Enhanced production of cellulases byCellulomonas strains grown on different cellulosic residues

Cellulomonas strains consumed commercial cellulose, cellulosic residues, xylan, cellobiose and carboxymethyl cellulose (CMC) as carbon sources in liquid culture, the growth being the most on cellobiose medium. All three components of the cellulase complex ofCellulomonas were produced when the organisms utilized all substrates as sole carbon and energy sources. The filter-paper cellulase (FPase) and endo-glucanase (CMCase) activities were higher in media containing α-cellulose and cellulosic residues than in media containing CMC, cellobiose, and xylan. Cell-free supernatants of all organisms exhibited greater CMC hydrolyzing activity than filter paper and β-glucoside hydrolyzing activities. All strains synthesized β-glucosidase maximally on cellobiose followed by commercial cellulose and cellulosic residues.C. biazotea produced the highest FPase and CMCase activity during growth on α-cellulose. It was followed byC. flavigena, C. cellasea, andC. fimi. Endo-glucanase and FPase from all organisms were secreted into the medium; 10–13 % became adsorbed on the surface of the insoluble substrates and could be successfully eluted using Tween 80. β-Glucosidase was located in cell extracts from all organisms.C. biazotea produced FPase and β-glucosidase activities several-fold greater than those produced by many other strains ofCellulomonas and some other cellulolytic bacteria and fungi. These studies were supported byPakistan Atomic Energy Commission. Some chemicals were purchased from funds allocated byUnited States Agency for International Development, Washington (DC, USA), under PSTC proposal 6.163.     

Isolation of Cellulolytic Fungi from Waste of Castor (<Emphasis Type="Italic">Ricinus communis</Emphasis> L.)

This is the first report of isolation of fungi present in fatty and defatted castor bean meal as well as the first of crop’s selection to test the cellulolytic potential, in order to verify the diversity and potential of cellulolytic fungi in castor bean waste (Ricinus communis L.). For the screening on solid medium, it was used carboxymethylcellulose (CMC) as the sole carbon source. The microcrystalline cellulose (Avicel) was used as a substrate for submerged fermentation for production of cellobiohydrolase (FPase) and the CMC to produce endoglucanases (CMCase) and β-glycosidases (BG). 189 cultures of fungi were isolated, including 40 species of filamentous fungi and three yeasts. The Aspergillus was the most frequent found genus. Regarding the distribution of isolated species from defatted castor bean meal, the A. niger was the most frequent one; and within the fatty castor bean meal, the Emericela variecolor prevailed among other species. Among the 67 fungal cultures tested in the initial screening on solid media to assess the cellulolytic potential, 54 disclosed Cellulolytic Index (CI) ranging from 1.04 to 6.00 mm. The isolates were selected for enzyme production in liquid medium with values above 2.0 CI. They were obtained with A. japonicus URM5620 FPase activity (4.99 U/ml) and BG (0.05 U/ml), and Rhodotorula glutinis URM5724 activity of CMCase 3.58 U/ml. These cases occurred after 168 h of submersion for both species of fungi. In our study, we could conclude that the castor bean is a promising source of fungi capable of producing cellulolytic enzymes.     

Probiotic Potential of the Farmed Olive Flounder,Paralichthys olivaceus,Autochthonous Gut Microbiota

In recent years, considerable and growing attention has been given to the application of host-associated microorganisms as a more suitable source of probiotics in aquaculture sector. Herein, we isolated and screened the olive flounder gut microbiota for beneficial bacterial strains that might serve as potential probiotics in a low fishmeal extruded aquafeed. Among the ten identified isolates, Bacillus amyloliquefaciens SK4079 and B. subtilis SK4082 were screened out based on their heat-resistant ability as well as enzymatic and non-hemolytic activities. Although both strains were well able to utilize carboxymethyl cellulose (CMC), xylan, and soybean meal (SBM) as a single carbon source in the minimal nutrient M9 medium, B. subtilis exhibited significantly higher cellulase, xylanase, and protease activities than B. amyloliquefaciens. The two selected strains were well able to degrade the undesirable anti-nutritional component of the SBM, which would limit its utilization as protein source in aquafeed industry. Significantly higher biofilm formation capacity and notably stronger adhesive interactions with the flounder’s skin mucus were detected in B. subtilis than B. amyloliquefaciens. Immobilization of the spores from the selected strains, in a SBM complex carrier, remarkably enhances their thermal resistance at 120 °C for 5 min and different drying conditions. It was also interesting to learn that the B. subtilis spores could survive and remain viable after being sprayed onto extruded low-fish meal feed pellets for as long as 6 months. Overall, the findings of the present study could help the food/feed industries achieve their goal of developing cost-effective yet efficient products.

