Regulatory mutations affecting the synthesis of cellulase in Bacillus pumilus

A wild strain of Bacillus pumilus was investigated for cellulase production, and putative mutants of this strain were screened for catabolite repression insensitivity after chemical mutagenesis using ethyl methanesulphonate (EMS) as a mutagenic agent. Out of four classes of mutants studied and classified according to their cellulase induction rate and level of cellulase production in the presence of high concentrations of glucose (2.6%[w/v]), classes III and IV exhibited cellulase production up to 6.2 mg cellulase and 11.4 mg cellulase per gram of dry cell mass respectively. These mutants were referred to as catabolite repression-insensitive when compared to the wild strain which exhibited a total repression of cellulase synthesis under the same conditions. How EMS triggered the catabolite repression insensitivity in these mutants was not established. However this mutation brought out new strains of cellulase hyperproducers (mutants 6 and 11) in the presence of glucose when compared to other cellulase producers such as Aspergillus terreus, A. nidulans and Trichoderma reesei, which exhibited catabolite repression of cellulase synthesis. These mutants were selected as the most promising candidates for cellulase synthesis even at high glucose concentration.     

Strain improvement of Penicillium janthinellum NCIM 1171 for increased cellulase production

The strain of Penicillium janthinellum NCIM 1171 was subjected to mutation involving treatment of Ethyl Methyl Sulfonate (EMS) for 24h followed by UV-irradiation for 3min. Successive mutants showed enhanced cellulase production (EMS-UV-8), clearance zone on Avicel containing plate (SM2) and rapid growth on Walseth cellulose agar plates containing 0.2% 2-deoxy-d-glucose (SM3). These mutants were transferred to Walseth cellulose plates containing higher concentration (1.5%) of 2-deoxy-d-glucose (SM4) in which only five mutants showed clearance zone on SM4. All these mutants showed approximately two-fold increase in activity of both FPase and CMCase in shake flask culture when grown on basal medium containing CP-123 (1%) and wheat bran (2.5%). The enzyme preparations from these mutants were used to hydrolyze Avicel. Higher hydrolysis yields of Avicel were obtained with enzyme preparations of EU1. This is the first report on the isolation and selection of mutants based on hydrolysis of Avicel, which is the most crystalline substrate.     

Cellulase hyperproducers constructed from polyploids of Lentinus edodes

A mycelial mat of Lentinus edodes was treated with 0.01% (w/v) colchicine solution for 240 h at 26 degrees C and autopolyploidization occurred. The mycelia were treated subsequently with the haploidizing reagent, benomyl, and fanshaped sectors were produced from colonies. Among such sectors, cellulase hyperproducers could be selected. The cellulase productivity of the hyperproducer, L1, did not decrease through five generations.     

Bioinsecticidal activity of conidia and dry mycelium preparations of two isolates of Beauveria bassiana against the sugarcane borer Diatraea saccharalis

Two isolates of Beauveria bassiana (Bb₁ and Bb₅ strains) were grown in solid-state and submerged cultures (SSC and SbC, respectively) in order to obtain conidia and dry mycelium preparations. The samples obtained at laboratory scale were tested as alternatives for their further use as mycoinsecticides to control the sugarcane borer Diatraea saccharalis. The spore yields obtained in SSC indicated that the Bb₅ strain was able to produce around three times more conidia per gram of initial dry matter (3.7×10¹⁰) than the Bb₁ strain (1.3×10¹⁰) in a solid medium composed of wheat bran and bran husk. Under this culture conditions, the former showed not only satisfactory spore yields, but also a higher bioinsecticidal activity than the latter. Laboratory bioassays carried out with conidia of both strains against D. saccharalis larvae indicated that the Bb₅ strain promotes an insect mortality of 82.5% whereas only 21.3% was observed with the Bb₁ strain preparations. Concerning the samples obtained in SbC, in which a culture medium based on glucose and yeast extract was employed, it is interesting to point out that although both strains showed similar behaviors with yields of approximately 1.50×10¹⁰ conidia per gram of dry mycelium, both preparations failed in their bioinsecticidal activities. Despite satisfactory yields, bioinsecticidal activity of the Bb₁ and Bb₅ strains dropped drastically showing a larvae mortality below 2%.     

