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.

Stimulation of Selected Cellulases from Trichoderma reesei with White Linearly Polarized Light

The impact of the illumination with white linearly polarized light (WLPL) of two commercially available cellulases from Trichoderma reesei on their activity in hydrolysis of microcrystalline cellulose was studied. Enzymes were illuminated with WLPL for 60 min and 120 min and for each native and illuminated enzyme sample specific activity and kinetics of enzyme catalyzed hydrolysis of microcrystalline cellulose were established. Molecular weight Mw and radii if gyration Rg of protein chains of native and illuminated enzymes were measured by means of high pressure size exclu-sion chromatography coupled with multiangle laser light scattering and refractometric detectors (HPSEC-MALLS-RI). Conformations of protein chains of native and illuminated enzymes were evaluated on the basis of their circular dichroism (CD) spectra. Additionally, molecular weight Mw and radii of gyration Rg of polysaccharide chains of microcrystalline cellulose native and digested for 10 min, 480 min and 1440 min with original and WLPL stimulated enzymes WT and TR were taken. Illumination with WLPL of both cellulases studied did not change secondary structures of protein molecules of native enzyme. Molecular weight Mw and radii of gyration Rg of illuminated enzymes differed greatly from those found for native enzymes. Illumination of enzymes led to increase of specific activity and rate constants of reaction of hydrolysis microcrystalline cellulose catalyzed by illuminated enzymes as compared with native enzymes.

The activity and kinetic properties of cellulases in substrates containing metal ions and acid radicals

The effects of various metal ions (Na+, K+, Ca2+, Mg2+, Al3+, Co2+) and anions (Cl–, SO2-4 and CH3COO–) on two cellulases were investigated. Fitting of the data to Michaelis-Menten kinetics showed that Al3+ noncom-petitively inhibited cellulase (Km = 22.68 g/L;Vmax = 0.269 mg/min at 5.0 mmol/L AlCl3) and Mg2+ competitively inhibited cellulase (Km = 50.0 g/L;Vmax = 0.434 mg/min at 10.0 mmol/L MgCl2) Different metal ions increased or decreased inhibition of cellulase activity slightly below 1 mmol/L and strongly over 10 mmol/L. The results indicated that filter paper activeity (FPA) was suitable for analysis of enzymatic saccharification with various lignocellulosic substrates whereas crude cellulase was suitable for applications in the biomass industry. Some metal ions were proved to inhibit cellulase reversibly.

Evolution and impact of cellulose architecture during enzymatic hydrolysis by fungal cellulases

The enzymatic hydrolysis of cellulose is still considered as a main limiting step of the biological production of biofuels from ligno-cellulosic biomass. Glycoside hydrolases from Trichoderma reesei are currently used to produce fermentable glucose units from degradation of cellulose packed in a complex assembly of cellulose microfibrils. The present work describes the structural evolution of two prototypical samples of cellulose (a micro-crystalline cellulose and a bleached sulfite pulp) over 5 length scale orders of magnitude. The results were obtained through wide angle, small angle and ultra-small angles synchrotron X-ray scattering, completed by Small Angle Neutron Scattering and particle size analyzers. These structural evolutions were followed as a function of enzymatic conversion. The results show that whereas there is no change at the nanometer scale, drastic changes occur at micron. The observed decrease of the size of the cellulose particles is accompanied by a smoothing of the crystalline surfaces that can be explained by a two-step mechanism of the enzymatic hydrolysis.

Use of wastes from the tea and coffee industries for the production of cellulases using fungi isolated from the Western Ghats of India

In this study,coffee pulp(Coffea arabica)and green tea(Camellia sinensis)residues were characterized for use as a substrate of solid-state fermentation for cellulases production.The invasion rate was evaluated,as well as cellulases production by strains of Aspergillus niger and Trichoderma asperellum from the western Ghats of India,on coffee pulp,green tea,and a mixture of both substrates(50:50).T.asperellum(AFP)strain was found to have the highest growth rate(0.409±0.021 mm/h)using a mixture of both substrates.The production of cellulases by T.asperellum was unsatisfactory due to the presence of polyphenols in the supports to which A.nigger cellulases are more resistant.The production of cellulases by A.nigger was linked to the pH of the supports,favouring the use of T and TC.It was found that the extracts produced by A.niger(28A strain using a mixture substrate,28A,and 20A strains using only green tea as a substrate)presented the highest cellulase activities when evaluated using a plate technique producing degradation halos of 2.3±0.1 cm of diameter.Aspergillus 28A strain did not require mineral enrichment media for cellulase production using green tea residues as support of solid-state fermentation.

