绿色木霉ZY-1固态发酵产纤维素酶

利用筛选的绿色木霉ZY-1（Trichoderma viride ZY-1）固态发酵产纤维素酶,采用稻草和麸皮为底物,考察稻草与麸皮比例随发酵时间对产酶的影响。结果表明：底物中,在m（稻草）：m（麸皮）为0：5和1：4时,发酵48h,pH保持4．5左右,还原糖量急剧上升,胞外蛋白产量最低;仅以稻草作底物时,整个发酵过程中pH约为7,还原糖量最低,胞外蛋白产量较高而滤纸酶活、羧甲基纤维素酶（CMCase）和β-葡萄糖苷酶（β-Gase）酶活均较低;在m（稻草）：m（麸皮）为3：2时,发酵96h,滤纸酶活达最大值5．01U／g干曲;m（稻草）：m（麸皮）为1：4时,发酵96h,β-Gase酶活达最大值4．6U／g干曲;m（稻草）：m（麸皮）为4：1时,发酵72h,CMCase酶活达最大值6．01U／g干曲。因此,底物中存在适量的稻草和麸皮有利于Trichoderma viride ZY—1产纤维素酶。     

Cost-effective production of cellulose hydrolysing enzymes from <Emphasis Type="Italic">Trichoderma</Emphasis> sp. RCK65 under SSF and its evaluation in saccharification of cellulosic substrates

Trichoderma sp. is a potential cellulase producing mesophilic fungi which grow under mild acidic condition. In this study, growth and nutritional conditions were manipulated for the maximum and cost-effective production of cellulase using lab strain Trichoderma sp. RCK65 and checked for its efficiency in hydrolysis of Prosopis juliflora (a woody substrate). Preliminary studies suggested that when 48 h old secondary fungal culture (20 % v/w) was inoculated in wheat bran moistened with mineral salt solution (pH 4.5 and 1:3 solid to moisture ratio), incubated at 30 °C and after 72 h, it produced maximum cellulase (CMCase 145 U/gds, FPase 38 U/gds and β-glucosidase 105 U/gds). However, using statistical approach a S:L ratio (1:1) was surprisingly found to be optimum that improved cellulase that is CMCase activity by 6.21 %, FPase activity by 23.68 % and β-glucosidase activity by 37.28 %. The estimated cost of crude enzyme (Rs. 5.311/1000 FPase units) seems to be economically feasible which may be due to high enzyme titre, less cultivation time and low media cost. Moreover, when the crude enzyme was used to saccharify pretreated Prosopis juliflora (a woody substrate), it resulted up to 83 % (w/w) saccharification.     

Production of thermostable hydrolases (cellulases and xylanase) from <Emphasis Type="Italic">Thermoascus aurantiacus</Emphasis> RCKK: a potential fungus

Thermophilic fungi are potential sources of thermostable enzymes and other value added products. Present study has focused on optimization of different physicochemical parameters for production of thermostable cellulases and xylanase by Thermoascus aurantiacus RCKK under SSF. Enzyme production was supported maximally on wheat bran fed with 20 % inoculum, at initial pH 5, temperature 45 °C and moisture ratio 1:3. The supplementation of wheat bran with yeast extract, Tween-80 and glycine further improved enzyme titres (CMCase 88 IU/g, FPase 15.8 IU/g, β-glucosidase 25.3 IU/g and xylanase 6,543 IU/g). The crude enzymes hydrolyzed phosphoric acid-swollen wheat straw, avicel and untreated xylan up to 74, 71 and 90 %, respectively. In addition, T. aurantiacus RCKK produced antioxidants as fermentation by-products with significant %DPPH^? scavenging, FRAP and in vivo antioxidant capacity against H₂O₂-treated Saccharomyces cerevisiae. These capabilities show that it holds potential to exploit crop by-products for providing various commodities.     

Production of cellulases and hemicellulases by Aspergillus niger KK2 from lignocellulosic biomass

To investigate the production of cellulases and hemicellulases from Aspergillus niger KK2, solid state fermentation (SSF) was performed by using different ratios of rice straw and wheat bran. When A. niger KK2 was grown on rice straw alone as a solid support in SSF, the maximum FPase activity was 19.5 IU g(-1) in 4 days. Also, CMCase (129 IU g(-1)), beta-glucosidase (100 IU g(-1)), xylanase (5070 IU g(-1)) and beta-xylosidase (193 IU g(-1)) activities were concurrently obtained after 5-6 days of fermentation. The higher enzyme activities produced by A. niger KK2 is a significant advantage from the viewpoint of practical saccharification reaction. Cellulases and hemicellulases produced by A. niger KK2 might be applied to pulp and paper industry, feed industry and chemical industry.     