Characteristics of the saprophytic reference strain FA ofClaviceps paspali

Growth properties and morphological features of the saprophytic collection cultureClaviceps paspali strain FA were investigated. The strain was characterized by a range of utilization of 13 carbon sources in a basic synthetic and a peptone medium. A temperature of 23 °C was more favourable for growth than 28 °C. Stages of changes of the culture cultivated for a long time on Sabouraud’s medium were detected by electron microscopy. The white culture was characterized by true septated mycelium with different types of terminal parts of hyphae. Arthrospores occurred after a 14-d cultivation, small spherical conidia released individually from hyphae were quite rare. As compared with other strains of the same species, strain FA did not form spherical clusters of conidia. On the other hand, a spontaneous rupture of the surface cell wall in different parts of hyphae and release of the cytoplasm were observed. In corn-steep containing media the formation of individual, pair and chain-like forms of arthritic conidia was stimulated. Destructive autolytic changes of hyphae were detected in the medium with potato extract.     

Cellulase production on glucose-based media by the UV-irradiated mutants of Trichoderma reesei

From 22,791 mutants of a cellulase hyper-producing strain of Trichoderma reesei (Hypocrea jecorina), ATCC66589, as the parent, we selected two mutants, M2-1 and M3-1, that produce cellulases in media containing both cellulose and glucose. The mutation enabled the mutants to produce cellulases, which were measured as p-nitrophenyl β-d-lactopyranoside-hydrolyzing activities, in media with glucose as a sole carbon source, although M2-1 exhibited different sensitivities to glucose from M3-1. When the mutants were grown for 8 days on a medium with cellulose as a sole carbon source, the filter-paper-degrading activities (FPAs) per gram of cellulose were 257 and 281 U for M2-1 and M3-1, respectively, values that were 1.1–1.2 times higher than that of the parental strain. Cellulase production by M2-1 and M3-1 on a medium with a continuously fed mixture of glucose and cellobiose resulted in 214 and 210 U of FPA/gram carbon sources, respectively, whereas less efficient production (140 U of FPA/gram carbon source) was achieved by the parental strain. The improved cellulase productivity of the mutants allows us to use glucose as a carbon source for efficient on-site production of cellulases with quality/quantity-controlled feeding of soluble carbon sources and inducers.     

Autopolyploid formation of Trichoderma reesei QM9414 by colchicine treatment

Conidia of Trichoderma reesei QM9414 were treated with colchicine. Nuclei in colchicine-treated conidia enlarged. When the concentration of colchicine or the treatment time with colchicine increased, the diameter of nuclei became larger. Colchicine-treated conidia generated sectors on a medium containing benomyl. Some sectors formed many conidia or could not produce clear zones on the plate assay medium for cellulase production. According to the DNA assay of conidia, colchicine-treated strains were autopolyploid.     

Production of cellulase by Aspergillus niger biofilms developed on polyester cloth

AIMS: To compare cellulase production by Aspergillus niger ATCC 10864 biofilms on polyester cloth and freely suspended cultures in shaken flasks and microbioreactors of bubble column type. METHODS AND RESULTS: Both shaken flasks and oxygenated microbioreactors containing 40 ml of production medium were used to compare cellulase secretion by free mycelium and biofilm cultures. Free mycelium cultures grew better in flasks than in microbioreactors producing compact and fluffy pellets, respectively, while the opposite was found for biofilm cultures without any visible change in biofilm morphology. Cellulase activities and volumetric productivities attained by biofilms in flask cultures were 70% higher than that produced by free mycelium cultures and threefold higher when biofilms were grown in microbioreactors. CONCLUSIONS: Fungal biofilms developed on polyester cloth in both flasks and microbioreactors produce higher cellulase yields and volumetric productivities than free mycelium cultures at lower biomass levels. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of the present study are of commercial and biological interest. All productivity parameters revealed that fungal biofilms may be used for the production of cellulase and other proteins in various types of bioreactors. Moreover, they may be used as model systems to study differential gene expression related to cell adhesion.     