     

白蚁肠道内纤维素酶产生菌的筛选及其酶活性

从黑翅土白蚁(Odontotermes formosanus Shiraki)肠道内筛选具有纤维素降解能力的细菌,并研究其酶活性。结果表明:筛选得到5株菌株,活力较高的菌株CMC-4被鉴定为土白蚁特拉布尔希氏菌Z-4(Trabulsiella odontotermitis ZJSRU-4)。同时对菌株T.odontotermitis ZJSRU-4进行了系统的研究,它具有完整的纤维素酶系统,主要产羧甲基纤维素酶(CMCase)和β-葡萄糖苷酶,滤纸酶的活力较低。在以羧甲基纤维素钠为碳源的培养基中培养36 h,发酵液中CMCase的比酶活达到20.8 U/m L,培养44 h,β-糖苷酶的比酶活达到18.2 U/m L。CMCase和β-葡萄糖苷酶作用的p H分别为6.0和6.5,它们作用的最适温度都为40℃。该菌对纤维质原料具有降解能力,具有潜在的应用价值。     

Characterization of cellulose degrading bacteria from the larval gut of the white grub beetle Lepidiota mansueta (Coleoptera: Scarabaeidae)

The goal of this study is to identify and characterize the cellulose degrading microorganisms in the larval gut of the white grub beetle, Lepidiota mansueta. Thirty bacterial strains were isolated and tested for cellulolytic activity using soluble carboxymethyl cellulose (CMC) degrading assays. Of these strains, five (FGB1, FB2, MB1, MB2, and HB1) degrade cellulose. Cellulolytic activity was determined based on formation of clear zone and cellulolytic index on CMC plate media. The highest cellulolytic index (2.14) was found in FGB1. Partial 16S rDNA sequencing, morphological, and biochemical tests were used to identify and characterize the five isolates, all Citrobacter sp. (Enterobacteriaceae). This study identifies new cellulose degrading microorganisms from the larval gut of L. mansueta. The significance of identifying these strains lies in possible application in cellulose degradation.     

Comparison of Cellulolytic Activities in Clostridium thermocellum and Three Thermophilic, Cellulolytic Anaerobes

Avicelase, carboxymethyl cellulase (CMCase), and β-glucosidase activities have been compared between Clostridium thermocellum and three extremely thermophilic, cellulolytic anaerobes, isolates TP8, TP11, and KT8. The three isolates were all small, gram-negative staining, oval-ended rods which occurred singly and, at exponential phase, in long chains. They were nonflagellated and no spores were visible. The KT8 and TP11 isolates caused clumping of the cellulose during growth. In all four organisms the CMCase activity paralleled cell growth; however, in C. thermocellum and TP8 the avicelase activity did not increase until early stationary phase. Total CMCase activity in C. thermocellum was significantly higher than in the three isolates; however, avicelase activities were much more comparable among the four organisms. C. thermocellum produced higher levels of ethanol, and all four organisms produced similar concentrations of acetate. The amounts of free and bound CMCase and avicelase activities were investigated. In C. thermocellum and TP8 most of the CMCase and avicelase activities were bound to the cellulose in the medium. In contrast, most of the CMCase activity in TP11 and KT8 was free in the culture supernatant; a significant percentage of avicelase activity was also free. The TP8 isolate was also grown on a defined medium with urea as sole nitrogen source and cellulose serving as the carbon source. Under these conditions the pattern of enzyme production was the same as that in the enriched medium, although the level of that production was considerably reduced.     