Enhancement of <Emphasis Type="Italic">Penicillium echinulatum</Emphasis> glycoside hydrolase enzyme complex

The enhancement of enzyme complex produced by Penicillium echinulatum grown in several culture media components (bagasse sugarcane pretreated by various methods, soybean meal, wheat bran, sucrose, and yeast extract) was studied to increment FPase, xylanase, pectinase, and β-glucosidase enzyme activities. The present results indicated that culture media composed with 10 g/L of the various bagasse pretreatment methods did not have any substantial influence with respect to the FPase, xylanase, and β-glucosidase attained maximum values of, respectively, 2.68 FPU/mL, 2.04, and 115.4 IU/mL. On the other hand, proposed culture media to enhance β-glucosidase production composed of 10 g/L steam-exploded bagasse supplemented with soybean flour 5.0 g/L, yeast extract 1.0 g/L, and sucrose 10.0 g/L attained, respectively, 3.19 FPU/mL and 3.06 IU/mL while xylanase was maintained at the same level. The proteomes obtained from the optimized culture media for enhanced FPase, xylanase, pectinase, and β-glucosidase production were analyzed using mass spectrometry and a panel of GH enzyme activities against 16 different substrates. Culture medium designed to enhance β-glucosidase activity achieved higher enzymatic activities values (13 measured activities), compared to the culture media for FPase/pectinase (9 measured activities) and xylanase (7 measured activities), when tested against the 16 substrates. Mass spectrometry analyses of secretome showed a consistent result and the greatest number of spectral counts of Cazy family enzymes was found in designed β-glucosidase culture medium, followed by FPase/pectinase and xylanase. Most of the Cazy identified protein was cellobiohydrolase (GH6 and GH7), endoglucanase (GH5), and endo-1,4-β-xylanase (GH10). Enzymatic hydrolysis of hydrothermally pretreated sugarcane bagasse performed with β-glucosidase enhanced cocktail achieved 51.4 % glucose yield with 10 % w/v insoluble solids at enzyme load of 15 FPU/g material. Collectively the results demonstrated that it was possible to rationally modulate the GH activity of the enzymatic complex secreted by P. echinulatum using adjustment of the culture medium composition. The proposed strategy may contribute to increase enzymatic hydrolysis of lignocellulosic materials.     

γ-ray induced mutagenesis ofCellulomonas biazotea for improved production of cellulases

A rifampin-resistant mutant ofCellulomonas biazotea secreted elevated levels of cellulasesin vivo. The cellulase production in the mutant was not inhibited in the presence of 5% glucose, cellobiose or glycerol in the solid medium. The mutant exhibited approximately two- to three-fold enhanced product yields and productivity of cellular β-glucosidase over the wild parent in shake-flask culture studies when grown on either cellulosic or lignocellulosic substrates. Extracellular production of filter paper cellulase (FPase) and endo-glucanase (CMCase) were also significantly (p≤0.05) altered. During growth of the mutant on α-cellulose, the maximum volumetric productivities for CMCase, FPase and β-glucosidase were 52, 23.3, and 15.2 IUL⁻¹ h⁻¹,i.e 118, 121, and 229% their respective values for the parental strain. Some enzyme properties of the mutant cellulases were altered. Mutant-derived cellulases produced higher yields of glucose arising by degradation of bagasse, wheat straw, and α-cellulose (1.53-, 1.57-, and 1.75-fold, respectively).     

培养条件对棘孢曲霉SM-L22所产纤维素酶系组成的影响*

本文研究了影响棘孢曲霉SM-L22纤维素酶系组成的培养条件。研究结果表明,碳源、氮源和初始pH对棘孢曲霉所产生纤维素酶的内、外切酶组分的比例有明显的影响。在2%麸皮,1%CF11,0.5%尿素或含尿素的复合氮源为氮源,初始pH为4.5时,28℃培养120h后,内、外切酶的比值最大,内切酶活可达到3.1 IU/ml,FPA为0.105 IU/ml,CMCase/FPase的比值为30.6。     

Optimization and characterization of extracellular cellulase produced by Bacillus pumilus MGB05 isolated from midgut of muga silkworm (Antheraea assamensis Helfer)

