Screening for a Novel Trichoderma vride Strain Highly Producing Cellulase via Ultraviolet Mutagenesis

[Objective] The aim of this study was to find out a new Trichoderma vride K strain highly producing cellulase.[Method] Ultraviolet（UV） was used to induce mutagenesis on T.vride K and to select out a new Trichoderma vride strain highly producing cellulase from the first round and further selection.[Result] A new T.vride strain K6 with high yield of cellulase was obtained with the enzyme production amount of 1.39 times over that of starting strain K.This strain showed highest cellulase yield under the culture condition of 28 ℃ for 96 h.[Conclusion] The strain K6 selected out from induced mutation is endowed with better capacity of producing cellulase,which provides a new method for the utilization of straw.

Characteristics of Cellulases from Anoplophora glabripennis Motsch (Coleoptera: Cerambycidae)

The property of major cellulases from the guts of Anoplophora glabripennis larvae have been characterized. The optimal temperatures of both β 1,4 glucosidase (β glucosidase) and endo β 1,4 endoglucanase (endoglucanase, Cx) are 40℃. The β glucosidase was optimally active at pH 4\^8, while the optimal activity of the endoglucanase occurred at pH 4 4 5 6 The endoglucanase was active with a wide range of pH and temperature, the levels of activity from 25℃ to 50℃ were more than 80%, and the activity remained 60% between pH 3 2 and pH 7 2. The endoglucanase exhibited higher thermal stability than β glucosidase. Both enzymes lose their activities by heat treatment at 60℃. Two isozymes of endoglucanase were detected in sodium carboxymethylcellulose polyacrymide gels (CMC gel) by chemical colorization, and purified by elution from the gel slices. The molecular weights of the two isozymes were estimated as 26kD and 39kD respectively. Moreover molecular characteristics of the two isozymes are currently underway.

Improvement of Cellulase Producing Capacity of Aspergillus niger by Ultraviolet Mutation

[Objective] The research aimed to breed the high-yield production strain of cellulase.[Method] Aspergillus niger was used as the starting strain,and a high-yield production strain of cellulase was selected after UV mutation treatment.[Result] Under the suitable condition,the strain 2（15） with the highest CMC production capacity was selected,which nearly increased 50% than that of the starting strain.[Conclusion] The research provided the foundation for its appliation in the feed production in the future.

Salt-tolerant mechanism of marine Aspergillus niger cellulase cocktail and improvement of its activity

The cellulase cocktail produced by marine Aspergillus niger exhibits a property of salt-tolerance,which is of great potential in cellulose degradation in high salt environment.In order to explain the mechanism on the salttolerance of the cellulase cocktail produced by marine A.niger,six cellulase components(AnCel6,AnCel7A,AnCel7B,AnEGL,AnBGL1 and AnBGL2)were obtained by directed expression.Studies on their enzymatic properties revealed that oneβ-glucosidase(AnBGL2)and one endoglucanase(AnEGL)exhibited an outstanding salttolerant property,and one cellobiohydrolase(AnCel7B)exhibited a certain salt-tolerant property.Subsequent study revealed that the salt-tolerant An EGL and AnCel7B endowed the cellulase cocktail with stronger salttolerant property,while the salt-tolerant An BGL2 had no positive effect.Moreover,after overexpression of AnCel6,AnCel7A,AnCel7B and AnEGL,the activity of cellulase cocktail increased by 80%,70%,63%and 68%,respectively.However,the activity of cellulase cocktail was not improved after overexpression of AnBGL1 and AnBGL2.After mixed-strain fermentation with cellobiohydrolase recombinants(cel6 a,cel7a and cel7b recombinants)and endoglucanase recombinant(egl recombinant),the the activity of cellulase cocktail increased by 114%,102%and91%,respectively.