Production of cellulases from alkali-treated bagasse in Trichoderma reesei

Summary Most of the mutants of Trichoderma reesei had good cellulase productivity on Avicel but this was low on alkali-treated bagasse, which could be a most promising cellulosic biomass to use as an inexpensive carbon source for cellulase production. Two T. reesei mutants, PC-3-7 and X-31, in which strong cellulase activity is inducible by l-sorbose, were, however, found to produce cellulase on alkali-treated bagasse. They produced about 100 units of CMCase per ml in 5-1 jar fermentor culture with 4% alkali-treated bagasse as carbon source. They also showed higher cellulase productivity than other mutants on other easily saccharified substrates, such as alkali-treated rice straw and Walseth's cellulose.Production of Ethanol from Biomasses Part IV.Production of Ethanol from Biomasses Part IV.     

Multinucleation in the conidia of polyploids derived fromTrichoderma reesei QM 9414 by colchicine treatment

Conidia ofTrichoderma reesei QM 9414 were treated with colchicine in order to obtain polyploids (diploids; tetraploids). Cellulase production by diploids (mononucleate conidia) was almost twice as great as that of the original strain, but that of tetraploids (binucleate conidia) was not increased. When these latter conidia were re-treated with 2.0% (w/v) colchicine, multiple nuclei were produced in each conidium, and their diameter was almost the same as that of the original nucleus. Cellulase production of the diploid was almost the same in either mononucleate or multinucleate nature. However, cellulase production by the tetraploid which produced multinucleate conidia was greater than that of the binucleate tetraploid and that of the diploid. The multinucleation technique can contribute to enhancing cellulase production.     

草酸青霉纤维素酶基因和木聚糖酶基因的调控基因POX05145的鉴定

草酸青霉能产生完整的纤维素酶和木聚糖酶酶系,其纤维素酶基因的表达主要受转录因子的调控。前期工作中,通过对草酸青霉菌株HP7-1在不同碳源培养基培养条件下转录组的比较分析,获得了调控纤维素酶和木聚糖酶产量的候选调控基因集。本研究以草酸青霉ΔPoxKu70为出发菌株,通过同源重组法,构建并获得了其中一个候选调控基因POX05145的缺失突变株ΔPOX05145。在微结晶纤维素Avicel诱导培养条件下,与出发菌株ΔPoxKu70相比,ΔPOX05145的纤维素酶产量和木聚糖酶产量发生了显著改变。其中,在诱导第2天时,ΔPOX05145对硝基苯-β-D-纤维二糖苷酶产量和木聚糖酶产量分别上升43.4%和164.7%,对硝基苯-β-D-半乳糖吡喃葡萄糖苷酶产量下降92.8%,但是,滤纸酶产量和羧甲基纤维素酶产量没有显著变化。然而,在诱导第4天时,所有纤维素酶产量和木聚糖酶产量上升100.4%~294.0%。实时荧光定量PCR检测表明POX05145在不同的时间不同程度的调控主要的纤维素酶基因和木聚糖酶基因的表达。序列分析表明POX05145含有一个GAL4类锌指结构的DNA结合功能域和一个保守的真菌特有的转录因子结构域(Fungal_TF_MHR)。     

以耐赖氨酸为标记的金霉素产生菌抗噬菌体变株的选育

本文以金霉素产生菌Ｆ－３０３作为出发菌株,通过ＵＶ＋ＥＭＳ复合处理,双层法不同梯度耐赖氨酸作标记,进行抗噬菌体的金霉素菌株选育,获得Ｆｓ－４８－１４菌株。该菌株不但表达了抗噬菌体的特性,而且产素效价超过Ｆ－３０３。通过无机盐正交条件试验,取得抗性变株最佳的发酵培养基配方。     

Production and characterization of cellulolytic enzymes from the thermoacidophilic fungal Aspergillus terreus M11 under solid-state cultivation of corn stover

The production of extracellular cellulases by a newly isolated thermoacidophilic fungus, Aspergillus terreus M11, on the lignocellulosic materials was studied in solid-state fermentation (SSF). The results showed that the high-level cellulase activity was produced at 45 degrees C pH 3 and moisture 80% with corn stover and 0.8% yeast extract as carbon and nitrogen sources. 581 U endoglucanase activity, 243 U filter paper activity and 128 U beta-glucosidase activity per gram of carbon source were obtained in the optimal condition. Endoglucanase and beta-glucosidase exhibited their maximum activity at pH 2 and pH 3, respectively, and both of them showed remarkable stability in the range of pH 2-5. The activities of endoglucanase and beta-glucosidase were up to the maximum at 70 degrees C and maintained about 65% and 53% of their original activities after incubation at 70 degrees C for 6h. The enzyme preparations from this strain were used to hydrolyze Avicel. Higher hydrolysis yields of Avicel were up to 63% on 5% Avicel (w/v) for 72 h with 20 U FPase/g substrate.     