Isolation and selection of cellulolytic fungi from palm oil mill effluent

Cellulolytic fungi, 34 strains, were isolated from samples taken from palm oil mill residues and effluent, and high cellulase producers selected in comparison with nine known reference strains. Although 13 isolates showed good filter paper distintegration within 14 days, only eight isolates exhibited clearing zones around their colonies on carboxymethylcellulose (CMC) agar medium. Quantitative cellulase activity measurements, using CMC as carbon source, selected three of the eight isolates as potential cellulase producers. Using dried palm oil mill condensate as carbon source, only one of the isolates (F 11) showed similar results on both carbon sources. During media optimization for CMCase production, a four-fold increase from 0.058 to 0.275 U/ml was obtained using a medium, containing 0.1% (v/v) Tween 80 0.02% (w/v) NH₄NO₃, 0.025% (w/v) proteose-peptone and 0.1% (w/v) CMC dissolved in undiluted condensate from the sterilization of oil palm bunches, with an initial pH of 5.5.     

Cellulase production from <Emphasis Type="Italic">Pseudoalteromonas</Emphasis> sp. NO3 isolated from the sea squirt <Emphasis Type="Italic">Halocynthia rorentzi</Emphasis>

Pseudoalteromonas sp. NO3 was isolated from the hemolymph of diseased sea squirts (Halocynthia rorentzi) with symptoms of soft tunic syndrome. The strain was found to produce an extracellular cellulase (CelY) that consisted of a 1,476 bp open reading frame encoding 491 amino acid residues with an approximate molecular mass of 52 kDa. Homologies of the deduced amino acid sequence of celY with the products of the celA, celX, celG and cel5Z genes were 92.6, 93.3, 92.6, and 59.1%, respectively. Additionally, CelY had 50–80% remnant catalytic activity at temperatures of 10–20°C. Highest carboxymethyl cellulose (CMC) hydrolysis was observed at pH 8.0 and 40°C. CMC activity was determined by zymogram active staining and different degraded product profiles for CelY were obtained when cellotetraose, cellopentaose, and CMC were used as substrates. This study identified a transglycosylation activity in CelY that allows the enzyme to digest G4 to G2 and G3 without the production of G1.     

Saccharification of Parthenium hysterophorus biomass using cellulase from Streptomyces sp. NAA2

Parthenium hysterophorus biomass can be used as a non-conventional renewable feedstock for the production of bioethanol. Therefore, the present work was designed to hydrolyze P. hysterophorus biomass using cellulase enzyme produced from an actinomycete, i.e., Streptomyces sp. NAA2 using P. hysterophorus biomass as a substrate. The isolate NAA2 was identified by molecular characterization of 16SrDNA. The enzyme production by strain NAA2 was enhanced by optimization studies conducted under submerged fermentation conditions using P. hysterophorus as a substrate. The crude enzyme produced under optimized conditions was used to hydrolyze alkali-acid pretreated P. hysterophorus biomass. The highest CMCase production was achieved in 4–5 days when steam-pretreated P. hysterophorus biomass was used at 1% (w/v) concentration, using 2 discs (1 disc = 5 × 10⁷ spores/ml) of inoculum, an initial pH 6.5, temperature at 40 °C, an agitation speed of 120–150 rpm, and by supplementing fermentation medium with 1.5% (w/v) carboxymethyl cellulose (CMC) as additional carbon source. Under optimized conditions, the actinomycete strain NAA2 showed production of 0.967 ± 0.016 U/ml CMCase, 0.116 ± 0.08 FPU/ml FPase, and 0.22 ± 0.012 U/ml β-glucosidase enzymes. On utilizing the cellulase enzyme for biomass hydrolysis, maximum 18.2% saccharification yield (of cellulose 0.202 g/g) was achieved in 96 h when enzyme and substrate levels were 30 FPU/100 ml and 2% (w/v) respectively. Parthenium hysterophorus biomass can be hydrolyzed enzymatically yielding considerable amounts of total reducing sugars. It can, therefore, be used as a feedstock for the production of bioethanol. Also, it has the potential to act as a substrate for the production of cellulases. Furthermore, the improved cellulolytic potential of Streptomyces sp. NAA2 can be exploited in various industrial applications.

     

Degradation of cellulose by nitrogen-fixing strain of Bacillus polymyxa.

During an intensive screening programme, several strains of cellulolytic bacteria were isolated. One nitrogenase-positive strain able to degrade filter paper, Avicel cellulose, carboxymethyl cellulose and cellobiose was selected for further study. On the basis of biochemical characteristics and Mol % of G+C content, the selected strain was identified as Bacillus polymyxa. The highest production of the enzymes degrading filter paper (FP-ase) and carboxymethyl cellulose (CMCase) by B. polymyxa was observed in Park's medium suplemented with Avicel cellulose. The investigated strain of bacteria produced cellulosome-like structures as was shown by transmission electron microscopy.     