Recent developments in activities, utilization and sources of cellulase

One of the latest sources of alternative energy, bioethanol, has been the focus of modem research, The production of bioethanol is commonly restricted by the activity of cellulase. Therefore, cellulase has become one of the critical issues in the conversion of lignocelluloses to bioethanol. This article is an overview of the sources and factors affecting enzyme activity, as well as methods of evaluation and utilizations of cellulase. We conclude that a combination of cellulases from various strains can enhance hydrolysis of substrates. Large enough amounts of cellobiase or sufficient cellobiase activity can reduce the inhibition to exoglucanase activity of cellobiose. Characterization and exploitation of cellulase should focus on a definite substrate. Promotion and mixed incubation of strains can reduce the cost of industrial utilization of cellulase.

Cellulase Extraction and Gas Chromatography Detection Technology of Swainsonine from Astragalus strictusin in Tibet

Locoweed is a poisonous plant wildly distributed in most areas of the world,which causes livestock poisoning or death with serious economic loss.The Astragalus strictus belongs to a species of Iocoweed.It is mainly distributed in Tibet of China and is a serious hazard to the local livestock industry.The objective of this study is extracting and purifying condition of Swainsonine from Astragalus strictus with the cellulase extraction method.An optimum extracting technology of SW from Astragalus strictus was investigated through the orthogonal experiment under the cellulose assistance conditions,and then the content of swainsonine was analyzed with gas chromatography （GC） method.The optimized extraction conditions were as follow：The crushed mesh is 40; solid-liquid ratio is 1∶40 （g/ml）; enzyme dosage is 3.5％; enzymatic time is 3 h.Under the above conditions,the extraction percentage of the swainsonine was 0.003 941％.The conditions for extracting swainsonine with cellulase extraction method are mild,it is easy to utilize industrial production.It is benefit for producing swainsonine from Astragalus strictus.

Optimization of Some Culture Conditions for Biosynthesis of Cellulase by Trichoderma vivide

[ Objective] To determine the best culture time and inducer for the biosynthesis of cellulase by Trichoderma vivide and thus provide the conditions for its practical application. [ Method] Within the 7 d after the inoculation of Trichoderma vivide ZJ strain, the cultures were collected once every day, and the enzyme yield was respectively determined by 3,5-dinitresalicylic acid assay. The Trichodernm vivide ZJ strain was inoculated into basal medium added by different types of carbon sources or nitrogen sources, and the growth of Trichoderma viride was observed. And the mycalium weight as well as the yield of CMCase enzyme after different culture time was determined. [Result] The optimal culture time for Trichoderma viride ZJ strain was 72-96 h; it grew rapidly in the medium added by monosaccharide or disaccharide as carbon sources, and the production of CMCase enzyme reached a peak after 3 -4 d post inoculation. Cellulose powder was the best carbon inducer. The compound nitrogen source composed of 1 g/L ammonium sulfate and 2 g/L yeast extract was the most suitable for the growth of ZJ strain and produced the highest enzyme activity. [ Condusion] The largest enzyme yield should be obtained after 3-4 d post the inoculation of Trichoderma viride ZJ strain. With cellulose powder as a carbon source and the complex substance composed of ammonium sulfate and yeast extract as a nitrogen source, Trichoderma viride has the highest enzyme activity.

Influence of lactic acid bacteria, cellulase, cellulase-producing Bacillus pumilus and their combinations on alfalfa silage quality

This study assessed the effects of lactic acid bacteria(LAB), cellulase, cellulase-producing Bacillus pumilus and their combinations on the fermentation characteristics, chemical composition, bacterial community and in vitro digestibility of alfalfa silage. A completely randomized design involving a 8(silage additives)×3 or 2(silage days) factorial arrangement of treatments was adopted in the present study. The 8 silage additive treatments were: untreated alfalfa(control); two commercial additives(GFJ and Chikuso-1); an originally selected LAB(Lactobacillus plantarum a214) isolated from alfalfa silage; a cellulase-producing Bacillus(CB) isolated from fresh alfalfa; cellulase(C); and the combined additives(a214+C and a214+CB). Silage fermentation characteristics, chemical composition and microorganism populations were analysed after 30, 60 and 65 days(60 days followed by exposure to air for five additional days). In vitro digestibility was analysed for 30 and 60 days. Compared with the other treatments, selected LAB a214, a214 combined with either C or CB, and Chikuso-1 had the decreased(P<0.001) pH and increased(P<0.001) lactic acid concentrations during the ensiling process, and there were no differences(P>0.05) among them. Fiber degradation was not significant(P≥0.054) in any C or CB treatments. The a214 treatment showed the highest(P=0.009) in vitro digestibility of dry matter(595.0 g kg–1DM) after ensiling and the highest abundance of Lactobacillus(69.42 and 79.81%, respectively) on days 60 and 65, compared to all of other treatments. Overall, the silage quality of alfalfa was improved with the addition of a214, which indicates its potential as an alfalfa silage inoculant.