Advances in the study of directed evolution for cellulases

If cellulose can be effectively hydrolyzed intoglucose by cellulase,the production costs of hydrogen,ethanol or other chemicals from cellulosic materials will begreatly decreased,and economically viable production ofbiohydrogen and bioethanol will become feasible.Celluloseis degraded into glucoses by multi-component enzymesystems.Nowadays cellulases are widely used in brewing,food,bioenergy,fodder,textiles,paper,pharmaceuticals,environmental protection and other industries.However,existing cellulases have several problems that limit theirwider applications,including the low turnover number forsolid cellulosic materials,and low stability in adapting tovarious application conditions.For example,high temperature,low pH,and so on.Application of directedevolution technology may be one of the most effectiveways for improving the characteristics of cellulases.Thispaper presents a brief review of the cellulases hydrolysismechanism by cellulase,advances in cellulases(endoglucanaseandβ-glucosidase)improvement by directedevolution for several characteristics(for instance,thermalstability,pH adaptability and enzyme activity),limitationsof directed evolution for cellulases,and the outlook fordirected evolution for cellulase.

Large-Scale Analyses of Glycosylation in Cellulases

Cellulases are important glycosyl hydrolases （GHs） that hydrolyze cellulose polymers into smaller oligosaccharides by breaking the cellulose β （1→4） bonds, and they are widely used to produce cellulosic ethanol from the plant biomass. N-linked and O-linked glycosylations were proposed to impact the catalytic efficiency, cellulose binding affinity and the stability of cellulases based on observations of individual cellulases. As far as we know, there has not been any systematic analysis of the distributions of N-linked and O-linked glycosylated residues in cellulases, mainly due to the limited annotations of the relevant functional domains and the glycosylated residues. We have computationally annotated the functional domains and glycosylated residues in cellulases, and conducted a systematic analysis of the distributions of the N-linked and O-linked glycosylated residues in these enzymes. Many N-linked glycosylated residues were known to be in the GH domains of cellulases, but they are there probably just by chance, since the GH domain usually occupies more than half of the sequence length of a cellulase. Our analysis indicates that the O-linked glycosylated residues are significantly enriched in the linker re- gions between the carbohydrate binding module （CBM） domains and GH domains of cellulases. Possible mechanisms are discussed.

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.

Isolation,Screening and Identification of Cellulose Decomposing Strains in Straw-amended Soil

[Objective]The aim was to isolate cellulose decomposing strains with high activity from straw-amended soil.[Method]Screening of CMC solid culture and shaking culture rescreening were carried out to obtain cellulose decomposing strains with high activity from the corn straw amended soil,which were analyzed by 16rDNA sequence analysis.[Result]A bacterium and a fungus with higher endonucleases activity were obtained through preliminary screening and secondary screening,a fungus with higher filter paper enzyme activity,and a bacterium（NO.5 strain） with higher of both filter paper enzyme activity and CMC enzyme activity were obtained.The result of 16S rDNA sequence analysis showed that the similarity was 100% between NO.5 strain and Bacillus subtilis.[Conclusion]NO.5 strain was identified as Bacillus subtilis.

益母草中挥发性组分的酶提取及分析

The cellulase was used for extracting the volatile constituents of the Leonurus japonicus Houtt.The effects of the pH,the amounts and the fineness of cellulase were discussed.The average yield obtained was 4.05%.16 chemical components were identified by gas chromatography-mass spectrometry (GC-MS)method.The cellulase extraction (CE)method was made a camparison with distillation extraction (DSE)method.