Penicillium verruculosum WA 30 a new source of cellulase

Summary Of fungi 110 strains were screened for extracellular cellulase production in shake flask experiments. Twelve strains produced the enzyme in significant quantity. Since the enzyme activity was assayed by different methods (liberation of reducing sugar from cotton, filter paper, carboxymethylcellulose and cellobiose), the estimation of the productivity of the strains differed according to the substrate used. The best cotton degrading activity per fermentation volume as well as per mg of secreted soluble protein was achieved by Penicillium verruculosum WA 30, a wild-type strain, for which the cellulase productivity has not yet been published. The cotton degrading (so-called C₁) activity was successfully enhanced nearly threefold in medium experiments. Analyses of saccharification digests showed that glucose was the predominant product, with negligible amounts of cellobiose. The pH and temperature optima for WA 30 cellulase complex were pH 4.2 and 60°C.     

Oszillierende-fed-batch-Technik bei der Cellulasegewinnung

From different fungal genera some mutant strains were selected which in cellulase formation exhibit reduced catabolite-repression related to several soluble carbon sources. The cellulase production by these mutant strains on the basis of soluble carbon sources is possible if a fed-batch-technique is used for feeding the substrate. The optimum procedure is an oscillating-fed-batch-technique (OFB) with feed-back-controlled intermittent addition of the substrate. The substrate concentration in the medium is adjusted in such a manner to realize oscillations between a limitation for mycelium growth and non-limiting conditions under a substrate concentration which is yet below the repressing concentration for cellulase formation. A suitable feed-back-parameter for the control of substrate addition is the specific CO₂-formation of the mycelium. Using OFB the growth in cellulase activity is nearly proportional to the feeded substrate whereas the growth in mycelium is reduced. The productivity of cellulase formation is enhanced in comparison to continuous substrate addition as well as under limiting and non-limiting conditions for growth of mycelium.     

Production of cellulase enzymes during the solid-state fermentation of empty palm fruit bunch fiber

Penicillium verruculosum COKE4E is a fungal strain isolated from bituminous coal. The microorganism cultivated in a minimal medium supplemented with Avicel, carboxymethylcellulose, and oat spelt xylan produced cellulase enzymes as exhibiting carboxymethylcellulase (CMCase), Avicelase, xylanase, and cellobiosidase activities. In this study, the productivity of the extracellular enzymes in the strain was evaluated by using empty palm fruit bunch fiber (EPFBF), a lignocellulosic biomass, as a substrate for solid-state bioconversion. The highest cellulase activities were observed after 6 days of fermentation at pH 6.0 and 30 °C. The enzymes were secreted as cellulosomes for the degradation of EPFBF as a sole carbon source. Focused ion beam analysis showed that P. verruculosum COKE4E produced cellulolytic enzymes that were able to effectively biodegrade EPFBF during solid-state fermentation. In this process, 6.5 U of CMCase, 6.8 U of Avicelase, and 8.8 U of xylanase per gram of dry solid EPFBF were produced. These results demonstrate that EPFBF may be a potential raw material in solid-state fermentation for the production of cellulase enzymes to be used for biofuel production.     

A new Penicillium echinulatum strain with faster cellulase secretion obtained using hydrogen peroxide mutagenesis and screening with 2-deoxyglucose

Aims: The aim of this study is to improve cellulase production and secretion by Penicillium echinulatum using mutagenesis and selection in association with microfermentation and microanalysis methods. Methods and Results: A new genetic variant was isolated from strain 9A02S1 and named S1M29. It was obtained by mutagenesis with H₂O₂ and two screening steps, which involved selection in Petri dishes using the medium supplemented with 2‐deoxyglucose and microfermentations in submerged culture. The mutant showed higher cellulase productivity than 9A02S1 based on the Filter Paper Activity assay and endoglucanase; the peak activities for these enzymes were reached significantly faster than for the parent strain. Conclusions: The mutant obtained after mutagenesis and selection could produce and secrete cellulase faster than the parent strain. Significance and Impact of the Study: Mutagenesis followed by selection is a useful tool for rapidly generating new cellulase‐producing phenotypes in fungi. Faster production and higher titers of cellulases in mutant strains contribute to reduce the production costs for enzymatic complexes that hydrolyse lignocellulose residues and form fermented syrups, thus contributing to the economic production of bioethanol.     

