Preparation of mutants of Trichoderma reesei with enhanced cellulase production.

The development of an agar plate screening technique has allowed the isolation of a range of mutants of Trichoderma reesei capable of synthesizing cellulase under conditions of high catabolite repression. The properties of one of these mutants (NG-14) is described to illustrate the use of this technique. NG-14 produced five times the filter paper-degrading activity per ml of culture medium and twice the specific activity per mg of excreted protein in submerged culture when compared with the best existing mutant, QM9414. NG-14 also showed enhanced endo-beta-glucanase and beta-glucosidase production. Although these mutants were isolated as cellulase producers in the presence of 5% glycerol on agar plates, in similar liquid medium, NG-14 exhibits only partial derepression of the cellulase complex. Since the proportions of filter paper activity, endo-beta-glucanase, and cellobiase were not the same in mutants NG-14 and QM9414, and the yields of each enzyme under conditions repressive for cellulase synthesis were different, differential control of each enzyme of the cellulase complex is implied. These initial results suggest that the selective technique for isolating hyper-cellulase-producing mutants of Trichoderma will be of considerable use in the development of commercially useful cellulolytic strains.     

Evaluation of culture conditions for cellulase production by two Trichoderma reesei mutants under solid-state fermentation conditions

Response surface methodology (RSM) was used to evaluate the effects of fermentation parameters for cellulase production by Trichoderma reesei QM9414 and T. reesei MCG77 in solid-state fermentation using rice bran as substrate. Initial pH, moisture content and temperature were optimized using filter paper activity (FPA) as response. Statistical analysis of the results for T. reesei QM9414 showed that only moisture content had significant effect on cellulase activity and had a linear effect on enzyme activity (maximum enzyme activities were obtained at 70% moisture content). The results for T. reesei MCG77 showed that temperature and moisture content were the most significant parameters for cellulase activity. The optimum cellulase production was in the temperature range of 25-30 degrees C and moisture content between 55% and 70%. After the optimization, the FPA in T. reesei MCG77 was increased by 2.5 folds compared to that of T. reesei QM9414.     

敲除组蛋白去乙酰化酶基因hdac对里氏木霉纤维素酶表达的调控作用

[背景]里氏木霉(Trichoderma reesei)是木霉属中产纤维素酶最具代表性的真菌之一,表观遗传调控是不涉及DNA序列变化的可遗传变化,组蛋白去乙酰化是其中一种。组蛋白去乙酰化酶(histone deacetylase,HDAC)负责脱乙酰化,敲除去乙酰化酶基因可引起菌株孢子、菌丝及纤维素酶活性等的一系列改变。[目的]通过敲除里氏木霉组蛋白去乙酰化酶基因(histone deacetylase,hdac)建立了里氏木霉hdac缺失突变株(T.reesei△hdac),以研究对纤维素酶基因表达的调控作用。[方法]利用Split-Maker技术构建了组蛋白去乙酰化酶基因敲除表达盒,并转化了里氏木霉T.reesei QM9414。经PCR及Southern blotting验证正确后,对突变体T.reesei△hdac连续7 d检测滤纸酶活(filter paper activity,AFP)、羧甲基纤维素钠酶活(carboxymethyl cellulase activity,CMCA),利用RT-qPCR检测纤维素酶及其相关基因cbh1、egl1和xyr1的表达。[结果]突变体T.reesei△hdac两种酶活力均显著高于出发菌株,分别高出8.00、30.00 IU/mL。突变体T.reesei△hdac纤维素酶及其相关基因cbh1、egl1和xyr1的转录水平分别为出发菌株T.reesei QM9414的6.50、6.01和4.51倍。[结论]里氏木霉中纤维素酶的基因表达明显受到组蛋白去乙酰化酶基因(hdac)的调控,这为研究里氏木霉表观遗传调控对纤维素酶的影响提供了新的证据。     

Enhanced cellulase production from Trichoderma reesei QM 9414 on physically treated wheat straw

