Binding of Extracellular Carboxymethylcellulase Activity from the Marine Shipworm Bacterium to Insoluble Cellulosic Substrates

The binding of extracellular endoglucanase, a carboxymethylcellulase (CMCase), produced by the marine shipworm bacterium to insoluble cellulose substrates was investigated. Up to 70% of CMCase activity bound to cellulosic substrates, and less than 10% bound to noncellulosic substrates. CMCase binding to cellulose was enhanced in basal salt medium or sodium phosphate buffer containing 0.5 M NaCl. Increased cellulose particle size correlated with decreased CMCase binding. Also, cellulose treated with either 5 N NaOH or commercial cellulase reduced the CMCase binding to these surfaces. Pretreatment of CMCase preparations with 0.01% sodium dodecyl sulfate, 5% β-mercaptoethanol, and 5 mM EDTA or ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) had little effect on binding to cellulose. While pretreatment of CMCase with trypsin, chymotrypsin, and pronase had little effect on CMCase enzymatic activity, the ability to bind to cellulose was greatly diminished by these treatments.     

Biosynthesis,optimization and potential textile application of fungal cellulases/xylanase multifunctional enzyme preparation from Penicillium sp. SAF6

The main task of the present work is to search for fungal strains isolated from agricultural soil with the potential to produce cellulases/xylanase enzyme preparation for bio-finishing of textiles. The most potent fungal strain (SAF6) was subjected to molecular identification using 18 SrRNA and was identified as Penicillium sp. SAF6 with the novel accession number of KM222497. Factors affecting the produced mixed enzyme activity were investigated. The optimum conditions for achieving maximum activity of the cellulases (FPase, CMCase and β-glucosidase) in addition to xylanase were the initial culture pH media 5, yeast extract (1.5gN/L), medium-to-air ratio (1:5) for FPase and CMCase and (1:10) for β-glucosidase, at 30?°C for 8 days incubation period. Potential application of the prepared crude enzyme in bio-finishing of cellulosic substrates, namely, bleached cotton, linen and indigo dyed fabrics were explored. Using the multi-component enzyme at appropriate dosage and conditions brought about a significant improvement and surface modification of the treated cotton substrates.     

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 four major subunits from Clostridium thermocellum cellulosome and their synergism in the hydrolysis of crystalline cellulose

The cellulosome of Clostridium thermocellum, purified by affinity chromatography, was dissociated under mild conditions and separated by SDS-PAGE. Two major p-nitrophenylcellobiosidases (PNPCases I and II) corresponding to the S₅ (103 kDa) and S₈ (78 kDa) subunits and one major carboxymethylcellulase (CMCase) coinciding with the S₁₁ (60.5 kDa) subunit were isolated and characterized using carboxymethylcellulose (CMC), H₃PO₄-swollen cellulose and cello-oligosaccharides. Both PNPCases showed little effect on the viscosity of CMC and released twice as much total sugar as reducing sugar from H₃PO₄-swollen cellulose. The CMCase released ten times more total sugar than reducing sugar from H₃PO₄-swollen cellulose and reduced the viscosity of CMC rapidly. None of these enzymes was active on cellotriose. Both PNPCases released cellobiose from cellotetraose, and cellobiose and cellotriose from cellopentaose. In contrast, CMCase was active only on cellopentaose and released mainly glucose. Use of MeUmb(Glc)_n revealed that both PNPCases cleaved preferentially either the second or fourth linkage from the non-reducing end while the CMCase was specific for the internal glycosidic bonds. Thus, the PNPCases and CMCase behaved as typical exo- and endoglucanases, respectively. When tested individually, all three enzymes degraded Avicel only to a small extent. A 1.5–2.0-fold increase in sugar release was observed when CMCase was combined with either PNPCase I, II or both. Combining S₁ with either PNPCase II or CMCase resulted in fourfold synergism in the hydrolysis of Avicel. Synergism was sevenfold when all three enzymes were combined with S₁.     

