 (A2), A3 and A4, with Aspergillus oryzae AS3.951 (A5) via enzyme assays and zymography and determined their correlation to the content of umami-tasting amino acids in high-salt diluted-solid state growth stage leading to soy sauce. In A2 koji, xylanase had the highest activity (28.75 U/g dry weight). Zymogram analysis revealed two different bands of CM-cellulase activity (termed Cel-1 and Cel-2) and one band of pectinase activity (Pec-1) in all koji samples. The intensity of these bands was comparable among all five strains. Interestingly, the same analysis revealed six different xylanase activity bands (termed Xyl-1 to Xyl-6) in all koji samples. After 60 days of cultivation, the free amino acid concentration was highest in soy sauce generated by A4 at 6709.41 mg/100 ml and contained 1690.82 mg/100 ml umami-tasting amino acids (including glutamic acid and aspartic acid), followed by soy sauce generated by A2. Pearson’s coefficient analysis indicated that Xyl-1 and Xyl-2 activities were positively associated with the umami-tasting amino acid content of soy sauce. In conclusion, these data suggested that the xylanases Xyl-1 and Xyl-2 in koji could be important contributors to the umami taste formation of soy sauce.

Identification and characterization of putative xylose and cellobiose transporters in Aspergillus nidulans.

BackgroundThe conversion of lignocellulosic biomass to biofuels (second-generation biofuel production) is an environmentally friendlier alternative to petroleum-based energy sources. Enzymatic deconstruction of lignocellulose, catalyzed by filamentous fungi such as Aspergillus nidulans, releases a mixture of mono- and polysaccharides, including hexose (glucose) and pentose (xylose) sugars, cellodextrins (cellobiose), and xylooligosaccharides (xylobiose). These sugars can subsequently be fermented by yeast cells to ethanol. One of the major drawbacks in this process lies in the inability of yeast, such as Saccharomyces cerevisiae, to successfully internalize sugars other than glucose. The aim of this study was, therefore, to screen the genome of A. nidulans, which encodes a multitude of sugar transporters, for transporters able to internalize non-glucose sugars and characterize them when introduced into S. cerevisiae.ResultsThis work identified two proteins in A. nidulans, CltA and CltB, with roles in cellobiose transport and cellulose signaling, respectively. CltA, when introduced into S. cerevisiae, conferred growth on low and high concentrations of cellobiose. Deletion of cltB resulted in reduced growth and extracellular cellulase activity in A. nidulans in the presence of cellobiose. CltB, when introduced into S. cerevisiae, was not able to confer growth on cellobiose, suggesting that this protein is a sensor rather than a transporter. However, we have shown that the introduction of additional functional copies of CltB increases the growth in the presence of low concentrations of cellobiose, strongly indicating CltB is able to transport cellobiose. Furthermore, a previously identified glucose transporter, HxtB, was also found to be a major xylose transporter in A. nidulans. In S. cerevisiae, HxtB conferred growth on xylose which was accompanied by ethanol production.ConclusionsThis work identified a cellobiose transporter, a xylose transporter, and a putative cellulose transceptor in A. nidulans. This is the first time that a sensor role for a protein in A. nidulans has been proposed. Both transporters are also able to transport glucose, highlighting the preference of A. nidulans for this carbon source. This work provides a basis for future studies which aim at characterizing and/or genetically engineering Aspergillus spp. transporters, which, in addition to glucose, can also internalize other carbon sources, to improve transport and fermentation of non-glucose sugars in S. cerevisiae.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0611-1) contains supplementary material, which is available to authorized users.

Production and Partial Characterization of Cellulase from Pseudomonas Isolates Obtained from Cow Dung and Municipal Solid Wastes

Cellulase producing bacteria were isolated from cow dung and municipal solid wastes using enrichment technique. Cellulase activity was determined on carboxymethyl cellulose (CMC) agar medium supplemented with 1% CMC. Production of clear zones by the bacterial isolates on CMC agar was considered as indicative of extracellular cellulase activity. The greater size of transparent zone diameter was found proportional to the higher amount of cellulase production. Two bacterial isolates producing significant clear zone were identified as Pseudomonas spp based on morphological, cultural and biochemical characteristics. Fermentation was carried out under shake flask conditions for production of cellulase from Pseudomonas isolates in a basal medium containing CMC, KH2PO4, K2HPO4, MgSO4, NH4SO4, CaCl2 and FeSO4 at pH 7.0. The assay of cellulase viz. endoglucanase and exoglucanase in terms of CMCase and FPase, respectively was done by measuring the release of the reducing sugar. Some parameters influencing the production of cellulase by these bacterial isolates were investigated. Optimum level of cellulase was produced after 48 h of fermentation at 37°C with 5% inoculums size under continuous agitation at 120 rpm in the growth medium of pH 7.0. Optimum temperature and pH for the activity of cellulase from these bacterial isolates was 40°C and 7.0, respectively. Cellulase from these isolates was found almost stable at 25-40°C and pH 6.0-8.0 for 1 h. Furthermore, the cellulase activity was stimulated by Ca2+ and K+ but inhibited by Hg2+ and Zn2+, whereas, Mg2+ showed very low effect. Results reported herein indicated that the Pseudomonas isolates can be used as producer of extracellular cellulase.Bangladesh J Microbiol, Volume 30, Number 1-2,June-Dec 2013, pp 11-16

The Endo-β-1,4-Glucanase of Bacillus amyloliquefaciens Is Required for Optimum Endophytic Colonization of Plants.