Studies on a wild strain of Schizophyllum commune: Cellulase and xylanase production and formation of the extracellular polysaccharide Schizophyllan

A wild strain of schizophyllum commune (Fr:Fr:) isolated in Bangladesh produced cellulase and xyianase in high yields as well as the exobiopolymer schizophyllan. It was found experimentally that concentrations of 4% Avicel, 3.5% peptone, and 0.5% Ca(NO(3))(2).4H(2)O were optimal for growth and product formation. Bacto-peptone was found to be the most suitable substrate of a number of casein, mycological, and meat peptone preparations for enzyme production. Young plate-culture inocula (4 days) were found to be better than comparatively aged fungal cultures (14 days). With the optimized medium, 5 units filter paper (FP) cellulase, 1244 units xylanase, 108 units beta-glucosidase, and 65 units of carboxymethyl (CM) cellulase per mL culture filtrate were obtained in shake flasks. In a laboratory fermentor the respective enzyme activities were 4.5 units FP-cellulase, 1200 units xylanase, 100 units beta-glucosidase, and 60 units CM-cellulase per mL culture filtrate. A biopolymer, reported to be active against can cerous cells, was an additional product in addition to the enzymes.     

Preparation of Mutants Resistant to Catabolite Repression of Trichoderma reesei

The development of agar plate screening techniques has allowed the isolation of mutants of Trichoderma reesei capable of synthesizing cellulase under the conditions of a high concentration of glucose. Mutants resistant to catabolite repression by glycerol or glucose were isolated on Walseth’s cellulose (WC) agar plates containing 5% glycerol or 5% glucose, respectively. Mutants resistant to catabolite repression by glycerol were not derepressed enough for the production of cellulase on WC agar plates containing 5% glucose or in flask cultures with a mixture of 1% Avicel and 3% glucose. On the contrary, two mutant strains resistant to catabolite repression by glucose (KDD-10 and DGD-16) produced large clearing zones on WC agar plates containing 5% glucose. Both strains could begin to produce CMCase even in the presence of residual glucose and finally produced 1.5 times the CMCase activity, in flask cultures on 1% Avicel and 3% glucose, than that with 1% Avicel alone. These results suggest that KDD-10 and DGD-16 are comparatively derepressed by glucose for cellulase production.     

核、质基因对肺形侧耳菌丝生长、营养成分及基因表达的影响

本研究通过杂交构建肺形侧耳同核异质菌株N1M1、N1M2和同质异核菌株N1M1、N2M1,比较菌丝形态与生长速度、营养成分以及常见的细胞核基因与线粒体基因的表达量,分析线粒体基因对肺形侧耳菌丝的影响,探讨线粒体基因与核基因的相互作用。由菌丝生长情况可知N1M1和N1M2菌丝形态相似,生长速度差异不显著,N1M1和N2M1菌丝形态差异大,生长速度差异极显著,在菌丝形态与生长速度上细胞核基因作用大于线粒体基因。进一步检测菌株中的主要营养成分发现必需氨基酸与总水解氨基酸含量差异显著,菌株N1M2蛋白含量显著高于N1M1,N1M1维生素C含量是N1M2的1.67倍,菌株N2M1多糖和蛋白含量显著高于N1M1,铁和维生素C含量显著低于N1M1。所以细胞核基因、线粒体基因都能影响肺形侧耳营养成分含量。检测同核异质菌株N1M1、N1M2的7个细胞核常见基因的表达情况发现,N1M2菌丝中6个细胞核基因的表达量都显著高于N1M1,这表明肺形侧耳线粒体基因的不同会影响核基因的表达;同质异核菌株N1M1、N2M1的14个线粒体普通编码蛋白基因表达差异显著,这说明线粒体基因的表达量会因核基因的不同有所差异。综上,肺形侧耳线粒体基因和细胞核基因能够相互影响,共同作用于生命活动。