Solid state fermentation for cellulases and β-glucosidase production by Aspergillus niger

Production of cellulases and β-glucosidase was studied using locally-isolated Aspergillus niger on various cheap sources of cellulose like bagasse, corn corbs, computer cards and sawdust, by solid state fermentation (SSF) and by liquid state fermentation (LSF). Enzyme activities were increased about 30–80% by SSF in comparison with conventional LSF. Enzyme production was further improved by various pretreatments, making cellulosic material easily accessible. The best results were obtained with 5 M NaOH treatment.     

Solid state fermentation for production of α-amylase byBacillus megaterium 16M

Summary The ratio of buffer to wheat bran, incubation temperature and initial pH influence -amylase production byBacillus megaterium 16M under solid state fermentation. The enzyme, with pH and temperature optima at 6.0 and 70°C, is formed at a level of 30,000 units/g dry bacterial bran without coproduction of proteases and cellulases.     

A simple and inexpensive method for cellulase and β-glucosidase production by Neurospora crassa

Summary Elevated levels of cellobiohydrolase, carboxymethylcellulase (CMCase) and -glucosidase were produced by strain STA of Neurospora crassa, grown in solid-state fermentation on untreated wheat straw supplemented with simple mineral salts. Yields as high as 6.1 units of cellobiohydrolase, 969.2 units of CMCase and 169.4 units of -glucosidase per gram of straw were obtained at optimum growth and enzyme assay conditions.     

Studies of Antarctic yeast for β-glucosidase production

Moss and lichen samples from the region of the Bulgarian base on Livingston Island, Antarctica were examined for the presence of yeasts. Six pure cultures were obtained. They were screened for -glucosidase production and two of them were selected. These were identified as Cryptococcus albidus AL₂ and C. albidus AL₃, according to their morphology, reproductive behaviour, and growth at different temperatures, salt concentrations, nutritional characteristics and various biochemical tests. These strains were examined for biosynthesis of -glucosidase on different carbon sources under aerobic conditions. High exocellular and endocellular activities were obtained when they were grown on cellobiose, methyl--D-glucopyranoside and salicin. The time course of growth and -glucosidase production of the yeast was examined by cultivation in a medium with cellobiose under aerobic conditions at temperatures 18 and 24 °C for 96 h. Cryptococcus albidus AL₂ and C. albidus AL₃ synthesized exocellular enzyme, respectively 58.33 and 55.83 U/ml and endocellular enzyme 137.75 and 205.34 U/ml at 24 °C for 72 h of the cultivation.     

Location and contribution of individual β-glucosidase from Neurospora crassa to total β-glucosidase activity

This study investigated the cellular location and the contribution of individual β-glucosidase (BGL) to total BGL activity in Neurospora crassa. Among the seven bgl genes, bgl3, bgl5, and bgl7 were transcribed at basal levels, whereas bgl1, bgl2, bgl4, and bgl6 were significantly up-regulated when the wild-type strain was induced with cellulose (Avicel). BGL1 and BGL4 were found to be contributors to intracellular BGL activity, whereas the activities of BGL2 and BGL6 were mainly extracellular. Sextuple bgl deletion strains expressing one of the three basally transcribed bgls did not produce any detectable BGL activity when they were grown on Avicel. BGL6 is the major contributor to overall BGL activity, and most of its activity resides cell-bound. The sextuple bgl deletion strain containing only bgl6 utilized cellobiose at a rate similar to that of the wild type, while the strain with only bgl6 deleted utilized cellobiose much slower than that of the wild type.     

Improvement of β-glucosidase production by co-culture of Aspergillus niger and A. oryzae under solid state fermentation through feeding process

Eleven different Aspergillus strains were evaluated for their ability to produce β-glucosidase using sugar cane bagasse as a sole carbon source under solid state fermentation (SSF). The most potent strains, A. niger NRC 7 (674.6 U/g ds) and A. oryzae NRRL 447 (83 U/g ds), were used in a mixed culture to enhance β-glucosidase production by co-culturing under SSF. In mixed culture, β-glucosidase of the two strains (814 U/g ds) was nearly 1.2- and 9.8-fold than that of monocultures of A. niger NRC 7A and A. oryzae NRRL 447, respectively. Optimization of the culture parameters, initial pH value, moisture content, inoculum size and ratios of the two strains. and incubation time exhibited a significant increase in β-glucosidase production (1,893 U/g ds) than before optimization. Single feeding with citrate-phosphate buffer, succinate buffer, casein. and soybean flour individually after the third day of the fermentation time and controlling the moisture content at 90 % (w/w) induced β-glucosidase production. Maximum enzyme production increased up to 2.1-fold compared to 2,188 U/g ds during normal batch culture. Among nitrogen sources, soybean flour gave the highest β-glucosidase (4,578 U/g ds). while urea reduced β-glucosidase production (1,693 U/g ds). However, the combination of buffers with soybean flour through two fed cycles resulted in a decrease of the enzyme than single fed with buffers or soybean flour alone.     