Screening and identification of newly isolated cellulose-degrading bacteria from the gut of xylophagous termite Microcerotermes diversus (Silvestri)

The aim of the present study was to isolate and characterize the cellulose-degrading bacteria from the gut of the local termite, Microcerotermes diversus (Silvestri), inhabiting the Khuzestan province of Iran. The microorganisms capable of growing in the liquid medium containing cellulose as the only source of carbon were isolated and their cellulolytic activity on CMC-containing media was confirmed by the congo red clearing zone assay. The isolates were identified based on biochemical characteristics and the phylogenetic analysis of 16S rRNA gene fragments. The results of the present study show that three cellulose-degrading bacteria isolated from local termite guts belonged to the genera Acinetobacter, Pseudomonas and Staphylococcus and four cellulose-degrading bacteria belonged to Enterobacteriaceae and Bacillaceae families. Several isolates recovered from separate termite Microcerotermes diversus samples closely clustered in phylogenetic trees indicating high similarity and the abundance of particular cellulolytic strains. Bacillus B5B and Acinetobacter L9B hydrolyzed cellulose faster than the other isolates (with CMCase activity of 1.47 and 1.22 U/mL, respectively). The stability of CMCase produced by Bacillus B5B over a broad range of pH and high temperature indicated that the enzyme may be of great commercial value.     

Screening of Rhizobacteria for Their Plant Growth Promotion Ability and Antagonism Against Damping off and Root Rot Diseases of Broad Bean (<Emphasis Type="Italic">Vicia faba</Emphasis> L.)

Development of microbial inoculants from rhizobacterial isolates with potential for plant growth promotion and root disease suppression require rigorous screening. Fifty-four (54) fluorescent pseudomonads, out of a large collection of rhizobacteria from broad bean fields of 20 different locations within Imphal valley of Manipur, were initially screened for antifungal activity against Macrophomina phaseolina and Rhizoctonia solani, of diseased roots of broad bean and also three other reference fungal pathogens of plant roots. Fifteen fluorescent pseudomonas isolates produced inhibition zone (8–29 mm) of the fungal growth in dual plate assay and IAA like substances (24.1–66.7 μg/ml) and soluble P (12.7–56.80 μg/ml) in broth culture. Among the isolates, RFP 36 caused a marked increase in seed germination, seedling biomass and control of the root borne pathogens of broad bean. PCR–RAPD analysis of these isolates along with five MTCC reference fluorescent pseudomonas strains indicated that the RFP-36 belonged to a distinct cluster and the PCR of its genomic DNA with antibiotic specific primers Phenazine-1-carboxylic acid and 2, 4-diacetyl phloroglucinol suggested possible occurrence of gene for the potent antibiotics. Overall, the result of the study indicated the potential of the isolate RFP 36 as a microbial inoculant with multiple functions for broad bean.     

The noncellulosomal family 48 cellobiohydrolase from Clostridium phytofermentans ISDg: heterologous expression, characterization, and processivity

Family 48 glycoside hydrolases (cellobiohydrolases) are among the most important cellulase components for crystalline cellulose hydrolysis mediated by cellulolytic bacteria. Open reading frame (Cphy_3368) of Clostridium phytofermentans ISDg encodes a putative family 48 glycoside hydrolase (CpCel48) with a family 3 cellulose-binding module. CpCel48 was successfully expressed as two soluble intracellular forms with or without a C-terminal His-tag in Escherichia coli and as a secretory active form in Bacillus subtilis. It was found that calcium ion enhanced activity and thermostability of the enzyme. CpCel48 had high activities of 15.1 U μmol⁻¹ on Avicel and 35.9 U μmol⁻¹ on regenerated amorphous cellulose (RAC) with cellobiose as a main product and cellotriose and cellotetraose as by-products. By contrast, it had very weak activities on soluble cellulose derivatives (e.g., carboxymethyl cellulose (CMC)) and did not significantly decrease the viscosity of the CMC solution. Cellotetraose was the smallest oligosaccharide substrate for CpCel48. Since processivity is a key characteristic for cellobiohydrolases, the new initial false/right attack model was developed for estimation of processivity by considering the enzyme's substrate specificity, the crystalline structure of homologous Cel48 enzymes, and the configuration of cellulose chains. The processivities of CpCel48 on Avicel and RAC were estimated to be ∼3.5 and 6.0, respectively. Heterologous expression of secretory active cellobiohydrolase in B. subtilis is an important step for developing recombinant cellulolytic B. subtilis strains for low-cost production of advanced biofuels from cellulosic materials in a single step.     