The eglS gene in Bacillus amyloliquefaciens encodes an endo-β-1,4-glucanase that belongs to glycosyl hydrolase family 5. In this study, a disruption mutant of gene eglS was constructed to examine its role in bacterial adaptation in plants. The mutant TB2k, eglS gene inactivated bacterial strain, was remarkably impaired in extracellular cellulase activity. When inoculated on Brassica campestris, the TB2k population was reduced by more than 60% compared with the wild-type strain in the root, stem, and leaf tissues. Overexpression of eglS in the wild-type strain increased the bacteria population in the plant tissues. Further studies revealed that the transcription level of eglS was correlated with bacterial population. These data demonstrate that endo-β-1,4-glucanase of B. amyloliquefaciens is required for its optimal endophytic colonization.

Isolation, Screening and Characterization of Cellulase Producing Bacterial Isolates from Municipal Solid Wastes and Rice Straw Wastes

Cellulolytic bacteria were isolated and screened from municipal solid wastes and rice straw wastes using Carboxy Methyl Cellulose (CMC) agar medium as a selective medium. Production of clear zones by the bacterial isolates on CMC agar medium supplemented with 1% CMC was considered as indicative of extracellular cellulase activity. The size of transparent zone diameter was considered as proportional to the level of cellulase production. These bacterial isolates were identified as Bacillus sp., Pseudomonas sp. and Serratia sp. based on morphological, cultural and biochemical characteristics. A basal medium containing CMC, KH2PO4, K2HPO4, MgSO4, (NH4)2SO4, CaCl2 and FeSO4 at pH 7.0 was used for cellulase production. The assay of cellulase in term of CMCase was performed by measuring the release of reducing sugar. Different physicochemical parameters were optimized for cellulase production at shake flask fermentation. Time course study revealed that maximum level of cellulase was produced by Bacillus and Serratia isolates after 24 h of cultivation and by Pseudomonas isolates after 42 h of cultivation. Optimum level of cellulase was produced by Pseudomonas and Bacillus isolates at 37°C, and that by Serratia isolate was at 35°C. Optimum pH for cellulase production by these bacterial isolates was 7.0. Optimum temperature and pH for the activity of cellulase from these isolates were 40°C and 7.0, respectively. The cellulase from these isolates was found almost stable up to 55°C and at pH 7.0 for 1 h. The crude cellulase could liberate reducing sugar from filter paper through hydrolysis. Results showed that bacterial isolates produced significant level of cellulase with cellulose degrading capability.

Improvement of Aspergillus oryzae NRRL 3484 by mutagenesis and optimization of culture conditions in solid-state fermentation for the hyper-production of extracellular cellulase.

Spore suspensions of Aspergillus oryzae NRRL 3484 were subjected to mutagenesis using ultraviolet-irradiation followed by chemical treatments to improve the biosynthesis of cellulase. Ten mutant strains namely UEAC7, UEAR5, UNAC4, UNAC16, UNAR19, UNBC7, UNBR3, UNBR10, UNBR23 and UNBR25 were selected and their extracellular cellulase activities were assayed. Mutant UNAC4 gave the highest cellulase production [2,455±28U/g-dry substrate (ds) for filter paper-ase (FP-ase)] in a yield 4-fold exceeding that of the wild type strain (578±5.0U/g-ds for FP-ase). Rice straw (RS) was used as a sole carbon source for the enzyme production at a concentration of 10% (w/v). Maximum cellulase production was achieved at initial medium pH 5.5, initial moisture content 77% and an incubation temperature 28°C on the fifth day of growth. NH4Cl proved to be the suitable added nitrogen source for maximum enzyme production followed by peptone. These results clearly indicate the cost-effectiveness of solid state fermentation technology in the economic production of extracellular cellulase. The hyper-production of cellulase by mutant strain UNAC4 has potential for industrial processes that convert lignocellulosic material (e.g. RS) into products of commercial value such as glucose and biofuels.

A comparison of plate assay methods for detecting extracellular cellulase and xylanase activity

Identification of microorganisms for the production of carbohydrolytic enzymes is extremely important given the increased demand for these enzymes in many industries. To this end, dye–polysaccharide interactions which provide a visual indication of polymer hydrolysis (clear zones or halos) have been used for decades. For the detection of extracellular cellulase or xylanase activity many laboratories use Gram's iodine as the chromogenic dye, as it is a more rapid initial screening method compared to the use of other dyes. Here, we compared Gram's iodine and Congo red as indicators of polysaccharide hydrolysis. We attempted to detect cellulase activity using carboxymethylcellulose, and xylanase activity using birchwood xylan, in fourteen uncharacterized bacteria isolated from wood chips. Our results indicate that Gram's iodine may lead to identification of false positives in a typical screening protocol and that Congo red allows for avoidance of such pitfall. Congo red allowed detection of cellulase activity from live microbial colonies but not Gram's iodine. To confirm this, detection of enzymatic activity was also assessed using cell-free enzyme preparations. Congo red was found to be reliable in detecting cellulase activity with isolated enzymes preparations. Under the same conditions, neither of these dyes detected xylanase activity, despite independent evidence of xylanase activity for one of the preparations. We detected xylanase activity for this particular enzyme preparation using a coloured derivative of xylan (Remazol Brillant Blue R-xylan adduct) that respond to xylan hydrolysis. Our results suggest that methods that rely on interactions between a dye (Congo red or Gram's iodine) and a polymeric substrate (carboxymethylcellulose or birchwood xylan) for indirect detection of hydrolysis may require the use of relevant controls and independent confirmation of enzymatic activities.