Mature larvae of Antheraea assamensis were collected from different locations of Assam to isolate the cellulolytic gut microflora. Altogether sixty cellulase degrading bacteria were isolated on agar plates containing microcrystalline cellulose as the sole carbon source. Among them, ten isolates showed hydrolyzing zone on agar plates containing carboxy methyl cellulose (CMC) after staining with Congo-red. Isolate MGB05 exhibited the highest CMCase activity (0.262?U/mL) at 72?h of incubation under submerged condition. FPase and β-glucosidase activity were 0.012?U/mL and 3.71?U/mL respectively. It showed maximum FPase (0.022?U/mL) activity on the 3rd day of incubation in the media containing wheat bran as a carbon source. β-glucosidase production was also found to be highest with wheat bran (20.03?U/mL) at 48?h of incubation. The optimum pH and temperature of FPase activity of MGB05 were found at 6.0 and 50?°C respectively while for β-glucosidase activity, it was maximum at pH?6.0 under 50?°C. In addition, metal ion Mg⁺⁺ and Ca⁺⁺ enhanced FPase activity up to 110.92% (0.026?U/mL) and 105.31% (0.025?U/mL) respectively. In-vitro antimicrobial bioassay of the most potent cellulolytic bacteria (MGB05) also showed high antimicrobial activity against Escherichia coli (2.9?cm) and Pseudomonas aeruginosa (3.0?cm). The isolate MGB05 has been identified based on 16S rDNA homology as Bacillus pumilus MGB05 with accession KP298708.2. Results encompass the prospective beneficial role of gut-microflora on digestion and disease resistance, which might be a potential probiotic component to enhance silk productivity.     

The use of lignocellulosic wastes for production of cellulase by Trichoderma reesei

Summary The feasibility of cellulase production by Trichoderma reesei using inexpensive lignocellulosic material was examined. Sulfite pulp used as standard substrate yielded 3.7 IU/ml filter paper units (FPU) and 2.15 IU/ml -glucosidase. The yield was 185 FPU per gram total carbohydrate (CH) in the fermentation medium. Steam treated wheat straw (2%) gave 1.9 FPU/ml, 0.83 IU/ml -glucosidase and 151 FPU/g CH, whereas the spent fibres remaining after enzymatic hydrolysis of steamed wheat straw gave 2.4 FPU/ml, 1.55 IU/ml -glucosidase and 147 FPU/g CH. A good substrate (3%) was also the combustible fraction of municipal waste (BRAM) treated with NaOH, which gave 2.5 FPU/ml, 0.86 IU/ml -glucosidase and 130 FPU/g CH. A further increase in the final enzyme titer is obtainable by increasing the substrate concentration. In shake cultures 5% steamed wheat straw gave 3.8 FPU/ml and 1.95 IU/ml -glucosidase. Untreated wheat straw gave only low final enzyme titers and low yields of FPU/g CH. In the case of lignocellulosic substrates a constant pH-value of pH 6.0 during the fermentation gave optimal yields.     

培养条件对棘孢曲霉SM-L22所产纤维素酶系组成的影响

本文研究了影响棘孢曲霉SM-L22纤维素酶系组成的培养条件。研究结果表明,碳源、氮源和初始pH对棘孢曲霉所产生纤维素酶的内、外切酶组分的比例有明显的影响。在2%麸皮,1%CF11,0.5%尿素或含尿素的复合氮源为氮源,初始pH为4.5时,28℃培养120h后,内、外切酶的比值最大,内切酶活可达到3.1 IU/ml,FPA为0.105 IU/ml,CMCase/FPase的比值为30.6。     

Concomitant production of cellulase and xylanase by thermophilic mould <Emphasis Type="Italic">Sporotrichum thermophile</Emphasis> in solid state fermentation and their applicability in bread making

Sporotrichum thermophile BJAMDU5 secreted high titres of xylanolytic and cellulolytic enzymes in solid state fermentation using mixture of wheat straw and cotton oil cake (ratio 1:1) at 45?°C, pH 5.0 after 72 h inoculated with 2.9?×?10⁷ CFU/mL conidiospores. Supplementation of solid medium with lactose and ammonium sulphate further enhanced the production of hydrolytic enzymes. Among different surfactants studied, Tween 80 enhanced the production of all enzymes [3455 U/g DMR (dry mouldy residue), 879.26 U/g DMR, 976.28 U/g DMR and 35.10 U/g DMR for xylanase, CMCase (Carboxymethylcellulase), FPase (Filter paper activity) and β-glucosidase, respectively] as compared to other surfactants. Recycling of solid substrate reduced the production of all these enzymes after second cycle. End products analysis by TLC showed the ability of hydrolytic enzymes of S. thermophile to liberate monomeric (xylose and glucose) as well as oligomeric (xylobiose, cellobiose and higher ones) sugars. Supplementation of enzyme resulted in improved nutritional properties of the bread. Formation of oligomeric sugars by xylanase enzyme of S. thermophile BJAMDU5 make it a good candidate in food industry.     

