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Abstract Background Low-cost cellulase production is vital to sustainable second-generation biorefineries. The catabolically derepressed strain of Penicillium funiculosum NCIM1228 (PfMig188 or ∆Mig1) secretes a superior set of cellulolytic enzymes, that are most suitable for 2G biorefineries. At a 3% (w/w) load, the ∆Mig1 secretome can release > 80% of fermentable sugars from lignocellulose at a 15% (w/v) biomass load, irrespective of the type of biomass and pretreatment. The robustness of the secretome can be further increased by improving the cellulase production capacity of the fungal strain. Results We began by identifying the transcription factor responsible for cellulase production in NCIM1228. An advanced RNA-seq screen identified three genes, clr-2, ctf1a and ctf1b; the genes were cloned under their native promoters and transformed into NCIM1228. Of the three, clr-2 overexpression led to twofold higher cellulase production than the parent strain and was thus identified as the transcriptional activator of cellulase in NCIM1228. Next, we overexpressed clr-2 in ∆Mig1 and expected an exponential increase in cellulolytic attributes accredited to the reinforced activation mechanisms, conjoint with diminished negative regulation. Although clr-2 overexpression increased the transcript levels of cellulase genes in ∆Mig1, there was no increase in cellulase yield. Even a further increase in the transcript levels of clr-2 via a stronger promoter was ineffective. However, when the CaCO3 concentration was increased to 5 g/l in the growth medium, we achieved a 1.5-fold higher activity of 6.4 FPU/ml in the ∆Mig1 strain with clr-2 overexpression. Enthused by the calcium effect, a transcriptomic screen for genes encoding Ca2+-activated kinase identified ssp1, whose overexpression could further increase cellulase yield to ~ 7.5 FPU/ml. Investigation of the mechanism revealed that calcium signaling exclusively enhances the translation and secretion of cellulase in Penicillium funiculosum. Conclusions Our study identifies for the first time that cellulose activates two discrete signaling events to govern cellulase transcription and posttranscriptional processes (translation, processing and secretion) in P. funiculosum NCIM1228. Whereas Clr-2, the transcriptional activator of cellulase, governs transcription, calcium signaling specifically activates cellulase translation and secretion.

Introduction[1]: Sunflower meal is considered as an attractive alternative to soybean meal due to its adaptation to dry and temperate climates as well as its lower anti-nutrient content than other oilseeds. Sunflower meal has a limiter that restricts its use in broiler diets. The fiber in sunflower meal has limited its use in the diet of broilers due to reduced metabolic energy as well as production. Fermentation, which uses beneficial microorganisms, can reduce the chemical composition of the feed and increase the yield of broilers by producing enzymes and using fiber as a feed source. In this study, in order to investigate the effect of fermentation on sunflower meal, growth yield, microbial population composition, blood parameters in broiler chickens were performed. Materials and methods: The research was conducted at the research farm of Sari University of Agricultural Sciences and Natural Resources in 2020. In this study, 200 commercial male Ross 308 broilers were used in a completely randomized design with five treatments, five replications, and eight chickens per replication. The treatments included: 1) Control diet, 2) Diet containing sunflower meal (negative control), 3) Diet containing sunflower meal fermented with Aspergillus Oryzae, 4) Diet containing sunflower meal fermented with Penicillium Funiculosum, and 5) Diet containing sunflower meal fermented with Aspergillus Oryzae and Penicillium Funiculosum. Sunflower meal was obtained from Behpak Behshahr (Mazandaran) company, ground in the animal nutrition laboratory of Sari University of Agricultural Sciences and Natural Resources, and sieved to a size of 2 mm. Aspergillus Oryzae (PTCC5010) and Penicillium Funiculosum (PTCC5301) were purchased as lyophilized vials from the Fungus and Bacteria Collection Center of the Iranian Research Organization for Science and Technology (IROST). During the fermentation process, 1.1 liters of the mixture of distilled water and primer culture (containing at least 105 colony forming units per ml) was added to each kilogram of sunflower meal, and the resulting mixture was thoroughly mixed by hand for 15 minutes. The mixture was fermented in special tanks (with a one-way valve to remove the produced gases and prevent air from entering) for 7 days at 30 °C, and finally, the fermented sunflower meal was dried for three days at room temperature. All chickens were kept under the same breeding conditions during the 39-day period and had free access to feed. Experimental diets were adjusted to three periods: initial (1-10 days), growth (11-24), and final (25-39) using Table 1 of the Ross 308 Catalog of Nutritional Requirements for Broilers. The pH of fermented sunflower meal was determined using the Chiang et al. method. Sunflower meal was sampled before and after fermentation to measure dry matter, ash, crude protein by the Kjeldahl method, crude fat by Soxhlet device, and insoluble fibers in acidic detergent and neutral detergent using a fibrotec device. Total amino acid was measured by the ninhydrin method. Soluble sugar was measured by the intron method. Total phenol content was measured using the Folin-Siocalcu reagent and spectrophotometry. Soluble proteins were measured by the Bradford method. Results and discussion: During the fermentation of sunflower meal, pH, crude fat, soluble protein and soluble sugar decreased. The amount of crude protein, total amino acid, insoluble fiber in acidic detergent, and insoluble fiber in neutral detergent increased. In the whole breeding period, body weight gain was improved in treatments fed with fermented feed. In the treatment of Aspergillus­ Oryza + Penicillium funiculosum, feed consumption decreased compared to control, negative control and Penicillium funiculosum treatments. The feed conversion ratio increased in the negative control treatment at the age of 1-39 days compared to other treatments. In the ileum, coliform bacteria decreased in negative control treatments, Aspergillus Oryzae, Aspergillus Oryzae + Penicillium funiculosum compared to the control treatment, but Penicillium funiculosum treatment increased compared to other treatments. At 39 days of age, the use of fermented and raw feed increased the level of HDL-c in the negative control and Aspergillus Oryzae + Penicillium funiculosum treatments. The amount of LDL-c increased in the treatments of Aspergillus Oryzae and Penicillium funiculosum Conclusion: Based on the results, the use of fermented feed in the feeding of broiler chickens during the entire breeding period has improved weight gain and feed conversion ratio.