Comparative characterization of a recombinant Volvariella volvacea endoglucanase I (EG1) with its truncated catalytic core (EG1-CM), and their impact on the bio-treatment of cellulose-based fabrics

Recombinant Volvariella volvacea endoglucanase 1 (EG1) and its catalytic module (EG1-CM) were obtained by expression in Pichia pastoris, purified by two-step chromatography, and the catalytic activities and binding capacities were compared. EG1 and EG1-CM exhibited very similar specific activities towards the soluble substrates carboxymethyl cellulose, lichenan and mannan, and insoluble H₃PO₄ acid-swollen cellulose, whereas the specific activities of EG1-CM towards the insoluble substrates -cellulose, Avicel and filter paper were approximately 58, 43 and 38%, respectively compared to EG1. No increase in reducing sugar release was detected in the reaction mixture supernatants after 50 h exposure of filter paper, Avicel or -cellulose to EG1-CM, whereas increases in the total reducing sugar equivalents (i.e. reducing sugar released into solution together with new reducing ends generated in the cellulosic substrates) in reaction mixtures were observed after 1 h. In reaction mixtures containing EG1, soluble reducing sugar equivalents were detected in supernatants after 3 h incubation with the insoluble cellulosic substrates. EG1-CM did not adsorb to Avicel, and the binding capacities of EG1-CM towards filter paper and H₃PO₄ acid-swollen cellulose were 27.9–33.3% and 29.6–60.6%, respectively of values obtained with EG1 within the range of total added protein. In enzymatic deinking experiments, the ink removal rate in EG1-CM-treated samples was only slightly higher (8%), than that of untreated controls, whereas that of the EG1-treated samples was 100% higher. Bio-stoning of denim with EG1-CM resulted in increases of 48% and 40% in weight loss and indigo dye removal, respectively compared with untreated controls. These increases were considerably lower than the corresponding values of 219% and 133% obtained when samples were treated with EG1.     

Production of cellulases and saccharification of lignocellulosics by A. Micromonospora sp.

A locally isolated strain of Micromonospora sp. when grown on different natural cellulosic substrates gave the highest activity of carboxymethylcellulase (34 U/ml) and Avicelase (0.9 U/ml) on rice straw. Sugar cane bagasse was also a good substrate for growth and cellulase production. With commercial cellulosic substrates, highest carboxymethylcellulase (90 U/ml) and Avicelase (2.8 U/ml) activities were when the organism grew on xylan. Saccharification of sugar cane bagasse and rice straw by enzyme preparations of the organism grown on the respective substrates released 5.6 and 5.8 mg reducing sugar/ml. With all enzyme preparations, bagasse was more easily saccharified than rice straw.The authors are with the Atomic Energy Research Establishment, GPO Box 3787, Dhaka 1000, Bangladesh; N.A. Chowdhury, M. Moniruzzaman, and N. Choudhury in the Institute of Food and Radiation Biology, and N. Nahar in the Institute of Nuclear Science and Technology.     

Isolation of cellulose-hydrolytic bacteria and applications of the cellulolytic enzymes for cellulosic biohydrogen production

Nine cellulolytic bacterial strains were isolated from soil sample taken in southern Taiwan. Through 16S rRNA sequence matching; eight of those isolates belong to Cellulomonas sp., while the other one belongs to Cellulosimicrobium cellulans. The activity of cellulolytic enzymes (cellulases and xylanase) produced from those strains was mainly present extracellularly and the enzyme production was dependent on cellulosic substrates (xylan, rice husk and rice straw) used for growth. HPLC analysis confirmed the bacterial hydrolysis of these cellulosic substrates for soluble sugars production. The efficiency of fermentative H₂ production from the enzymatically hydrolyzed rice husk was examined with seven H₂-producing pure bacterial isolates. With an initial reducing sugar concentration of 0.36 g l⁻¹, only Clostridium butyricum CGS5 exhibited efficient H₂ production from the rice husk hydrolysates with a cumulative H₂ production and H₂ yield of 88.1 ml l⁻¹ and 19.15 mmol H₂ (g reducing sugar)⁻¹ (or 17.24 mmol H₂ (g cellulose)⁻¹), respectively.     