Effect of Cellulase and Lactic Acid Bacteria on Fermentation Quality and Chemical Composition of Wheat Straw Silage

The object of this study was to determine the effect of cellulase and lactic acid bacteria (LAB) on fermentation quality and chemical composition of wheat straw silage. Silages were prepared using a small-scale fermentation system and the moisture level was adjusted to 60% of fresh matter (FM) with deionized water. Treatments were designed as: control silage without additives, LAB inoculant Lactobacillus casei Z3-1 (1.0 × 106 cfu·g-1 of FM), commercial inoculant L. plantarum FG 1 (1.0 × 106 cfu·g-1 of FM), Z3-1 + cellulase and FG 1 + cellulase. The neutral detergent fiber (NDF), acid detergent fiber (ADF) and crude protein (CP) contents of the wheat straw prior to ensiling were 76.93%, 48.52% and 4.63% of dry matter (DM), respectively. After 30 days of fermentation, the silages treated with LAB and LAB + cellulase had a lower (P < 0.05) pH and higher (P < 0.05) lactic acid content than the control, and the coliform bacteria, yeast and mold were inhibited at the early stage of fermentation. Besides, silages treated with cellulase had lower (P < 0.05) values of ADF and NDF than the control. The results confirmed that the addition of cellulase and LAB contributed to improving the fermentation quality of wheat straw silage.

Soil Cellulase Activity and Fungal Community Responses to Wetland Degradation in the Zoige Plateau, China

Four soil types(peat, marsh, meadow, and sandy) in the Zoige Plateau of China are associated with the severity of wetland degradation. The effects of wetland degradation on the structure and abundance of fungal communities and cellulase activity were assessed in these 4 soil types at 3 depths using DGGE(Denatured Gradient Gel Electrophoresis), q PCR(Quantitative Real-time PCR),and 3,5-dinitrosalicylic acid assays. Cellulase activity and abundance of the fungal community declined in parallel to the level of wetland degradation(from least to most disturbed). DGGE analysis indicated a major shift in composition of fungal communities among the4 soil types consistent with the level of degradation.Water content(WC), organic carbon(OC), total nitrogen(TN), total phosphorus(TP), available nitrogen(AN), and available phosphorus(AP) were strongly correlated with cellulase activity and the structure and abundance of the fungal community.The results indicate that soil physicochemical properties(WC, OC, TN, TP, AN, and AP), cellulase activity, and diversity and abundance of fungal communities are sensitive indicators of the relative level of wetland degradation. WC was the major factorinvolved in Zoige wetland degradation and lower WC levels contributed to declines in the abundance and diversity of the fungal community and reduction in cellulase activity.

Effects of cellulase and xylanase enzymes mixed with increasing doses of Salix babylonica extract on in vitro rumen gas production kinetics of a mixture of corn silage with concentrate