Effect of media composition and growth conditions on production of cellulase and β-glucosidase by a mixed fungal fermentation

The effect of growth temperature, medium composition and pH were examined in shake-flask-scale studies to determine the optimum conditions for cellulase production in mixed cultures of Trichoderma reesei Rut C30 and Aspergillus phoenicis. The optimum temperature and pH were 27°C and pH 4.6. Decreases in medium complexity and cost were achieved through reductions in the concentration of Ca, Mg, and K salts. Ten litre fermentations were implemented to study the kinetics of substrate utilization and product formation. The pH at which the fermentation was controlled appeared to be the critical parameter for batch growth of mixed cultures of Trichoderma reesei Rut C30 and Aspergillus phoenicis.     

Immobilized β-glucosidase on magnetic chitosan microspheres for hydrolysis of straw cellulose

β-Glucosidase immobilized on magnetic chitosan microspheres for potential recycling usage in hydrolysis of cellulosic biomass was investigated. The immobilized enzyme had an activity of 6.4 U/g support under optimized condition when using cellobiose as substrate. Immobilization resulted in less increase of the apparent K_m, low drift of the optimal pH, as well as improved stability relative to the free enzyme. The immobilized β-glucosidase was applied to enzymatic hydrolysis of corn straw to produce 60.2 g/l reducing sugar with a conversion rate of 78.2% over the course of a 32-h reaction. This conversion rate was maintained above 76.5% after recycling the enzyme for use in eight batches (total 256 h), showing favorable operational stability of the immobilized enzyme.     

Solid state fermentation—An overview

Since ancient times many solid state fermentations have utilized fungi and bacteria, almost always in mixed culture. The discovery and development of penicillin led to extensive use of liquid fermentations using actinomycetes and fungi and to subsequent neglect of research on solid state fermentations and the use of mixed cultures. This paper reviews the types of solid state fermentations, equipment used, products made, as well as the advantages and disadvantages of solid state fermentations. Products resulting from old and new solid and liquid substrate fermentations are enumerated.     

Solid state fermentation for the production of α-amylase from Penicillium chrysogenum using mixed agricultural by-products as substrate

Production of α-amylase from local isolate, Penicillium chrysogenum, under solid-state fermentation (SSF) was carried out in this study. Different agricultural by-products, such as wheat bran (WB), sunflower oil meal (SOM), and sugar beet oil cake (SBOC), were used as individual substrate for the enzyme production. WB showed the highest enzyme activity (750 U/gds). Combination of WB, SOM, and SBOC (1:3:1 w/w/w) resulted in a higher enzyme yield (845 U/gds) in comparison with the use of the individual substrate. This combination was used as mixed solid substrate for the production of α-amylase from P. chrysogenum by SSF. Fermentation conditions were optimized. Maximum enzyme yield (891 U/gds) was obtained when SSF was carried out using WB + SOM + SBOC (1:3:1 w/w/w), having initial moisture of 75%, inoculum level of 20%, incubation period of 7 days at 30°C. Galactose (1% w/w), urea and peptone (1% w/w), as additives, caused increase in the enzyme activity.     

Comparative study of the production of extracellular β-glucosidase by four different strains of Aspergillus using submerged fermentation

Four strains of Aspergillus (Aspergillus niger CDBB-H-176, A. niger CDBB-H-175, A. niger ATCC 9642, and Aspergillus terreus CDBB-H-194) were used to produce extracellular β-glucosidase. Using an orthogonal experimental design (L₉), we optimized the parameters of culture medium to maximize the activity of β-glucosidase. The optimal conditions (same for the four strains) were as follows: temperature, 30°C; pH, 6.0; orbital agitation, 200?rpm; concentration of sucrose, 0.5% (w/v). The most productive strain was A. niger CDBB-H-175, with a yield of 701.2?U/mL. In a second stage, we optimized (L₁₈) the concentration of nutrients in the culture medium to determine whether this modification would increase the production of β-glucosidase. The optimal conditions for A. niger CDBB-H-175 were as follows (%, w/v): NaNO₃, 0.3; KCl, 0.3; KH₂PO₄, 0.15; NH₄NO₃, 0.1; NH₄H₂PO₄, 0.1; MgSO₄?·?7H₂O, 0.05; yeast extract, 0.1. The production of β-glucosidase under these conditions was 1207.9?U/mL. Enzymatic assays were used to characterize the enzyme; the optimum temperature and pH of β-glucosidase produced by the four selected micro-organisms were found to be 65°C and 5.0, respectively. We determined the Michaelis–Menten constants (K_m) only for A. niger CDBB-H-175 and CDBB-H-176; the values were 2.7 and 2.2?mM, respectively.     