Summary Trichoderma reesei QM 9414 was grown on wheat straw as the sole carbon source. The straw was pretreated by physical and chemical methods. The particle size of straw was less than 0.177 mm. Growth of T. reesei QM 9414 was maximal with alkali-pretreated straw whereas cellulase production was optimal when physically pretreated straw was used as substrate. Cellulase yields expressed as IU enzyme activity/g cellulose present in the cultures were considerably higher when alkali pretreatment of wheat straw was omitted. Cellulase yields of 666 IU/g cellulose for filter paper activity (FPA) are the highest described for cultures of T. reesei QM 9414 carried out in analogous conditions. Crystallinity index of the cellulose contained in wheat straw increased slightly after alkali pretreatment. This increase did not decrease cellulose accessibility to the fungus. Delignification of wheat straw was not necessary to achieve the best cellulase production.     

Intraspecific hybridization of Trichoderma reesei by protoplast fusion

Protoplasts obtained from mycelia of a single auxotrophic mutant of Trichoderma reesei QM 9414 were fused with those of T. reesei QM 9136 in the presence of 0.5 M glycine-NaOH buffer, pH 7.5, containing 0.05 M CaCl₂ · 2H₂O and 35% polyethylene glycol 4,000. The regeneration frequency of these protoplasts was 8.9–12.0% on a solid culture medium with soft agar overlay. The fused protoplasts successfully formed heterokaryons showing 3.33% of the fusion frequency. A heterozygous diploid was obtained from conidia of the heterokaryon by treatment with 0.1% d-camphor. The diploid showed a 1.9 fold DNA content per conidial nucleus compared to T. reesei QM 9414. The frequency of diploid formation was about 1.9 × 10⁻⁴ per conidium. Cellulase activities, such as filter paper degrading and CM-cellulose and Avicel saccharifying activities, and the xylanase activity of the diploid showed intermediate values between those of T. reesei QM 9414 and T. reesei QM 9136. However, the β-glucosidase, β-1,3-glucanase and chitinase activities of the diploid increased to levels equal to on above those of T. reesei QM 9414 and T. reesei QM 9136. The existence of a parasexual cycle of T. reesei and the possibility of its application to enhanced enzyme productivity were confirmed using the protoplast fusion technique.     

Spent brewery grains as substrate for the production of cellulases byTrichoderma reesei QM9414

Summary The cellulolytic fungusTrichoderma reesei QM9414 can be cultivated on spent brewery grains for the production of cellulases. The levels of the cellulase components endoglucanase and exoglucanase synthesized, and the complexes filter paper cellulase and grain-hydrolyzing cellulase synthesized by the organism on spent grains were as high as 287, 182, 187, and 449 units per g available cellulose, respectively. Scaling up the spent grains fermentation system by up to 40-fold (200g dry substrate/tray) demonstrated that cellulase production was comparable to laboratory scale (5g dry substrate/flask) yields. Cultivation of the fungus was feasible on spent grains without pretreatment or further adjustment, although the enzyme yield was somewhat lower than that on dried grains moistened with water orTrichoderma medium. This suggested the possible reutilization of spent grains, with minimal pretreatment, in the cultivation ofT.reesei QM9414 for cellulase synthesis and for future incorporation into animal feed.     

Formation and release of cellulolytic enzymes during growth of Trichoderma reesei on cellobiose and glycerol

Summary Production and release of cellulolytic enzymes by Trichoderma reesei QM 9414 were studied under induced and non-induced conditions. For that purpose, a method was developmed to produce cellulases by Trichoderma reesei QM 9414 using the soluble inducer, cellobiose, as the only carbon source. The production was based on continuous feeding of cellobiose to a batch culture. For optimum production, the cellobiose supply had to be adjusted according to the consumption so that cellobiose was not accumulated in the culture. With a proper feeding program the repression and/or inactivation by cellobiose could be avoided and the cellulase production by Trichoderma reesei QM 9414 was at least equally as high as with cellulose as the carbon source.During the cultivation, specific activities against filter paper, carboxymethyl cellulose (CMC) and p-nitrophenyl glucoside were analyzed from the culture medium as well as from the cytosol and the cell debris fractions. There was a base level of cell debris bound hydrolytic activity against filter paper and p-nitrophenyl glucoside even in T. reesei grown non-induced on glycerol. T. reesei grown on cellobiose was induced to produce large amounts of extracellular filter paper and CMC hydrolyzing enzymes, which were actively released into the medium even in the early stages of cultivation. -Glucosidase was mainly detected in the cell debris and was not released unless the cells were autolyzing.     