Abstract The food enzyme cellulase (4‐(1,3;1,4)‐β‐d‐glucan 4‐glucanohydrolase; EC 3.2.1.4) is produced with the non‐genetically modified Penicillium funiculosum strain Lzc35 by Danisco US Inc. The cellulase is intended to be used in distilled alcohol production, baking and brewing processes. Since residual amounts of total organic solids (TOS) are removed by distillation, dietary exposure was only calculated for baking and brewing processes. Based on the proposed maximum use levels, dietary exposure to the food enzyme–TOS was estimated to be up to 0.416 mg TOS/kg body weight (bw) per day. Genotoxicity tests did not raise a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 84 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 200. Similarity of the amino acid sequence of the food enzyme to those of known allergens was searched and no match was found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood is considered to be low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Lân là một trong những nguyên tố chính quyết định năng suất cây trồng và nấm hòa tan lân đóng vai trò quan trọng trong gia tăng lượng lân hữu dụng cho cây trồng. Mục tiêu của nghiên cứu nhằm phân lập và định danh một số dòng nấm hòa tan lân cao từ nền đất lúa tưới nước ngập khô xen kẽ kết hợp bón phân hữu cơ ở nhà lưới. Môi trường NBRIP dùng để phân lập và đánh giá khả năng hòa tan lân. Lân hòa tan được xác định bằng phương pháp hiện màu molybdate. Kết quả cho thấy tổng cộng có 37 dòng nấm có khả năng hòa tan lân được phân lập từ 7 mẫu đất. Trong đó, hai dòng nấm ký hiệu B1 và B10 hoà tan lân lần lượt đạt 2104 mg.L-1 và 2618 mg.L-1 sau 3 và 4 ngày thí nghiệm. Hai dòng nấm này đều hòa tan lân tốt ở mức pH từ 5-7, nhiệt độ 25oC-35oC, độ mặn lên đến 0,5-1% NaCl và hòa tan tốt các dạng lân khó tan như AlPO4 và FePO4. Kết quả định danh thông qua đoạn ITS cho thấy hai dòng nấm này được định danh khoa học lần lượt là Penicillium funiculosum B1 và Aspergillus tubingensis B10. Tóm lại, hai dòng nấm này có vai trò và tiềm năng quan trọng trong việc sản xuất chế phẩm vi sinh giúp gia tăng độ phì nhiêu đất, kích thích sinh trưởng cây trồng.

The aim of this work was to optimize the growth conditions and continuous production of the enzyme using free and immobilized cells of inulinase by Penicillium funiculosum. The highest yield of enzyme (163.5U/mL) was obtained when the culture was incubated at 27oC and 200 rpm for 96h in a fermentation medium containing both inulin and peptone as sole carbon and nitrogen source, respectively. When the cells of the P. funiculosum were immobilized on different carriers, especially linen fibers, their production ability was successfully maintained for seven successive batches. When the fermentation was carried out using inulin juice prepared from Jerusalem artichoke tubers (in place of pure inulin), inulinase production could be sustained till the second cultivation batch of the P. funiculosum immobilized on linen fibers, yielding 122 U/mL enzyme. Results proved the feasibility of using crude inulin juice as a simple and economic carbon source for the production of inulinase.