纤维素酶降解-离子色谱法测定魔芋中的亚硝酸盐

建立了以纤维素酶降解魔芋葡甘聚糖释放阴离子,然后采用离子色谱测定亚硝酸盐含量的方法。考察了纤维素酶降解葡甘聚糖的最佳条件,结果表明：在50℃、pH5.5条件下酶解24h,可以释放出被包埋的亚硝酸盐。NO2-的检出限为0.039mg/L,回收率为96.7%-97.8%。运用本法对施用稀土微肥后魔芋中亚硝酸盐的含量进行测定,发现施用稀土微肥能够在一定程度上降低魔芋中亚硝酸盐的含量。

Identification of novel catalytic features of endo-β-1,4-glucanase produced by mulberry longicorn beetle Apriona germari

Mulberry longicorn beetle, Apriona germari, has been reported to produce two endo-β-1,4-glucanases or AgEGases (accession Nos. Q6SS52 and Q5XQD1). AgEGase sequence contains catalytic motif (amino acid residues 37~48), which is the characteristic of family Glycohydrolase 45 and is identified as the substrate binding site. The application of bioinformatics ap-proaches includes sequence analysis, structural modeling and inhibitor docking to relate the structure and function of AgEGases. We have dissected the sequence and structure of AgEGase catalytic motif and compared it with crystal structure of Humicola insolens endoglucanases V. The results show an involvement of sulfur containing amino acid residues in the active site of the enzyme. Cys residues and position of disulfide bonds are highly conserved between the two structures of endoglucanases of A. germari. Surface calculation of AgEGase structure in the absence of Cys residues reveals greater accessibility of the catalytic site to the substrate involving Asp42, a highly conserved residue. For the inhibition study, tannin-based structure was docked into the catalytic site of AgEGase using ArgusLab 4.0 and it resulted in a stable complex formation. It is suggested that the inhibition could occur through formation of a stable transition state analog-enzyme complex with the tannin-based inhibitor, as observed with other insect cellulases in our laboratory.

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.

Cellulase Production by Endophytic Strains of <i>Trichoderma reesei</i>from <i>Baccharis dracunculifolia</i>D. C. (Asteraceae)

Cellulases are enzymes responsible for the degradation of cellulose, the major compound in plant cells. Cellulose is a polysaccharide composed of several glucose units linked together by chemical bonds. Cellulases, such as endoglucanases, beta-glucosidase and exoglucanases, break the chemical bonds between the glucose units. Fungi, including the endophytic species, can be great cellulase producers. This study aimed to evaluate cellulase production by four endophytic strains of Trichoderma reesei semi-solid media containing sugarcane bagasse, supplemented or not with salts. Two fermentations were carried out for 43 days. Samples were taken every seven days to obtain production peaks. The enzymes were characterized by their optimum pH and temperature of activity and stability upon incubation in the presence of ions, pH and temperature variations. The results showed that the endophytic strains FB1, FB2, FB3 and FB4 of Trichoderma reesei produce cellulases in a sugarcane bagasse medium, supplemented or not with salts, at pH 5.5 and 30°C. The supplemented medium proved to be more appropriate to induce cellulase production after 29 days of fermentation, with FB4 having the best yield: 16.32 ± 2.65 IU/gram of fermented substrate.

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.

Use of Hypocrea jecorina (anamorph Trichoderma reesei) as a model system for Trichoderma biocontrol of Pythium blight identifies new targets for genetic strain improvement

Biocontrol by Trichoderma has been studied mainly with selected isolates of T. harzianum, T. atroviride and T. asperellum, which are members of sections Pachybasium and Trichoderma. In contrast, species from section Longibrachiatum have only rarely been studied. On the other hand, one taxon from this section-Hypocrea jecorina (anamorph: Trichoderma reesei)-has been widely used for the production of cellulolytic and hemicellulolytic enzymes and recombinant proteins. As far as Trichoderma is concerned, molecular genetic methods and tools are most advanced in H. jecorina, and its genome has recently been fully sequenced, thus making this taxon a model organism for the genus. Here we will demonstrate that H. jecorina is able to antagonize plant pathogenic fungi in plate confrontation tests, and can protect tomato and cucumber plants against Pythium ultimum blight. Using this as a model case, we made use of available H. jecorina mutants to investigate (a) whether carbon catabolite repression via the Cre1-regulator protein has an impact on biocontrol, and (b) whether cellulase gene expression is necessary for biocontrol of P. ultimum. In the first case, plate confrontation tests and in planta experiments yielded opposite results, i.e. while a Cre1 mutant was more active in antagonization of fungi on plates, the survival rates of P. ultimum-inoculated cucumber plants was lower than with the H. jecorina wild-type strain. Mutants of H. jecorina, unable to form cellulases, were still able to antagonize fungi on plates and provided similar protection of tomatos against P. ultimum as the wild type, indicating that the pronounced biocontrol ability of H. jecorina against fungi with cellulose-containing cell-walls is not due to its high cellulolytic activity. A strain disrupted in the light-modulator gene envoy (Schmoll et al., ms submitted) exhibited in planta biocontrol activity strongly exceeding that of the wild-type strain, thereby providing a first link between Trichoderma biocontrol and light. In view of the numerous other metabolic and regulatory mutants of H. jecorina available, we suggest that this fungus should increasingly be used in basic studies on the biochemistry and genetics of biocontrol.