In situ identification of carboxymethyl cellulose-digesting bacteria in the rumen of cattle fed alfalfa or triticale

A method was developed and used to arrest and stain reducing sugars (glucose) produced by bacteria with cell-surface-associated carboxymethyl cellulase (CMCase) and endoglucanase activities (CMC bacteria) in the rumen of cows fed alfalfa or triticale. Precipitation of silver oxide on the surface of individual cells was observed using cellulolytic bacterial pure cultures with known CMCase activity and rumen mixed cultures. The CMC bacteria in the liquid and solid fractions of the rumen digesta were identified using fluorescence in situ hybridization (FISH) with currently available and newly designed oligonucleotide probes. The CMC bacteria contributed between 8.2% and 10.1% to the total bacterial cell numbers. Most of the CMC bacteria (75.2-78.5%) could be identified by FISH probing. The known cellulolytic populations Ruminococcus flavefaciens, R.?albus, and Fibrobacter succinogenes constituted 44.5-53.1% of the total. Other CMC bacteria identified hybridized with the probe Clo549 (11.2-23.0%) targeting members of an uncharacterized genus in Clostridia, the probe Inc852 (8.9-10.7%) targeting members of the family Incertae Sedis III and unclassified Clostridiales, and the probe But1243 (相似文献   

Cloning and expression of cellulase gene EG1 from Rhizopus stolonifer var. reflexus TP-02 in Escherichia coli

A novel gene, EG encoding enzymes involved in carboxymethyl cellulose (CMC) degradation was isolated, sequenced from the filamentous fungus Rhizopus stolonifer var. reflexus TP-02, and expressed in Escherichia coli BL21. The results showed that the gene amplified from the cDNA of the strain could be classified as the family of endoglucanase. During the fermentation process, the maximum endoglucanase activity (i.e. 0.715 IU/ml) of the recombinant bacteria was obtained at 36 h. The SDS–PAGE analysis on purified samples showed that a band with apparent molecular weight of about 40 kDa was detected after staining with Coomassie brilliant blue.     

In vitro fibrolytic potential of anaerobic rumen fungi from ruminants and non-ruminant herbivores

In the present study, anaerobic fungi were isolated from different ruminants and non-ruminants; i.e., cattle, buffalo, sheep, goats, wild bluebulls, elephants, deer, and zebras; and were identified as Anaeromyces, Orpinomyces, Caecomyces, Piromyces, and Neocallimastix sp., based on their morphological characteristics. These isolates possessed significant in vitro hydrolytic enzyme activities; however, an isolate of Caecomyces sp. from elephant was found to exhibit maximum activity, i.e., filter paper cellulase (Fpase; 21.4 mIU/ml), carboxymethyl cellulose (CMCase; 15.1 mIU/ml), cellobiase (37.4 mIU/ml), and xylanase (26.0 mIU/ml). Besides, this isolate also showed the significantly highest ability to digest plant cell-wall contents in vitro. The in vitro dry matter digestibility increased from 45.1 to 48.9% after 48 h of incubation, and the plant cell-wall contents, in terms of neutral detergent fiber and acid detergent fiber, decreased from 64.2 to 61.3% and from 31.3 to 29.6%, respectively. These results indicate that such fibrolytic ruminal fungal strains are prevalent in wild herbivores such as elephants, as well as in other ruminants and non-ruminants, and could be exploited as microbial feed additives for improved nutrition and productivity in domesticated ruminants.     