An in vitro gas production （GP） technique was used to investigate the effects of combining different doses of Salix babylonica extract （SB） with exogenous fibrolytic enzymes （EZ） based on xylanase （X） and cellulase （C）, or their mixture （XC; 1：1 v/v） on in vitro fermentation characteristics of a total mixed ration of corn silage and concentrate mixture （50：50, w/w） as substrate. Four levels of SB （0, 0.6, 1.2 and 1.8 mL g-1 dry matter （DM）） and four supplemental styles of EZ （1 μL g-1 DM; control （no enzymes）, X, C and XC （1：1, v/v） were used in a 4×4 factorial arrangement. In vitro GP （mL g-1 DM） were recorded at 2, 4, 6, 8, 10, 12, 24, 36, 48 and 72 h of incubation. After 72 h, the incubation process was stopped and supernatant pH was determined, and then filtered to determine dry matter degradability （DMD）. Fermentation parameters, such as the 24 h gas yield （GY24）, in vitro organic matter digestibility （OMD）, metabolizable energy （ME）, short chain fatty acid concentrations （SCFA）, and microbial crude protein production （MCP） were also estimated. Results indicated that there was a SBxEZ interaction （P〈0.0001） for the asymptotic gas production （b）, the rate of gas production （c）, GP from 6 to 72 h, GP2 （P=0.0095）, and GP4 （P=0.02）. The SB and different combination of enzymes supplementation influenced （P〈0.001） in vitro GP parameters after 12 h of incubation; the highest doses of SB （i.e., 1.8 mL g-1 DM）, in the absence of any EZ, quadratically increased （P〈0.05） the initial delay before GP begins （L） and GP at different incubation times, with lowering b （quadratic effect, P〈0.0001 ） and c （quadratic effect, P〈0.0001 ; linear effect, P=0.0018）. The GP was the lowest （P〈0.05） when the highest SB level was combined with cellulose. There were SBxEZ interactions （P〈0.001） for OMD, ME, the partitioning factor at 72 h of incubation （PF72）, GY24, SCFA, MCP （P=0.0143）, and pH （P=0.0008）. The OMD, ME, GY24 and SCFA with supplementation of SB extract at 1.8 mL g-1 DM were higher （P〈0.001） than the other treatments, however,PF72 was lower （quadratic effect, P=0.0194） than the other levels. Both C and X had no effect （P〉0.05） on OMD, pH, ME, GY24, SCFA and MP. The combination of SB with EZ increased （P〈0.001） OMD, ME, SCFA, PFz2 and GP24, whereas there was no impact on pH. It could be concluded that addition of SB extract, C, and X effectively improved the in vitro rumen fermentation, and the combination of enzyme with SB extract at the level of 1.2 mL g-1 was more effective than the other treatments.

Screening and characterization of a novel ruminal cellulase gene(Umcel-1) from a metagenomic library of gayal(Bos frontalis)

Gayal is a rare semi-wild bovine species found in the Indo-China. They can graze grasses, including bamboo leaves, as well as reeds and other plant species, and grow to higher mature live weights than Yunnan Yellow cattle maintained in similar harsh environments. The aim of this study was to identify specific cellulase in the gayal rumen. A metagenomic fosmid library was constructed using genomic DNA isolated from the ruminal contents of four adult gayals. This library contained 38400 clones with an average insert size of 35.5 kb. The Umcel-1 gene was isolated from this library. Investigation of the cellulase activity of 24 random clones led to the identification of the Umcel-1 gene, which exhibited the most potent cellulase activity. Sequencing the Umcel-1 gene revealed that it contained an open reading frame of 942 base pairs that encoded a product of 313 amino acids. The putative gene Umcel-1 product belonged to the glycosyl hydrolase family 5 and showed the highest homology to the cellulase （GenBank accession no. YP_004310852.1 ） from Clostridium lentocellum DSM 5427, with 44% identity and 62% similarity. The Umcel-1 gene was heterologously expressed in Escherichia coil BL21, and recombinant Umcel-1 was purified. The activity of purified recombinant Umcel-1 was assessed, and the results revealed that it hydrolyzed carboxymethyl cellulose with optimal activity at pH 5.5 and 45~C. To our knowledge, this study provides the first evidence for a cellulase produced by bacteria in gayal rumen.

Brewing of low-alcoholic drink from corncobs via yeast-cellulase synchronous fermentation process

The present work focuses on the influence of various parameters, i.e., the dosage of cellulase, the inoculum concentration of yeast, the fermentation temperature and the fermentation time, on the alcohol content and sensory evaluation of the low-alcoholic health drink produced from corncob in a yeast-cellulase synchronous fermentation process. The fermentation was performed by inoculating the seed solution (containing corncob powder and yeast) and cellulase into the synchronous saccharification fermentation medium. Single-factor experiments and orthogonal experiments were performed, and the optimal processing conditions were obtained based on the characterizations of alcohol content and sensory evaluation. The results show that the alcohol content and sensory evaluation of the drink can reach 6.1 vol.% and 92, respectively, when the dosage of cellulase, inoculum concentration of yeast, the fermentation temperature and the fermentation time are 15 U/g, 7%, 32℃ and 84 h, respectively.