Effect of surfactant and particle size reduction on hydrolysis of deinking sludge and nonrecyclable newsprint

Disposal of sludge from deinking mills represents a significant proportion of operating costs. Bioconversion of the cellulosic fraction of deinking sludge (DIS) to ethanol greatly reduces disposal costs while producing an environmentally friendly fuel. In this study, the cellulosic fraction of newsprint and deinking sludge was hydrolysed to produce fermentable sugars. For newsprint, a particle size of 1 to 1.5 mm provided optimal reaction rates in batch reactors over practical hydrolysis times, and reducing sugar concentrations as high as 35 g/L could be achieved using a fed-batch reactor configuration. For both newsprint and DIS, the hydrolysis rate increased nonlinearly with enzyme loading. Tween-80 only marginally improved sugar production but was able to release sugars from cellulosic substrates in the absence of lytic enzymes, in an amount proportional to the surfactant concentration and the substrate particle size. DIS was relatively recalcitrant to enzymatic hydrolysis, possibly due in part to inhibition by hydrophobic constituents. (c) 1995 John Wiley & Sons, Inc.     

产纤维素酶黏细菌在生物转化的作用

目的:预处理对木质纤维素降解的影响.方法:从土壤中分离筛选到高纤维素酶活的黏细菌菌株So ce sh1008.该菌具有CMC酶活(CMCase)及微晶纤维素酶活性.研究NaOH联合黏细菌降解盐蒿、稻草、棉花秸秆和甘蔗渣四种木质纤维素的情况.结果:碱(2％ NaOH) -黏细菌处理的方法优于黏细菌-碱的方法,其中降解棉花秸秆降解效果最明显,以5.0g木质纤维素为原料,其最终干重损失达2.1g,溶液中总糖含量和还原糖含量均值分别为12.8 mg/mL和0.93 mg/mL.酵母菌发酵产乙醇的研究结果表明,最佳发酵时间为47h,碱-黏细菌甘蔗渣降解液发酵效果最好,乙醇产出达6.0％.结论:黏细菌联合2％ NaOH能有效降解甘蔗渣,提高乙醇产量.     

Multicomponent cellulase production by Cellulomonas biazotea NCIM‐2550 and its applications for cellulosic biohydrogen production

Among four cellulolytic microorganisms examined, Cellulomonas biazotea NCIM‐2550 can grow on various cellulosic substrates and produce reducing sugar. The activity of cellulases (endoglucanase, exoglucanase, and cellobiase), xylanase, amylase, and lignin class of enzymes produced by C. biazotea was mainly present extracellularly and the enzyme production was dependent on cellulosic substrates (carboxymethyl cellulose [CMC], sugarcane bagasse [SCB], and xylan) used for growth. Effects of physicochemical conditions on cellulolytic enzyme production were systematically investigated. Using MnCl₂ as a metal additive significantly induces the cellulase enzyme system, resulting in more reducing sugar production. The efficiency of fermentative conversion of the hydrolyzed SCB and xylan into clean H₂ energy was examined with seven H₂‐producing pure bacterial isolates. Only Clostridiumbutyricum CGS5 exhibited efficient H₂ production performance with the hydrolysate of SCB and xylan. The cumulative H₂ production and H₂ yield from using bagasse hydrolysate (initial reducing sugar concentration = 1.545 g/L) were approximately 72.61 mL/L and 2.13 mmol H₂/g reducing sugar (or 1.91 mmol H₂/g cellulose), respectively. Using xylan hydrolysate (initial reducing sugar concentration = 0.345 g/L) as substrate could also attain a cumulative H₂ production and H₂ yield of 87.02 mL/L and 5.03 mmol H₂/g reducing sugar (or 4.01 mmol H₂/g cellulose), respectively. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Biohydrogen production from cellulosic hydrolysate produced via temperature-shift-enhanced bacterial cellulose hydrolysis