Screening of highly cellulolytic fungi and the action of their cellulase enzyme systems

Over 100 strains of wood-rotting fungi were compared for their ability to degrade wood blocks. Some of these strains were then assayed for extracellular cellulase [1,4-(1,3;1,4)-β- -glucan 4-glucanohydrolase, EC 3.2.1.4] activity using a variety of different solid media containing carboxymethyl cellulose or acid swollen cellulose. The diameter of clearing on these plates gave an approximate indication of the order of cellulase activities obtained from culture filtrates of these strains. Trichoderma strains grown on Vogels medium gave the highest cellulase yields. The cellulase enzyme production of T. reesei C30 and QM9414 was compared with that of eight other Trichoderma strains. Trichoderma strain E58 had comparable endoglucanase and filter paper activities with the mutant strains while the β- -glucosidase [β- -glucoside glucohydrolase, EC 3.2.1.21] activity was approximately six to nine times greater.     

Mutants of the white-rot fungus Sporotrichum pulverulentum with increased cellulase and β-d-glucosidase production

This paper reports the isolation of mutants of the white-rot fungus Sporotrichum pulverulentum and the results of a survey of enzymic activity among these mutants. The strains were screened for extracellular cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] and β-d-glucosidase (β-d-glucoside glucohydrolase, EC 3.2.1.21) production in shake flask experiments. Apart from strain 63-2, strains 6, 63, 9, L5, E-1 and UV-18 showed equal or higher endo-1,4-β-d-glucanase (cellulase), filter paper-degrading and β-d-glucosidase activities than S. pulverulentum. The cellulase activity obtained, measured as filter paper activity, was comparable to that reported for Trichoderma reesei QM9414. However, the β-d-glucosidase activity was about six times higher than for the QM9414 strain. The pH and temperature-activity profiles of crude β-d-glucosidase preparations from the various strains were determined and were found to be identical. The thermal stability at pH 4.5 and 40°C was 5 days for all these preparations.     

青霉的纤维素酶抗降解物阻遏突变株的选育

从土样中筛出一株生长快,产纤维素酶较高的斜卧青霉(Penicillium decumbens114-2)。能在全纤维素(hlocellulose)双层平板上形成清晰的透明圈。摇瓶培养的滤纸酶活可达8.8mg 葡萄糖/ml.h。 114-2菌株(其纤维素酶合成可为葡萄糖所阻遏)经UV和NG诱变处理后,在含葡萄糖的全纤维素平板上筛选到多株仍能形成明显透明圈的突变株。其中JN15和JU1在含葡萄糖的全纤维素液体培养基中,在残余葡萄糖浓度为 1％左右时,滤纸酶活可分别达到7.3 和13.9mg葡萄糖ml·h。这是出发株114-2和纤维素酶高产菌株木霉EA_3-867和QM9414所不能的。在不加阻遏剂的对比试验中,两个突变株的纤维素酶产量都比较高。其中,JU1的滤纸、CMC和棉花酶活分别达到33mg葡萄糖/ml·h,234mg葡萄糖/ml·h和86mg葡萄糖/ml·24h。     

Effect of different carbon sources on cellulase production by Hypocrea jecorina (Trichoderma reesei) strains

