Purification and Characterisation of a Thermostable β-Xylosidase from Aspergillus niger van Tieghem of Potential Application in Lignocellulosic Bioethanol Production

Soybean straw is a renewable resource in agricultural residues that can be used for lignocellulosic bioethanol production. To enhance enzymatic digestibility and fermentability, the biomass was prepared with an alkali-thermal pretreatment (sodium hydroxide, 121°C, 60 min). The delignification yield was 34.1~53%, in proportion to the amount of sodium hydroxide, from 0.5 to 3.0 M. The lignin and hemicellulose contents of the pretreated biomass were reduced by the pretreatment process, whereas the proportion of cellulose was increased. Under optimal condition, the pretreated biomass consisted of 74.0±0.1% cellulose, 10.3±0.1% hemicellulose, and 10.1±0.6% lignin. During enzymatic saccharification using Cellic® CTec2 cellulase, 10% (w/v) of pretreated soybean straw was hydrolyzed completely and converted to 67.3±2.1 g/L glucose and 9.4±0.5 g/L xylose with a 90.9% yield efficiency. Simultaneous saccharification and fermentation of the pretreated biomass by Saccharomyces cerevisiae W303-1A produced 30.5±1.2 g/L ethanol in 0.5 L fermented medium containing 10% (w/v) pretreated biomass after 72 h. The ethanol productivity was 0.305 g ethanol/g dry biomass and 0.45 g ethanol/g glucose after fermentation, with a low concentration of organic acid metabolites. Also, 82% of fermentable sugar was used by the yeast for ethanol fermentation. These results show that the combination of alkaline pretreatment and biomass hydrolysate is useful for enhancing bioethanol productivity using delignified soybean straw.

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Characterisation of a novel thermostable endoglucanase from Alicyclobacillus vulcanalis of potential application in bioethanol production

Applied Microbiology and Biotechnology ◽

10.1007/s00253-015-6474-8 ◽

2015 ◽

Vol 99 (18) ◽

pp. 7515-7525 ◽

Cited By ~ 26

Author(s):

Angela Boyce ◽

Gary Walsh

Keyword(s):

Potential Application ◽

Bioethanol Production

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Cloning, characterization and directed evolution of a xylosidase from Aspergillus niger

10.51415/10321/1710 ◽

2016 ◽

Author(s):

◽

Bibi Khadija Khan

Keyword(s):

Enzyme Activity ◽

Aspergillus Niger ◽

Random Mutagenesis ◽

Thermomyces Lanuginosus ◽

Major Protein ◽

Bioethanol Production ◽

Xylanase Production ◽

Fluorescent Band ◽

Gap Promoter ◽

Major Protein Band

β-xylosidases catalyse the hydrolyses of xylooligosaccharides into the monosaccharide sugar, xylose. In this study we report the production of xylose under different conditions in Pichia pastoris and Saccharomyces. cerevisiae, and its conversion to bioethanol using Pichia stipitis. The aim of this study was to change the characteristics of the A. niger 90196 β-xylosidase through random mutagenesis and increase expression under the control of different promoter systems in yeasts P. pastoris and S. cerevisiae. The recombinant library created through random mutagenesis was screened for changes in activity and subsequently pH and temperature stability. One variant showed an increase in enzyme expression, thermostability, and a change in amino acid sequence at residue 226. The enzyme was then cloned, expressed and characterized in P. pastoris GS115 and S. cerevisiae. β-xylosidase was constitutively expressed in P. pastoris using the GAP promoter and the inducible AOX promoter. In S. cerevisiae the enzyme was expressed using the constitutive PGK promoter and inducible ADH2 promoter systems. Enzyme functionality with the different expression systems was compared in both hosts. The GAP system was identified as the highest-producing system in P. pastoris, yielding 70 U/ml after 72 hours, followed by the PGK system in S. cerevisiae, with 8 U/ml. A 12% SDS-PAGE gel revealed a major protein band with an estimated molecular mass of 120 kDA, and the zymogram analysis revealed that this band is a fluorescent band under UV illumination, indicating enzyme activity. Stability characteristics was determined by expressing the enzyme at different pH and temperatures. Under the control of the GAP promoter in P. pastoris, enzyme activity peaked at pH4 while retaining 80% activity between pH 3 – 5. Highest activity of 70 U/ml xylosidase was recorded at 60ºC. Due to the high enzyme production in P. pastoris, the co-expression of this enzyme with a fungal xylanase was evaluated. The xylanase gene from Thermomyces lanuginosus was cloned with the GAP promoter system and expressed together with the β-xylosidase recombinant in P. pastoris. Enzyme activities of the co-expressed recombinant revealed a decrease in enzyme activity levels. The co-expressed xylanase production decreased by 26% from 136 U/ml to 100 U/ml while the xylosidase expression decreased 86% from 70 U/ml to 10 U/ml. The xylose produced from the hydrolysis of birchwood xylan was quantified by HPLC. The monosaccharide sugar was used in a separate saccharification and fermentation strategy by P. stipitis to produce bioethanol, quantified by gas chromatography. Bioethanol production peaked at 72 h producing 0.7% bioethanol from 10 g/l xylose. In conclusion a β-xylosidase from Aspergillus niger was successfully expressed in P. pastoris and was found to express large quantities of xylosidase, that has not been achieved in any prior research to date. The enzyme was also successfully co-expressed with a Thermomyces xylanase and is now capable of bioethanol production through xylan hydrolysis. This highlights potential use in industrial applications in an effort to reduce the world dependence on petroleum and fossil fuels. However the technical challenges associated with commercialization of bioethanol production are still significant.