A “temperature-shift” strategy was developed to improve reducing sugar production from bacterial hydrolysis of cellulosic materials. In this strategy, production of cellulolytic enzymes with Cellulomonas uda E3-01 was promoted at a preferable temperature (35 °C), while more efficient enzymatic cellulose hydrolysis was achieved under an elevated culture temperature (45 °C), at which cell growth was inhibited to avoid consumption of reducing sugar. This temperature-shift strategy was shown to markedly increase the reducing sugar (especially, monosaccharide and disaccharide) concentration in the hydrolysate while hydrolyzing pure (carboxymethyl-cellulose, xylan, avicel and cellobiose) and natural (rice husk, rice straw, bagasse and Napier-grass) cellulosic materials. The cellulosic hydrolysates from CMC and xylan were successfully converted to H₂ via dark fermentation with Clostridium butyricum CGS5, attaining a maximum hydrogen yield of 4.79 mmol H₂/g reducing sugar.     

Isolation and partial properties of cellulose-decomposing strain of Cytophaga sp. LX-7 from soil

A new facultatively anaerobic, Gram-negative bacterium, Cytophaga sp. LX-7, degrading crystalline cellulose completely, was isolated from soil by dilution plating on cellodextrin agarose plates. This strain could excrete extracellularly all three types of cellulase and cellulosic substrates were the strongest inducer of endocellulase with CMC-liquefying activity production. No reducing sugar was found in cultures of cellulose during incubation. An enzyme which degrades crystalline cellulose was detected in cultures of cellulose by measuring the formation of soluble carbohydrate but was not detected by determining the reducing sugar released. This strain also synthesized cell-bound cellobiose oxidizing enzyme which was previously noted only in fungi. Both cellulose and soluble sugars could promote the production of cellobiose oxidizing enzyme.     

Enhancement of cellulose accessibility and enzymatic hydrolysis by simultaneous wet milling

It has been shown that the rate of enzymatic saccharification of cellulosic materials including “pure” cellulose (Whatman CF?11 cellulose), newsprint, lignocellulose (prehydrolyzed to remove hemicelluloses), and wood can be substantially increased by simultaneous wet milling. An enhanced hydrolysis rate was sustained above that observed for ball milling: providing a more extensive saccharification. The cellulosic substrates were wet milled with a variety of grinding elements, such as sand, glass beads, and stainless-steel beads, agitated in a shaker bath. Simultaneous hydrolysis was achieved with a 2% substrate slurry in a 0.1M acetate buffer at 45°C and pH 5. The effectiveness of this process was dependent upon the lignified matrix of the cellulose microfibrils, the grinding elements, and the oscillation frequency of the shaker bath. Wet milling “pure” cellulose for 48 hr, with 3.5 mm glass beads and 200 oscillations/min (opm), yielded 1031 mg reducing sugar/g substrates (93% saccharification) as compared to 483 mg (44%) for the ball-milled sample and 253 mg (23%) for the unmilled material. With the lignified substrates stainless-steel beads (3.5 mm) were more effective than glass. For lignocellulose 529 mg sugar/g substrate (93% saccharification) could be obtained by wet milling with cellulase for 24 hr. This was about three times greater than that of the ball milled (169 mg, 30%) and 10 times greater than that of the unmilled (52 mg, 9%) substrates. The method was also effective for wood particles (60 mesh) giving 143 mg sugar/g wood (approximately 38% saccharification) in 48 hr, whereas the ball-milled sample gave only 79 mg (21%) and the unmlilled substrate 38 mg (10%). These observations can be explained on the basis of the current crystalline theory for the morphology of the cellulosic microfibrils. The advantage of wet milling and simultaneous hydrolysis apparently depends on a continuous generation of accessible sites and sustained rapid hydrolysis rate as the saccharification proceeds, where in the pretreated substrates the hydrolysis rate slow down as the active sites are reduced.     