Purification and characterization of the kinetic parameters of cellulase produced from wheat straw by Trichoderma viride under SSF and its detergent compatibility

This paper reports the purification and characterization of kinetic parameters of cellulase produced from Trichoderma viride under still culture solid state fermentation technique using cheap and an easily available agricultural waste material, wheat straw as growth supported substrate. Trichoderma viride was cultured in fermentation medium of wheat straw under some previously optimized growth conditions and maximum activity of 398±2.43U/mL obtained after stipulated fermentation time period. Cellulase was purified 2.33 fold with specific activity of 105U/mg in comparison to crude enzyme extract using ammonium sulfate precipitation, dialysis and Sephadex-G-100 column chromatography. The enzyme was shown to have a relative low molecular weight of 58kDa by sodium dodecyl sulphate poly-acrylamide gel electrophoresis. The purified enzyme displayed 6.5 and 55oC as an optimum pH and temperature respectively. Using carboxymethyl cellulose as substrate, the enzyme showed maximum activity (Vmax) of 148U/mL with its corresponding KM value of 68μM. Among activators/inhibitors SDS, EDTA, and Hg2+ showed inhibitory effect on purified cellulase whereas, the enzyme activated by Co2+ and Mn2+ at a concentration of 1mM. The purified cellulase was compatible with four local detergent brands with up to 20 days of shelf life at room temperature suggesting its potential as a detergent additive for improved washing therefore, it is concluded that it may be potentially useful for industrial purposes especially for detergent and laundry industry.

Cellulase production by Aspergillus unguis in solid state fermentation

Lignocellulosic substrates are a good carbon source and provide rich growth media for a variety of microorganisms which prodLuce industrially important enzymes. Cellulases are a group of hydrolytic enzymes such as filter paperase (FPase), carboxymethyl cellulase(CMCase) andβ-glucosidase-responsible for release of sugars in the bioconversion of the lignocellulosic biomass into a variety of value-added products. This study examined cellulase production by a newly isolated Aspergillus unguis on individual lignocellulosic substrates in solid state fermentation (SSF). The maximum peak production of enzymes varied from one substrate to another, however,based on the next best solid support and local availability of groundnut fodder supported maximum enzyme yields compared with other solid supports used in this study.Groundnut fodder supported significant production of FPase (5.9 FPU/g of substrate), CMCase (1.1 U/g of substrate) andβ-glucosidase activity (6.5 U/g of substrate) in SSF. Considerable secretion of protein (27.0 mg/g of substrate) on groundnut fodder was recorded. Constant increment of protein content in groundnut fodder due to cultivation of A. unguis is an interesting observation and it has implications for the improvement of nutritive value of groundnut fodder for cattle.

Research Progress of Cellulase

Cellulase is a complex enzyme that can decompose cellulose into glucose,and it could effectively treat cellulose waste. In this paper,it aims to explore development status and research progress of cellulase,and introduce concept and action mechanism of cellulase,research situation of cellulase in molecular aspect,application of cellulase,and development of cellulase is also prospected.

A Carboxymethyl Cellulase from a Marine Yeast(Aureobasidium pullulans 98): Its Purification, Characterization, Gene Cloning and Carboxymethyl Cellulose Digestion