Extracellular β-glucosidase production by a marine Aspergillus sydowii BTMFS 55 under solid state fermentation using statistical experimental design

A potential fungal strain producing extracellular β-glucosidase enzyme was isolated from sea water and identified as Aspergillus sydowii BTMFS 55 by a molecular approach based on 28S rDNA sequence homology which showed 93% identity with already reported sequences of Aspergillus sydowii in the GenBank. A sequential optimization strategy was used to enhance the production of β-glucosidase under solid state fermentation (SSF) with wheat bran (WB) as the growth medium. The two-level Plackett-Burman (PB) design was implemented to screen medium components that influence β-glucosidase production and among the 11 variables, moisture content, inoculums, and peptone were identified as the most significant factors for β-glucosidase production. The enzyme was purified by (NH₄)₂SO₄ precipitation followed by ion exchange chromatography on DEAE sepharose. The enzyme was a monomeric protein with a molecular weight of ∼95 kDa as determined by SDS-PAGE. It was optimally active at pH 5.0 and 50°C. It showed high affinity towards pNPG and enzyme has a K _m and V _max of 0.67 mM and 83.3 U/mL, respectively. The enzyme was tolerant to glucose inhibition with a K _i of 17 mM. Low concentration of alcohols (10%), especially ethanol, could activate the enzyme. A considerable level of ethanol could produce from wheat bran and rice straw after 48 and 24 h, respectively, with the help of Saccharomyces cerevisiae in presence of cellulase and the purified β-glucosidase of Aspergilus sydowii BTMFS 55.     

Optimization of β-glucosidase production from Aspergillus niger

本研究对Aspergillus niger Glu05生产β-葡萄糖苷酶的培养基组分及培养条件进行了优化.优化后的培养基组成和培养条件分别为:麸皮4％,tryptone 4％,1μmol MnSO4,1μmol NaCl,KH2PO40.2％,oH自然,摇床转速250 r/min,培养温度30℃,培养周期5d.优化后发酵液中酶活力达到44.11 IU/mL,与初始的产酶水平32.87 IU/mL相比,提高了36％.     

Simultaneous production of endoglucanase and β-glucosidase using synthetic two cistron genes

Summary Simultaneous production of endoglucanase and -glucosidase by using a synthetic two cistron system inEscherichia coli was attempted as a possible way of reducing production cost. The first cistron in this system we constructed is an endoglucanase gene fused to a tac promoter that provides for efficient expression. The second cistron is a -glucosidase structural gene. A ribosome binding site sequence of 33-base was inserted between the two cistron genes.E. coli cells transformed with the system produced 12.4 units/mg protein of endoglucanase and 327 units/mg protein of -glucosidase, which represent 15% and 22% of total cellular protein, respectively, in L medium within three hours after induction with IPTG.     

Cellodextrin utilization and β-glucosidase production by Bacteroides polypragmatus

Bacteroides polypragmatus, a mesophilic obligate anaerobe, was shown to simultaneously ferment glucose and cellobiose giving ethanol as a major metabolic end-product. A mixture of higher cellodextrins was also utilized. The bacterium produced a -glucosidase with a pI value of 4.2 and a molecular weight of approximately 100000 daltons. The enzyme was intracellular and functioned optimally at pH 7. The K _m values obtained with p-nitrophenyl--d-glucoside and cellobiose as substrates were 0.73 mM and 100 mM, respectively. The enzyme was quite stable at elevated temperatures; in the presence of 10% glycerol (v/v), it had a half-life of 4 h at 55°C. It was also stable during long-term storage at either 4°C or-20°C, provided that 10% (v/v) glycerol was added to preparations maintained at-20°C.Abbreviations HPLC high-performance liquid chromatography - IEF isoelectric focusing - pNPG p-nitrophenyl--d-glucoside NRCC No. 25676     

Exocellular β-glucosidase inducibility in a mutant strain ofCandida wickerhamii derepressed for endocellular β-glucosidase production

Summary Candida wickerhamii produces one endocellular and one exocellular -glucosidase. Both enzymes are repressed by glucose in the wild-type strain. In the M7 mutant ofC. wickerhamii, which was previously demonstrated to be derepressed for endocellular -glucosidase biosynthesis, the exocellular -glucosidase is derepressed and hyperproduced when cellodextrins are added to the culture medium. This enzyme, which was produced constitutively in the wildtype, has thus become inducible in the M7 mutant strain. The interest of this strain for industrial production of -glucosidases is discussed.