Production and localisation of carboxymethylcellulase,xylanase and β-glucosidase fromCellulomonas andMicrococcus spp.

Summary Cellulomonas and Micrococcus spp. grew well at 30°C, pH 7.0, and produced carboxymethylcellulase (CMCase) and xylanase enzymes. Only one species of Micrococcus was able to produce an appreciable amount of -glucosidase. This is the first report where Micrococcus sp., isolated from termite gut, was able to produce all three enzymes (i.e. CMCase, xylanase and -glucosidase) required for degradation of cellulosic and hemicellulosic substrates. Offprint requests to: A. Varma     

Enzymatic saccharification of sugar cane bagasse by continuous xylanase and cellulase production from cellulomonas flavigena PR‐22

Cellulase (CMCase) and xylanase enzyme production and saccharification of sugar cane bagasse were coupled into two stages and named enzyme production and sugar cane bagasse saccharification. The performance of Cellulomonas flavigena (Cf) PR‐22 cultured in a bubble column reactor (BCR) was compared to that in a stirred tank reactor (STR). Cells cultured in the BCR presented higher yields and productivity of both CMCase and xylanase activities than those grown in the STR configuration. A continuous culture with Cf PR‐22 was run in the BCR using 1% alkali‐pretreated sugar cane bagasse and mineral media, at dilution rates ranging from 0.04 to 0.22 1/h. The highest enzymatic productivity values were found at 0.08 1/h with 1846.4 ± 126.4 and 101.6 ± 5.6 U/L·h for xylanase and CMCase, respectively. Effluent from the BCR in steady state was transferred to an enzymatic reactor operated in fed‐batch mode with an initial load of 75 g of pretreated sugar cane bagasse; saccharification was then performed in an STR at 55°C and 300 rpm for 90 h. The constant addition of fresh enzyme as well as the increase in time of contact with the substrate increased the total soluble sugar concentration 83% compared to the value obtained in a batch enzymatic reactor. This advantageous strategy may be used for industrial enzyme pretreatment and saccharification of lignocellulosic wastes to be used in bioethanol and chemicals production from lignocellulose. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:321–326, 2016     

玉米秸秆分批补料获得高还原糖浓度酶解液的条件优化

木质纤维素高浓度还原糖水解液的获得是纤维乙醇产业化发展的方向。在发酵工业领域,分批补料法是实现这一目标的重要研究途径。本研究采用分批补料法对获得高浓度玉米秸秆酶解还原糖的条件进行了优化。以稀硫酸预处理的玉米秸秆为原料,考察了液固比、补加量与补加时间对分批补料糖化的影响。结果表明,秸秆高浓度酶解液条件的初始物料为20％ (重量/体积),木聚糖酶220 U/g (底物),纤维素酶6 FPU/g (底物),果胶酶50 U/g (底物),在24 h、48 h后分批补加8％预处理后的物料,同时添加与补料量相应的木聚糖酶20 U/g (底物),纤维素酶2 FPU/g (底物),72 h后,最终糖化结果与非补料法相比,还原糖浓度从48.5 g/L提高到138.5 g/L,原料的酶解率最终达到理论值的62.5％。试验结果表明补料法可以显著提高秸秆水解液还原糖浓度。     

Cellulolytic activity of Actinomycetes isolated from termites (Termitidae) gut

Abstract Cellulolytic actinomycetes were isolated from the hindgut of four different termites: Macrotermes, Armitermes, Odontotermes and Microcerotermes spp.
The isolated actinomycetes ( Streptomyces sp. and Micromonospora sp.) were grown on cellulosic substrates and their extracellular cellulase (C_l, C_x and cellobiase) activity evaluated; using filter paper as a substrate for C_l, carboxymethylcellulose (CMC) for C_x and d -cellobiose for cellobiase, all strains were shown to degrade soluble and insoluble cellulose; optimum pH for growth was 6.2–6.7 at 28°C; three strains could grow at 48°C on cellulosic substrates.
Some strains exhibited high cellulase activity, constant for 5–7 days, but inhibition by glucose was a common feature for almost all isolates.     