We have reported that A. pullulans 98 produces a high yield of cellulase. In this study, a carboxymethyl cellulase （CMCase） in the supematant of the culture ofA. pullulans 98 was purified to homogeneity, and the maximum production of CMCase was 4.51 U （mg protein）-1. The SDS-PAGE analysis showed that the molecular mass of the purified CMCase was 67.0kDa. The optimal temperature of the purified enzyme with considerable thermosensitivity was 40℃, much lower than that of the CMCases from other ftmgi. The optimal pH of the enzyme was 5.6, and the activity profile was stable in a range of acidity （pH 5,0-6.0）. The enzyme was activated by Na＋, Mg2＋, Ca2＋, K＋, Fe2＋ and Cu2＋, however, it was inhibited by Fe3＋, Ba2＋, Zn2＋, Mn2＋ and Ag＋. Km and Vmax values of the purified enzyme were 4.7mgmL-1 and 0.57 pmol L-1 min-1 （mg protein）-1, respectively. Only oligosaccharides with different sizes were released from carboxymethylcellulose （CMC） after hydrolysis with the purified CMCase. The putative gene encoding CMCase was cloned from A. pullulans 98, which contained an open reading flame of 954bp （EU978473）. The protein deduced contained the conserved domain of cellulase superfamily （glucosyl hydrolase family 5）. The N-terminal amino acid sequence of the purified CMCase was M-A-P-H-A-E-P-Q-S-Q-T-T-E-Q-T-S-S-G-Q-F, which was consistent with that deduced from the cloned gene. This suggested that the purified CMCase was indeed encoded by the cloned CMCase gene in this yeast.

Application of Statistically Based Experimental Designs to Optimize Cellulase Production and Identification of Gene

A natural bacterial strain identified as Bacillus amyloliquefaciens MBAA3 using 16S rDNA partial genome sequencing has been studied for optimization of cellulase production.Statistical screening of media components for production of cellulase by B.amyloliquefaciens MBAA3 was carried out by Plackett–Burman design.Plackett–Burman design showed CMC,MgSO4 and pH as significant components influencing the cellulase production from the media components screened by Plackett-Burman fractional factorial design.The optimum concentrations of these significant parameters were determined employing the response surface central composite design,involving three factors and five levels was adopted to acquire the best medium for the production of cellulase enzyme revealed concentration of CMC(1.84 g),MgSO4(0.275 g),and pH(8.5)in media for highest enzyme production.Response surface counter plots revealed that middle level of MgSO4 and middle level of CMC,higher level of CMC and lower level of pH and higher level of MgSO4 with lower level of pH increase the production of cellulase.After optimization cellulase activity increased by 6.81 fold.Presence of cellulase gene in MBAA3 was conformed by the amplification of genomic DNA of MBAA3.A PCR product of cellulase gene of 1500 bp was successfully amplified.The amplified gene was conformed by sequencing the amplified product and sequence was deposited in the gene bank under the accession number KF929416.

Isolation and characteristics of one marine psychrotrophic cellulase-generating bacterium Ar/w/b/75°/10/5 from Chuckchi Sea,Arctic

Microorganisms living in polar zones play an important part as the potential source of organic activity materials with low temperature characteristics in the bio-technological applications. A psychrotrophic bacterium (strain Ar/w/b/75°/10/5) , producing cellulose at low temperatures during late-exponential and early-stationary phases of cell growth, was isolated from sea ice-covered surface water in Chuckchi Sea, Arctic. This bacterium, with rod cells, was Gram-negative, slightly halophilic. Colony growing on agar plate was in black. Optimum growth temperature was 15℃. No cell growth was observed at 351 or above. Optimum salt concentration for cell growth was between 2 and 3 % of sodium chloride in media. Maximal cellulase activity was detected at a temperature of 35℃ and pH8. Cellulase was irreversibly inactivated when incubated at 55℃ within 30 min. Enzyme can be kept stable at the temperature no higher than 25℃. Of special interest was that this bacterium produced various extracellular enzymes including cellulase, amylase, agar hydrolase and protease, at low or moderate temperature conditions, which is certainly of it potential value for applications.

Response Relationship between the Cadmium Pollution and the Cellulase in Activity Change of Earthworm in Soils

The research explored response of cellulase in Eisenia fetida in soils with Cd at different concentrations, as per artificial soil test. The results indicated that as Cd concentration increased, the activity of cellulase was decreasing in a short peri- od, which suggested that cellulase activity was inhibited to certain extent. As Cd concentration and the treatment period grew, the activities increased in varying de- grees. With Cd at a high concentrate, Cd had a kind of induction effects on cellu- lase activity in earthworms.