The ascomycete Hypocrea jecorina, an industrial (hemi)cellulase producer, can efficiently degrade plant polysaccharides. At present, the biology underlying cellulase hyperproduction of T. reesei, and the conditions for the enzyme induction, are not completely understood. In the current study, three different strains of T. reesei, including QM6a (wild-type), and mutants QM9414 and RUT-C30, were grown on 7 soluble and 7 insoluble carbon sources, with the later group including 4 pure polysaccharides and 3 lignocelluloses. Time course experiments showed that maximum cellulase activity of QM6a and QM9414 strains, for the majority of tested carbon sources, occurred at 120 hrs, while RUT-C30 had the greatest cellulase activity around 72 hrs. Maximum cellulase production was observed to be 0.035, 0.42 and 0.33 µmol glucose equivalents using microcrystalline celluloses for QM6a, QM9414, and RUTC-30, respectively. Increased cellulase production was positively correlated in QM9414 and negatively correlated in RUT-C30 with ability to grow on microcrystalline cellulose.     

Antagonism of Pythium blight of zucchini by Hypocrea jecorina does not require cellulase gene expression but is improved by carbon catabolite derepression

Toward a better understanding of the biochemical events that lead to biocontrol of plant pathogenic fungi by Hypocrea/Trichoderma spp., we investigated the importance of carbon catabolite (de)repression and cellulase formation in the antagonization of Pythium ultimum by Hypocrea jecorina (Trichoderma reesei) on agar plates and in planta. Hypocrea jecorina QM9414 could antagonize and overgrow P. ultimum but not Rhizoctonia solani in plate confrontation tests, and provided significant protection of zucchini plants against P. ultimum blight in planta. A carbon catabolite derepressed cre1 mutant of H. jecorina antagonized P. ultimum on plates more actively and increased the survival rates of P. ultimum-inoculated zucchini plants in comparison with strain QM9414. A H. jecorina mutant impaired in cellulase induction could also antagonize P. ultimum on plates and provided the same level of protection of zucchini plants against P. ultimum as strain QM9414 did. We conclude that cellulase formation is dispensable for biocontrol of P. ultimum, whereas carbon catabolite derepression increases the antagonistic ability by apparently acting on other target genes.     

Mutants of the white-rot fungus Sporotrichum pulverulentum with increased cellulase and β- -glucosidase production

This paper reports the isolation of mutants of the white-rot fungus Sporotrichum pulverulentum and the results of a survey of enzymic activity among these mutants. The strains were screened for extracellular cellulase [see 1,4-(1,3;1,4)-β- -glucan 4-glucanohydrolase, EC 3.2.1.4] and β- -glucosidase (β- -glucoside glucohydrolase, EC 3.2.1.21) production in shake flask experiments. Apart from strain 63-2, strains 6, 63, 9, L5, E-1 and UV-18 showed equal or higher endo-1,4-β- -glucanase (cellulase), filter paper-degrading and β- -glucosidase activities than S. pulverulentum. The cellulase activity obtained, measured as filter paper activity, was comparable to that reported for Trichoderma reesei QM9414. However, the β- -glucosidase activity was about six times higher than for the QM9414 strain. The pH and temperature-activity profiles of crude β- -glucosidase preparations from the various strains were determined and were found to be identical. The thermal stability at pH 4.5 and 40°C was 5 days for all these preparations.     

Sensitivity of various yeasts to crude cellulolytic enzyme complexes fromTrichoderma reesei

Summary Twenty-six yeast strains, representative of different yeast genera, were tested for their sensitivity to crude extracellular cellulolytic enzyme complexes obtained from the fungusTrichoderma reesei QM 9414 and its mutants M 6 and MHC 22 (Microcrystalline cellulose was the sole carbon source.) Practically all the yeast strains tested were found to be sensitive, exhibiting signs of cellwall weakening and lysis during prolonged incubation with the emzymes fromTrichoderma. Under growth conditions, the effect of cellulolytic enzymes on yeast cells and their growth rates was much less pronounced. However, at increased cellulase concentrations (5 mg/ml) in the growth medium, lysis of stationary phase yeast cells was observed.     