Assimilation of Cellooligosaccharides by a Cell Surface-Engineered Yeast Expressing β-Glucosidase and Carboxymethylcellulase from Aspergillus aculeatus

Since Saccharomyces cerevisiae lacks the cellulase complexes that hydrolyze cellulosic materials, which are abundant in the world, two types of hydrolytic enzymes involved in the degradation of cellulosic materials to glucose were genetically co-immobilized on its cell surface for direct utilization of cellulosic materials, one of the final goals of our studies. The genes encoding FI-carboxymethylcellulase (CMCase) and β-glucosidase from the fungus Aspergillus aculeatus were individually fused with the gene encoding the C-terminal half (320 amino acid residues from the C terminus) of yeast α-agglutinin and introduced into S. cerevisiae. The delivery of CMCase and β-glucosidase to the cell surface was carried out by the secretion signal sequence of the native signal sequence of CMCase and by the secretion signal sequence of glucoamylase from Rhizopus oryzae for β-glucosidase, respectively. The genes were expressed by the glyceraldehyde-3-phosphate dehydrogenase promoter from S. cerevisiae. The CMCase and β-glucosidase activities were detected in the cell pellet fraction, not in the culture supernatant. The display of CMCase and β-glucosidase proteins on the cell surface was confirmed by immunofluorescence microscopy. The cells displaying these cellulases could grow on cellobiose or water-soluble cellooligosaccharides as the sole carbon source. The degradation and assimilation of cellooligosaccharides were confirmed by thin-layer chromatography. This result showed that the cell surface-engineered yeast with these enzymes can be endowed with the ability to assimilate cellooligosaccharides. This is the first step in the assimilation of cellulosic materials by S. cerevisiae expressing heterologous cellulase genes.     

Bioethanol production with carboxymethylcellulase of Pseudomonas poae using castor bean (Ricinus communis L.) cake

Increased consumption of fossil fuels is an emerging problem. Scientists look for the existence of other alternatives to fossil fuels, including so-called renewable energy. Accordingly, we report the production of bio-ethanol from the remnants of castor oil bean seed cake (CBC) by the carboxymethylcellulase enzyme (CMCase). A bacterial strain isolated from rice straw showing higher CMCase activity was identified. The 16S rRNA result showed a 93% homology with the 16SrRNA gene sequences of Pseudomonas poae RE11-1-14, the strain was identified as Pseudomonas poae AB3. In addition, our results showed that the highest enzyme activity was achieved after 48 h and inoculum size of 3.7 × 10⁵ CFU. The optimum temperature, pH and Carboxymethylcellulose (CMC) concentration for the highest enzyme activity was 25 °C, pH 7 and 10 g/l respectively. Furthermore, The CMCase was purified by ammonium sulphate at a concentration of 60%. The SDS-PAGE of the purified enzyme showed a molecular weight of 88 kDa. Additionally, the (CBC) was hydrolyzed by the purified CMCase at the enzyme optimum conditions. The results showed the liberation of 5.2 g/L of reducing sugar by using dinitrosalicylic acid (DNS) assay. Finally, the total sugar produces 35 g/L after 48 h when Saccharomyces cerevisiae was used as a fermentation agent. Hence for the first time, we have been successfully able to produce bioethanol from CBC with CMCase of Pseudomonas poae.     

Improved CMCase production by Spicellum roseum

Abstract CMCase enzyme production by Spicellum roseum NRRL 13104 was increased from 2.1 to 3.4 mg reducing sugar (RS) h⁻¹· ml⁻¹ broth in four experimental stages: (1) selection of important types of ingredients, (2) confirmation of their importance, (3) comparison of sources of the ingredient and (4) partial evaluation of concentrations.