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3-Methylxanthine production through biodegradation of theobromine by Aspergillus sydowii

10.21203/rs.3.rs-19407/v1 ◽

2020 ◽

Author(s):

Bin-Xing ZHOU ◽

Cunqiang Ma ◽

Chengqin Zheng ◽

Tao Xia ◽

Binsong Ma ◽

...

Keyword(s):

Uric Acid ◽

Aspergillus Niger ◽

Potential Application ◽

Metal Ion ◽

Liquid Culture ◽

Aspergillus Sydowii ◽

Aspergillus Tamarii ◽

Synthetic Compounds ◽

Aspergillus Ustus ◽

Bacteria And Fungi

Abstract Background Methylxanthines including caffeine, theobromine and theophylline are natural and synthetic compounds in tea. Theobromine and other methylxanthine could be metabolized by certain kinds of bacteria and fungi. Previous studies confirmed that several isolates from Pu-erh tea could degrade and convert caffeine and theophylline through N-demethylation and related oxidation, respectively. In this study, seven tea-derived fungi were inoculated into various theobromine agar mediums and liquid mediums to assess their capacity in theobromine utilization. Related metabolites with theobromine degradation were detected by using HPLC in the liquid culture inoculated by candidate isolates to investigate potential application in the production of 3-methylxanthine. Results Based on the extent of theobromine utilization, four isolates including Aspergillus niger, Aspergillus sydowii, Aspergillus ustus and Aspergillus tamarii have demonstrated the potential for theobromine biodegradation. Particularly, A. sydowii and A. tamarii could degrade theobromine significantly (p < 0.05) in all given theobromine liquid mediums. 3,7-Dimethyluric acid, 3-methylxanthine, 7-methylxanthine, 3-methyluric acid, xanthine, and uric acid were detected in A. sydowii and A. tamarii culture, respectively, confirming the existence of N-demethylation and oxidation in theobromine catabolism. 3-Methylxanthine was common and main demethylated metabolite of theobromine in A. sydowii and A. tamarii culture. 3-Methylxanthine in A. sydowii culture showed a linear relation with the initial theobromine concentrations that 177.12 ± 14.06 mg/L of 3-methylxanthine was accumulated in TLM-S with 300 mg/L of theobromine. Additionally, pH at 5 and metal ion of Fe 2+ promoted 3-methylxanthine production significantly (p < 0.05). Conclusions This study is the first to confirm that A. sydowii and A. tamarii degrade theobromine through N-demethylation and oxidation, respectively. A. sydowii showed the potential application in 3-methylxanthine production with theobromine as feedstock through the N-demethylation at position N-7.

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PENGARUH KADAR Aspergillus niger TERHADAP PRODUKSI BIOETANOL DARI BONGGOL PISANG KEPOK ( Musa paradisiaca L)

Alotrop ◽

10.33369/atp.v3i2.10496 ◽

2019 ◽

Vol 3 (2) ◽

Author(s):

Junaini Junaini ◽

Elvinawati Elvinawati ◽

Sumpono Sumpono

Keyword(s):

Saccharomyces Cerevisiae ◽

Aspergillus Niger ◽

Specific Gravity ◽

Fermentation Process ◽

Bioethanol Production ◽

Gravity Method ◽

Musa Paradisiaca ◽

Ethanol Content

This study aims to determine the effect of Aspergillus niger levels on bioethanol production in banana cobs using Saccharfication Simultation Fermentation (SSF) method. This research uses banana kepok (Musa paradisiaca L.) obtained from Enggano Island of Bengkulu Province. Enggano Island is one of the outermost islands of Bengkulu Province which has a coordinate point of 5023'25,000 '' LS - 102014'16,000 '' BT. Samples of banana done preparation before the hydrolysis and fermentation process by smoothing the banana cobs using a blender until it becomes mush. Samples in the form of slurry were then added by Aspergillus niger and Sccharomyces cerevisiae. Hydrolysis performed for 72 hours which then continued with the fermentation process for 5 days. In the study there were 5 treatments: addition of Aspergillus niger 107 CFU/mL, addition of 10 mL Saccharomyces cerevisiae, addition of 10 mL Saccharomyces cerevisiae + Aspergillus niger 106 CFU/mL, 10 mL Saccharomyces cerevisiae + Aspergillus niger 107CFU/mL and 10 mL Saccharomyces cerevisiae + Aspergillus niger 108CFU/mL. The fermentation results were distilled and then measured the ethanol content by the specific gravity method. Ethanol content obtained from each treatment were 3.995%, 6.218%, 6.825%, 9.065%, and 12.348%, respectively. From one-way analysis test can be obtained the value of Fcount and Ftabel respectively are 25.73 and 5.19, so the value of Ftable< Fcount which means each treatment has a different result significantly.

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