Copper,Zinc-Superoxide Dismutase from Clinically Isolated <Emphasis Type="Italic">Escherichia coli</Emphasis>: Cloning,Analysis of <Emphasis Type="Italic">sodC</Emphasis> and Its Possible Role in Pathogenicity

Superoxide dismutase has been discovered within the periplasm of several Gram-negative pathogens. We studied the Cu,Zn-SOD enzyme in Escherichia coli isolated from clinical samples (stool samples) collected from patients suffering from diarrhea. Antibiogram studies of the isolates were carried out to determine the sensitive and resistant strains. The metal co-factor present in the enzyme was confirmed by running samples in native gels and inhibiting with 2 mM potassium cyanide. A 519 bp sodC gene was amplified from resistant and sensitive strains of Escherichia coli. Cloning and sequencing of the sodC gene indicated variation in the protein and amino acid sequences of sensitive and resistant isolates. The presence of sodC in highly resistant Escherichia coli isolates from diarrheal patients indicates that sodC may play role in enhancing the pathogenicity by protecting cells from exogenous sources of superoxide, such as the oxidative burst of phagocytes. The presence of SodC could be one of the factors for bacterial virulence.     

Resuscitation of viable but non‐culturable bacteria to enhance the cellulose‐degrading capability of bacterial community in composting

Nowadays, much of what we know regarding the isolated cellulolytic bacteria comes from the conventional plate separation techniques. However, the culturability of many bacterial species is controlled by resuscitation‐promoting factors (Rpfs) due to entering a viable but non‐culturable (VBNC) state. Therefore, in this study, Rpf from Micrococcus luteus was added in the culture medium to evaluate its role in bacterial isolation and enhanced effects on cellulose‐degrading capability of bacterial community in the compost. It was found that Proteobacteria and Actinobacteria were two main phyla in the compost sample. The introduction of Rpf could isolate some unique bacterial species. The cellulase activity of enrichment cultures with and without Rpf treatment revealed that Rpf treatment significantly enhanced cellulase activity. Ten isolates unique in Rpf addition displayed carboxymethyl‐cellulase (CMCase) activity, while six isolates possessed filter paper cellulase (FPCase) activity. This study provides new insights into broader cellulose degraders, which could be utilized for enhancing cellulosic waste treatment.     

Purification and characterization of carboxymethylcellulase isolated from a marine bacterium, Bacillus subtilis subsp. subtilis A-53

The microorganism hydrolyzing carboxymethylcellulose (CMC) was isolated from seawater, identified as Bacillus subtilis subsp. subtilis by analyses of 16S rDNA and partial sequences of the gyrA gene, and named as B. subtilis subsp. subtilis A-53. The molecular weight of the purified carboxymethylcellulase (CMCase) was estimated to be about 56 kDa with the analysis of SDS-PAGE. The purified CMCase hydrolyzed carboxymethylcellulose (CMC), cellobiose, filter paper, and xylan, but not avicel, cellulose, and p-nitrophenyl-β-d-glucospyranoside (PNPG). Optimal temperature and pH for the CMCase activity were determined to be 50 °C and 6.5, respectively. More than 70% of original CMCase activity was maintained at relative low temperatures ranging from 20 to 40 °C after 24 h incubation at 50 °C. The CMCase activity was enhanced by EDTA and some metal ions in order of EDTA, K⁺, Ni²⁺, Sr²⁺, Pb²⁺, and Mn²⁺, but inhibited by Co²⁺ and Hg²⁺.     

An analysis of the extracellular xylanases and cellulases of Butyrivibrio fibrisolvens H17c

The extracellular xylanase and cellulase components of Butyrivibrio fibrisolvens H17c were investigated. Two major peaks of enzyme activity were eluted by hydroxylapatite chromatography and designated complex A (CA), having cellulase activity, and complex B (CB) having predominantly xylanase activity but with some activity on carboxymethyl cellulose (CMC). CB was further purified on a DE-52 column and subjected to gel filtration. The xylanase and CMCase activities eluted in a single peak with an apparent molecular mass greater than thyroglobulin (Mr 669,000). CMC xymograms of polyacrylamide gels electrophoresed under non-denaturing conditions indicated the presence of five bands with CMCase activity from CA and eight from CB. Xylan xymograms under the same conditions indicated the presence of four bands of activity in CB. Under mild denaturing conditions the xylanase activity in CB was found in 11 bands with molecular mass ranging from 45 to 180 and the CMCase activity in three bands with molecular mass ranging from 45 kDa to 60 kDa. This indicates that CB exists as a multi-subunit protein aggregate of xylanases, some of which also have cellulase activity.