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Resumen Candida wikerhamii produce dos -glucosidasas: una endocelular y otra exocelular. Ambos enzimas son reprimidos por glucosa en la cepa salvaje. Al añadir celodextrina al medio de cultivo del mutante M7 deC. wickerhamii, en el cual se ha demostrado ya la desrepresión de la síntesis de -glucosidasa endocelular, se desreprime la -glucosidasa extracelular obteniéndose una hiperproducción de este enzima. Dicho enzima que era producido de forma constitutiva en el tipo salvaje, se ha convertido en inducible en la cepa mutante M7. Se discute el interés de esta cepa para la producción industrial de -glucosidasas.

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Résumé Candida wickerhamii produit deux -glucosidases, l'une endo- et l'autre exocellulaire. Les deux enzymes de la souche sauvage sont réprimées par le glucose. Le mutant M7, chez qui il a été antérieurement constaté que la synthèse de la -glucosidase endocellulaire est déréprimée, l'enzyme exocellulaire est elle aussi déréprimée et hyper-produite lorsque des cellodextrines sont ajoutées au milieu de culture. Cette enzyme, qui est constitutive chez la souche sauvage, est donc devenue inductible chez le mutant M7. Cette souche est intéressante pour la production industrielle de -glucosidases.

     

Optimization of cellobiose dehydrogenase and β-glucosidase production by cellulose-degrading cultures of Phanerochaete chrysosporium

The effect of succinate, acetate, and phosphate on the production of cellobiose dehydrogenase (CDH), cellobiose: quinone oxidoreductase (CBQase), -glucosidase, and protease by Phanerochaete chrysosporium in media containing cotton linters, filterpaper, microcrystalline cellulose, or acid-treated cellulose was investigated. The succinate medium,with an initial pH of 4.5 and with cotton linters as the cellulose source, has been demonstrated to yield the highest levels of CDH (141 U/l) and -glucosidase (237 U/l), and the lowest levels of CBQase (53 U/l). The optimized culture conditions identified here permit isolation of milligram quantities of CDH and -glucosidase from P. chrysosporium.     

Bimutation breeding of Aspergillus niger strain for enhancing β-mannanase production by solid-state fermentation

A parent strain Aspergillus niger LW-1 was mutated by the compound mutagenesis of vacuum microwave (VMW) and ethyl methane sulfonate (EMS). A mutant strain, designated as A. niger E-30, with high- and stable-yield β-mannanase was obtained through a series of screening. The β-mannanase activity of the mutant strain E-30, cultivated on the basic fermentation medium at 32 °C for 96 h, reached 36,675 U/g dried koji, being 1.98-fold higher than that (18,501 U/g dried koji) of the parent strain LW-1. The purified E-30 β-mannanase, a glycoprotein with a carbohydrate content of 19.6%, had an apparent molecular weight of about 42.0 kDa by SDS–PAGE. Its optimal pH and temperature were 3.5 and 65 °C, respectively. It was highly stable at a pH range of 3.5–7.0 and at a temperature of 60 °C and below. The kinetic parameters K_m and V_max, toward locust bean gum and at pH 4.8 and 50 °C, were 3.68 mg/mL and 1067.5 U/mg, respectively. The β-mannanase activity was not significantly affected by an array of metal ions and EDTA, but strongly inhibited by Ag⁺ and Hg²⁺. In addition, the hydrolytic conditions of konjak glucomannan using the purified E-30 β-mannanase were optimized as follows: konjak gum solution 240 g/L (dissolved in deionized water), hydrolytic temperature 50 °C, β-mannanase dosage 120 U/g konjak gum, and hydrolytic time 8 h.     

Production and characterization of β-glucosidase from Aspergillus niger fermentation: Application for organic fraction of municipal solid waste hydrolysis and methane enhancement

The anaerobic digestion of the organic fraction of municipal solid waste (OFMSW) is currently an attractive treatment process with energy production in the form of biogas. Hydrolysis is the rate-limiting step for the anaerobic digestion of solid wastes. Thus, in the present study fungal enzymatic pretreatment of OFMSW was applied to enhance biogas production. Two enzyme cocktails rich on β-glucosidase were produced from submerged fermentation of Aspergillus niger on basal medium using OFMSW as carbon source and urea (Urea cocktail) and Ulva rigida as nitrogen source (Ulva cocktail). Ulva cocktail displayed an important effect on OFMSW solubilization. Therefore, an increase of reducing sugar concentration about 60% was obtained which was in correlation with chemical oxygen demand (COD) increase. The performance of enzymatic pretreatment on anaerobic digestion of OFMSW was studied by conducting biochemical methane potential tests. Results showed that the enzymatic pretreatment improved methane yield of OFMSW even at high solid concentration. High methane yield about 500 ml/g total volatile solid was obtained, which corresponds up to 68% enhancement over the control.