Action of Trichoderma reesei QM9414 and C30 cellulase on substrates with varying crystallinity

The action of cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] preparations from Trichoderma reesei QM9414 and C30 has been compared on Sigmacell, Solka Floc and alkali-treated bagasse in the presence and absence of added d-glucose and cellobiose. On the basis of equal filter paper activity the two preparations acted similarly on the two cellulosic substrates, while in the case of alkali-treated bagasse the C30 preparation gave greater d-glucose release. The relative levels of cellobiose produced from alkali-treated bagasse suggests that the non-cellobiose route was more important in d-glucose release by the C30 preparation compared to the QM9414 preparation.     

A high cellulase-producing mutant strain of Trichoderma reesei

A mutant strain with increased production of cellulolytic enzymes was induced from the good cellulase producer Trichoderma reesei QM 9414. Cellulase activities of the mutant in fermenter cultivations were increased two- to three-fold and β-glucosidase activity up to six-fold when compared to the corresponding activities produced by QM 9414.     

液态发酵条件下拟康宁木霉8985产纤维素酶能力初探

液态发酵条件下,以微晶纤维素为唯一碳源,比较了拟康宁木霉Trichoderma koningiopsis 8985和里氏木霉T. reesei QM9414产纤维素酶的能力。8985发酵12 h开始产生纤维素酶,36 h时酶活达到产酶峰值的50%,此时QM9414尚未诱导产酶。测定8985发酵84 h时上清液中滤纸纤维素酶、羧甲基纤维素酶、β-葡萄糖苷酶和木聚糖酶的酶活分别为1.06、3.62、1.80和6.67 IU/mL,分别是QM9414上述酶活的1.72、1.70、6.35和1.12倍。8985滤纸纤维素酶酶活的最适反应条件为pH 4.5,反应温度50 ℃,在Fe³⁺ (≤ 4 mmol/L)和Cu²⁺ (0-10 mmol/L)存在条件下酶活稳定。     

Purification and characteristics of xyloglucanase and five other cellulolytic enzymes from Trichoderma reesei QM9414

By combining anion-exchange chromatography with gel filtration, an effective method for purification of wild-type xyloglucanase and five other cellulolytic enzymes from strain QM9414 of Trichoderma reesei was established. Characterization by enzyme activity assay, SDS-PAGE, and mass spectrometry identified the purified proteins as cellobiohydrolases I and II, endoglucanases I and II, a xyloglucanase, and β-xylosidase, of which the xyloglucanase was purified for the first time from the mutant strain QM9414. This method holds great promise to study the mechanism of cellulolytic enzymes, to investigate the synergistic action between cellulase and other cellulolytic enzymes, and to better exploit enzyme preparations for degradation of lignocellulose.     

pH and thermal stability studies of carboxymethyl cellulase from intergeneric fusants of Trichoderma reesei/Saccharomyces cerevisiae

The combined effect of pH and temperature on carboxymethyl cellulase from two intergeneric fusants (M 14 and M 62) of Trichoderma reesei QM 9414/Saccharomyces cerevisiae NCIM 3288 was studied using response surface methodology. A central composite design for two variables was employed for the optimization studies. This study was compared with similar studies carried out with Trichoderma reesei QM 9414. The optimal pH and temperature for the enzymes derived from these organisms were: for the fusant M 14—pH 5.7 and 41.7°C, for the fusant M 62—pH 5.3 and 43°C, and for Trichoderma reesei QM 9414—pH 4.31 and 38.3°C. Received 5 May 1997/ Accepted in revised form 17 July 1998     

Synergism of cellulase enzymes in mixed culture solid substrate fermentation

Sugar cane bagasse was subjected to a mixed culture, solid substrate fermentation with Trichoderma reesei QM9414 and Aspergillus terreus SUK-1 to produce cellulase and reducing sugars. The highest cellulase activity and reducing sugar amount were obtained in mixed culture. The percentage of substrate degradation achieved employing mixed culture was 26% compared to 50% using separate cultures of the two molds. This suggests that the synergism of enzymes in mixed culture solid substrate fermentation have lower synergism than in pure culture.