ABSTRACT Two induced mutant strains, obtained from the fungus Penicillium funiculosum after UV treatments, were used for protoplast fusion. These mutants (12 and 18) were selected according to their dextranase activity and stability. Seven fusants having dextranase over-yield than their parental strain were obtained. The best dextranase producing fusant (No. 2) was selected for further studies. Dextranase was purified from cell-free culture of fusant 2 by consecutive column chromatography using Q-sepharose FF, Superose 12 prep. grade 60/600 and Mono Q-FPLC. The purification was estimated by SDS-PAGE as well as isoelectric focusing. Four dextranase components (I, II, III and IV) were separated with an estimated molecular weights of 67 kDa. The pI of the all components were found to be around 3.0 as estimated by gel electrophoresis using both broad and low pI calibration protein kits. The dxetranase components showed pH and optimum temperature of 5 and 55oC, respectively. Dextran was the sole carbon source for dextranase production by the dextranase components. The effect of the dextranase components on different types of dextrans were also studied.

The optimization of growth conditions for the production of inulinase by Penicillium funiculosum cells were studied as well as the continuous production of the enzyme using immobilized cells. The highest amount of enzyme (163.5U/mL) was obtained when the producing cells were incubated for 96 hours at 27oC and 200 rpm in a fermentation medium containing both inulin and peptone as sole carbon and nitrogen sources respectively. However, when the cells of the tested microorganism were adsorbed on different carriers, especially linen fibers, their production ability was also successfully maintained, to different extends, for seven successive batches. Moreover, commercially pure inulin is very expensive in only small quantities, this fermentation medium was later substituted by a crude inulin solution obtained from Jerusalem artichoke tubers (Helianthus tuberosus). The crude inulin juice was able to sustain inulinase production during the second batch cultivation of the P. funiculosum, immobilized by their adsorption on linen fibers, in a satisfactory level of about 122U/mL. Moreover, the use of the previously mentioned crude inulin preparation was also compared to the use of either complete or minimal media, composed solely of 1% pure inulin. The method, adopted in this study for inulinase production, is simple, economic, time saving, non-toxic to the microorganism and the loaded linen pads are reusable.

The optimization of growth conditions for the production of inulinase by Penicillium funiculosum cells were studied as well as the continuous production of the enzyme using immobilized cells. The highest amount of enzyme (163.5U/mL) was obtained when the producing cells were incubated for 96 hours at 27oC and 200 rpm in a fermentation medium containing both inulin and peptone as sole carbon and nitrogen sources respectively. However, when the cells of the tested microorganism were adsorbed on different carriers, especially linen fibers, their production ability was also successfully maintained, to different extends, for seven successive batches. Moreover, commercially pure inulin is very expensive in only small quantities, this fermentation medium was later substituted by a crude inulin solution obtained from Jerusalem artichoke tubers (Helianthus tuberosus). The crude inulin juice was able to sustain inulinase production during the second batch cultivation of the P. funiculosum, immobilized by their adsorption on linen fibers, in a satisfactory level of about 122U/mL. Moreover, the use of the previously mentioned crude inulin preparation was also compared to the use of either complete or minimal media, composed solely of 1% pure inulin. The method, adopted in this study for inulinase production, is simple, economic, time saving, non-toxic to the microorganism and the loaded linen pads are reusable.

Abstract Background The filamentous fungus Penicillium funiculosum produces a range of glycoside hydrolases (GH). The XynD gene, encoding the sole P. funiculosum GH10 xylanase described so far, was cloned into the pPICZαA vector and expressed in methylotrophe yeast Pichia pastoris, in order to compare the results obtained with the P. funiculosum GH11 xylanases data. Results High level expression of recombinant XynD was obtained with a secretion of around 60 mg.L-1. The protein was purified to homogeneity using one purification step. The apparent size on SDS-PAGE was around 64 kDa and was 46 kDa by mass spectrometry thus higher than the expected molecular mass of 41 kDa. The recombinant protein was N- and O-glycosylated, as demonstrated using glycoprotein staining and deglycosylation reactions, which explained the discrepancy in molecular mass. Enzyme-catalysed hydrolysis of low viscosity arabinoxylan (LVAX) was maximal at pH 5.0 with Km(app) and kcat/Km(app) of 3.7 ± 0.2 (mg.mL-1) and 132 (s-1mg-1.mL), respectively. The activity of XynD was optimal at 80°C and the recombinant enzyme has shown an interesting high thermal stability at 70°C for at least 180 min without loss of activity. The enzyme had an endo-mode of action on xylan forming mainly xylobiose and short-chain xylooligosaccharides (XOS). The initial rate data from the hydrolysis of short XOS indicated that the catalytic efficiency increased slightly with increasing their chain length with a small difference of the XynD catalytic efficiency against the different XOS. Conclusion Because of its attractive properties XynD might be considered for biotechnological applications. Moreover, XOS hydrolysis suggested that XynD possess four catalytic subsites with a high energy of interaction with the substrate and a fifth subsite with a small energy of interaction, according to the GH10 xylanase literature data.