Kinetic Characterization of Trichoderma reesei CL847 TR3002： An Engineered Strain Producing Highly Improved Cellulolytic Cocktail

Enzymatic hydrolysis of lignocellulose is often considered to be the major economic bottleneck of the production process of bioethanol from lignocellulose. It is generally admitted that the most efficient organism for the production ofcellulolytic enzymes is the fungus Trichoderma reesei, mostly due to its high secretion capacity. Unfortunately, this fungus secretes very low concentrations of β-glucosidase, thereby often requiring β-glucosidase supplementation for complete cellulose hydrolysis. It is especially important to have sufficient quantities of β-glucosidase in order to prevent inhibition of cellobiohydrolases by cellobiose. In order to optimize the produced cocktail, a more efficient β-glucosidase was cloned into T. reesei CL847 strain. The new strain, called CL847 TR3002, secretes the evolved β-glucosidase and was tested for cellulase production in laboratory-scale reactors. Its growth kinetics and cellulase production were characterized using fed-batch and chemostat modes under various culture conditions. The cellulase activities of the evolved strain were compared with activities of the parent strain. In addition, hydrolysis of a steam exploded wheat straw was performed at three different enzyme-loading levels （5 mg/g, 10 mg/g and 20 mg/g of dry matter） and a new kinetic model was developed.

Ethanol Production by Enzymatic Hydrolysis of Elephant Grass

The aim of this work is to evaluate the efficiency of ethanol production by fermentation of a hydrolysate obtained by the enzymatic hydrolysis of purple elephant grass （Pennisetum purpureum Schum.） using a blend of cellulases. Three hundred grams of 20-mesh granulated purple elephant grass was subjected to hot water pretreatment at 100 ℃ for a period of 25 min in a batch autoclave reactor. The pulp obtained from the pretreatment process was washed with water at 60 ℃ and submitted to enzymatic hydrolysis using a combination of exoglucanases, endoglucanases and beta-glucosidases or β-glucosidases from Novozymes. The pH of the system was kept constant at 4.8 by adjusting the levels of acetic acid or sodium acetate in the buffer solution. Enzymatic hydrolysis occurred at 50 ℃ upon agitation at 200 rpm on a shaker for 72 hours. The hydrolysate obtained after agitation was fermented using dried Saccharomyces cerevisiae yeast （manufactured by Dr. Oetker） at 30 ℃ for 10 hours. The liquids obtained after fermentation were analysed using HPLC to determine the quantity of ethanol produced. After 4 hours of fermentation, the maximum quantity of ethanol was 1.8 g/L. The stoichiometric yield of ethanol was approximately 95%. However, the step ofpretreatment was deemed unsatisfactory due to the loss of glucose during the pretreatment process.

Effects of Biochar on Soil Enzyme Activity and Mechanism of Action

[Objective] The aim was to detect effects of biochar on soil enzyme activity and mechanism. [Method] Soils were sampled from experimental fields of Shanxi Academy of Agricultural Sciences and added with charcoals in different types in order to analyze dynamic changes of alkaline phosphatase and cellulase activities and effects of charcoals on soil enzyme activities. [Result] By applying charcoal, enzyme activity of soil samples all improved and the increase degree was higher of alkaline phosphatase than cellulase activity. Mid-temperature charcoal at 8% is the best. [Conclusion] The research provides references for effects of biochar on physi- cal and chemical properties.