Cellulolytic enzymes on lignocellulosic substrates in solid state fermentation by <Emphasis Type="Italic">Aspergillus niger</Emphasis>

The production of cellulolytic enzymes by Aspergillus niger on lignocellulosic substrates groundnut fodder, wheat bran, rice bran and sawdust in solid state fermentation in a laboratory scale was compared. Czapek Dox liquid broth amended with cellulose (0.5%) was used to moisten lignocellulosic solid supports for cultivation of Aspergillus niger. The production of filter paperase, carboxymethyl cellulase and -glucosidase were monitored at daily intervals for 5 days. The peak production of the enzymes occurred within 3 days of incubation. Among solid supports used in the study, wheat bran was the best solid matrix followed by groundnut fodder in production of cellulolytic enzymes in solid state fermentation. Groundnut fodder supported significant production of FPase (2.09 FPU/g), CMCase (1.36 U/g) and -glucosidase activity (0.0117 U/g) in solid state fermentation. Considerable secretion of protein (5.10 mg/g) on groundnut fodder at peak time interval 1st day of incubation was recorded.     

Solid-State Fermentation with Trichoderma reesei for Cellulase Production

Cellulase yields of 250 to 430 IU/g of cellulose were recorded in a new approach to solid-state fermentation of wheat straw with Trichoderma reesei QMY-1. This is an increase of ca. 72% compared with the yields (160 to 250 IU/g of cellulose) in liquid-state fermentation reported in the literature. High cellulase activity (16 to 17 IU/ml) per unit volume of enzyme broth and high yields of cellulases were attributed to the growth of T. reesei on a hemicellulose fraction during its first phase and then on a cellulose fraction of wheat straw during its later phase for cellulase production, as well as to the close contact of hyphae with the substrate in solid-state fermentation. The cellulase system obtained by the solid-state fermentation of wheat straw contained cellulases (17.2 IU/ml), β-glucosidase (21.2 IU/ml), and xylanases (540 IU/ml). This cellulase system was capable of hydrolyzing 78 to 90% of delignified wheat straw (10% concentration) in 96 h, without the addition of complementary enzymes, β-glucosidase, and xylanases.     

Production of cellulases and xylanase by Aspergillus fumigatus SK1 using untreated oil palm trunk through solid state fermentation

Direct utilization of untreated oil palm trunk (OPT) for cellulases and xylanase production by Aspergillus fumigatus SK1 was conducted under solid-state fermentation (SSF). The highest activities of extracellular cellulases and xylanases were produced at 80% moisture level, initial pH 5.0, 1 × 10⁸ spore/g (inoculum) with 125 μm of OPT as sole carbon source. The cellulases and xylanase activities obtained were 54.27, 3.36, 4.54 and 418.70 U/g substrates for endoglucanase (CMCase), exoglucanase (FPase), β-glucosidase and xylanase respectively. The crude cellulases and xylanase required acidic condition to retain their optimum activities (pH 4.0). Crude cellulases and xylanase were more stable at 40 °C compared to their optimum activities conditions (60 °C for FPase and 70 °C for CMCase, β-glucosidase and xylanase). SDS-PAGE and zymogram analysis showed that Aspergillus fumigatus SK1 could secrete cellulases (endoglucanase, exoglucanase and β-glucosidase), xylanase and protease. Enzymatic degradation of alkaline treated OPT with concentrated crude cellulases and xylanases resulted in producing polyoses.     

Solid state fermentation of straw withNeurospora crassa for CMCase and β-glucosidase production

Summary CMCase and -glucosidase were produced by the mutantNeurospora crassa 40b cultivated on untreated wheat straw in a solid state fermentation. Best enzyme activities were observed when the growth medium was composed of wheat straw mixed with certain mineral solutions at a ratio 1:2 (w/v). A partially purified enzyme preparation showed optimum enzyme activities of CMCase and -glucosidase at pH 4.0 and 5.0 and temperature 50 and 60°C respectively. The apparent Km values for the same enzymes were 16.8 g/l and 1.03x10^–4 M respectively. At optimum growth and enzyme assay conditions yields as high as 586.2 U CMCase and 58.4 U -glucosidase per gram of straw were obtained.     

Secretome analysis of thermophilic mould Myceliophthora thermophila cultivated on rice straw and hydrolysis of lignocellulosic biomass for bioethanol production

Abstract

Myceliophthora thermophila encodes for large number of carbohydrate-active enzymes (CAZymes) involved in lignocellulosic biomass degradation. The mould was grown on rice straw in solid state fermentation at pH 5.0 and 45?°C that produced high levels of cellulolytic and xylanolytic enzymes i.e. 2218.12, 515.23, 478.23, 13.34?U/g DMR for xylanase, CMCase, FPase and β-glucosidase, respectively. The secretome analysis of M. thermophila BJAMDU5 by mass spectroscopy, described 124 different proteins with majority of CAZymes consisting of glycosyl hydrolases (GH), lytic polysaccharide mono-oxygenases (LPMO), carbohydrate esterases (CE) and polysaccharide lyases (PL). Furthermore, the enzyme cocktail of the mould was evaluated for hydrolysis of steam treated rice straw that produced 184.59?mg/g substrate reducing sugars after 24?h, which was used for production of bioethanol by using fast fermenting yeast Saccharomyces cerevisiae resulting in high production of bioethanol.     

Double Mutants of Cellulomonas biazotea for Production of Cellulases and Hemicellulases following Growth on Straw of a Perennial Grass

Summary Deoxyglucose-resistant mutants of Cellulomonas biazotea secreted elevated levels of cellulases and xylanases. The production of β-glucosidase in the constitutive mutant was increased 5-fold over its parent strain. This mutant showed an approximately 1.6-fold enhanced productivity of extracellular endo-glucanase following growth on Leptochloa fusca over the mutant parent. Extracellular production of xylanase, filter-paper cellulase (FPase) and endo-glucanase (CMCase) were also altered in the mutant. Maximum volumetric productivities for xylanase, β-xylosidase, FPase, β-glucosidase and endo-glucosidase were 451, 98, 80, 95, and 143 IU l⁻¹ h⁻¹ which were significantly more than their respective values from the parental strains. The enzyme preparation of the mutants exhibited improved saccharification of kallar grass straw.     

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.     

真空微波诱变结合化学诱变选育酸性纤维素酶高产菌株

以酸性纤维素酶产生菌绿色木霉（Trichoderma viride）WL0512作为原始出发菌株,首先经自然分离筛选出一株产酶较稳定的菌株TVN-18,其羧甲基纤维素酶活（CMC酶活）达2765．8U／g,滤纸酶活（FPA酶活）达48．5U／g。再经真空微波和甲基磺酸乙酯（EMS）逐级诱变处理,获得了一株高产、稳产酸性纤维素酶的E6—1菌株,其CMC酶活达4396．6U／g,FPA酶活达126．0U／g,分别是菌株TVN-18的1．59倍和2．60倍。通过对固态发酵培养基麸皮和稻草比例、料水比以及初始pH值的优化,突变株的产酶能力进一步得到提高,其产的CIVIC酶活和FPA酶活分别提高了22．3％和22．4％。     

Production of extracellular enzymes by two Pleurotus species using banana pseudostem biomass

The degradation of lignocellulosic biomass of banana pseudoste was investigated during solid state fermentation (SSF) by P. ostreatus and P. sajor-caju. Both organisms proved to be efficient degraders of banana pseudostem biomass. P.ostreatus degraded hemicellulose (40% of dry weight, d.w.) better than cellulose (17.5% of d.w.) and lignin (10% of d.w.). P. sajor-caju also degraded hemicellulose (31% of d.w.) better than cellulose (12.4% of d.w.) and lignin (6% of d.w.). In both cases, a preferential removal of hemicellulose during the initial growth period and a delayed degradation of lignin were observed. The kinetics of cellulolytic, hemicellulolytic and lignolytic enzyme production in liquid culture were also examined. The activities of CMCase and β-glucosidase were highest at 16 days of growth and avicelase activity was at its maximum after 24 days (CMCase - 1.1 IU/ml, β-glucosidase - 0.09 IU/ml in the case of P. ostreatus; CMCase - 1.0 IU/ml, β-glucosidase - 0.087 - IU/ml in the case of P. sajor-caju.). Xylanase and laccase activity reached their maximum after day 16 and day 24 of incubation, respectively. (Xylanase - 1.1 IU/ml and laccase 3.0 IU/ml in the case of P. ostreatus; xylanase - 1.0 IU/ml and laccase - 3.6 IU/ml in the case of P. sajor-caju.). The efficient degrading capacity of test fungi demonstrated their potential use in the conversion of banana pseudostem biomass into mycelial protein-rich fermented animal feed.