• Journal of Microbiology and Biotechnology
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• v.27 no.9
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• pp.1649-1656
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• 2017

In simultaneous saccharification and fermentation (SSF) for production of cellulosic biofuels, engineered Saccharomyces cerevisiae capable of fermenting cellobiose has provided several benefits, such as lower enzyme costs and faster fermentation rate compared with wild-type S. cerevisiae fermenting glucose. In this study, the effects of an alternative intracellular cellobiose utilization pathway-a phosphorolytic pathway based on a mutant cellodextrin transporter (CDT-1 (F213L)) and cellobiose phosphorylase (SdCBP)-was investigated by comparing with a hydrolytic pathway based on the same transporter and an intracellular ${\beta}$-glucosidase (GH1-1) for their SSF performances under various conditions. Whereas the phosphorolytic and hydrolytic cellobiose-fermenting S. cerevisiae strains performed similarly under the anoxic SSF conditions, the hydrolytic S. cerevisiae performed slightly better than the phosphorolytic S. cerevisiae under the microaerobic SSF conditions. Nonetheless, the phosphorolytic S. cerevisiae expressing the mutant CDT-1 showed better ethanol production than the glucose-fermenting S. cerevisiae with an extracellular ${\beta}$-glucosidase, regardless of SSF conditions. These results clearly prove that introduction of the intracellular cellobiose metabolic pathway into yeast can be effective on cellulosic ethanol production in SSF. They also demonstrate that enhancement of cellobiose transport activity in engineered yeast is the most important factor affecting the efficiency of SSF of cellulose.

• Journal of Forest and Environmental Science
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• v.27 no.3
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• pp.143-149
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• 2011

The possibility of employing biomass of Salix viminalis cv. Q683 as a resource of bio-energy was evaluated. The chemical analysis of S. viminalis cv. Q683 leaf biomass showed components such as, extractives (2.57%), lignin (39.06%), hemicellulose (21.61%), and cellulose (37.83%), whereas, its stem was composed of extractives (1.67%), lignin (23.54%), hemicellulose (33.64%), and cellulose (42.03%). The biomass of S. viminalis cv. Q683 was saccharified using two enzymes celluclast and viscozyme. The saccharification of S. viminalis cv. Q683 biomass was influenced by enzymes and their strengths. The optimal enzyme combination was found to be celluclast (59 FPU/g substrate) and viscozyme (24 FBG/g substrate). On saccharification the glucose from leaf and stem biomass was 7.5g/L and 11.7g/L, respectively after 72 hr of enzyme treatment. The biomass and enzyme-treated biomass served as the feedstock for ethanol production by fermentation. The ethanol production from stem and leaf biomass was 5.8 g/L and 2.2 g/L respectively, while the fermentation of the enzymatic hydrolysates yielded 5 g/L to 8 g/L bioethanol in 72 hours.

• Journal of Microbiology and Biotechnology
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• v.31 no.10
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• pp.1446-1454
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• 2021

Herein, we cloned and expressed an endo-β-1,4-glucanase gene (celA1805) from Bacillus subtilis B111 in Escherichia coli. The recombinant celA1805 contains a glycosyl hydrolase (GH) family 8 domain and shared 76.8% identity with endo-1,4-β-glucanase from Bacillus sp. KSM-330. Results showed that the optimal pH and temperature of celA1805 were 6.0 and 50℃, respectively, and it was stable at pH 3-9 and temperature ≤50℃. Metal ions slightly affected enzyme activity, but chemical agents generally inhibited enzyme activity. Moreover, celA1805 showed a wide substrate specificity to CMC, barley β-glucan, lichenin, chitosan, PASC and avicel. The K_m and V_max values of celA1805 were 1.78 mg/ml and 50.09 µmol/min/mg. When incubated with cellooligosaccharides ranging from cellotriose to cellopentose, celA1805 mainly hydrolyzed cellotetrose (G4) and cellopentose (G5) to cellose (G2) and cellotriose (G3), but hardly hydrolyzed cellotriose. The concentrations of reducing sugars saccharified by celA1805 from wheat straw, rape straw, rice straw, peanut straw, and corn straw were increased by 0.21, 0.51, 0.26, 0.36, and 0.66 mg/ml, respectively. The results obtained in this study suggest potential applications of celA1805 in biomass saccharification.

• Journal of Microbiology and Biotechnology
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• v.28 no.12
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• pp.2036-2045
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• 2018

An endo-${\beta}$-1,4-glucanase gene, cel5L, was cloned using the shot-gun method from Bacillus sp.. The gene, which contained a predicted signal peptide, encoded a protein of 496 amino acid residues, and the molecular mass of the mature Cel5L was estimated to be 51.8 kDa. Cel5L contained a catalytic domain of glycoside hydrolase (GH) family 5 and a carbohydrate-binding module family 3 (CBM_3). Chromatography using HiTrap Q and CHT-II resulted in the isolation of two truncated forms corresponding to 50 (Cel5L-p50) and 35 kDa (Cel5L-p35, CBM_3-deleted form). Both enzymes were optimally active at pH 4.5 and $55^{\circ}C$, but had different half-lives of 4.0 and 22.8 min, respectively, at $70^{\circ}C$. The relative activities of Cel5L-p50 and Cel5L-p35 for barley ${\beta}$-glucan were 377.0 and 246.7%, respectively, compared to those for carboxymethyl-cellulose. The affinity and hydrolysis rate of pNPC by Cel5L-p35 were 1.7 and 3.3 times higher, respectively, than those by Cel5L-p50. Additions of each to a commercial enzyme set increased saccharification of pretreated rice straw powder by 17.5 and 21.0%, respectively. These results suggest CBM_3 is significantly contributing to thermostability, and to affinity and substrate specificity for small substrates, and that these two enzymes could be used as additives to enhance enzymatic saccharification.

• Journal of Microbiology and Biotechnology
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• v.32 no.1
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• pp.117-125
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• 2022

Until recently, four types of cellobiose-fermenting Saccharomyces cerevisiae strains have been developed by introduction of a cellobiose metabolic pathway based on either intracellular β-glucosidase (GH1-1) or cellobiose phosphorylase (CBP), along with either an energy-consuming active cellodextrin transporter (CDT-1) or a non-energy-consuming passive cellodextrin facilitator (CDT-2). In this study, the ethanol production performance of two cellobiose-fermenting S. cerevisiae strains expressing mutant CDT-2 (N306I) with GH1-1 or CBP were compared with two cellobiose-fermenting S. cerevisiae strains expressing mutant CDT-1 (F213L) with GH1-1 or CBP in the simultaneous saccharification and fermentation (SSF) of cellulose under various conditions. It was found that, regardless of the SSF conditions, the phosphorolytic cellobiose-fermenting S. cerevisiae expressing mutant CDT-2 with CBP showed the best ethanol production among the four strains. In addition, during SSF contaminated by lactic acid bacteria, the phosphorolytic cellobiose-fermenting S. cerevisiae expressing mutant CDT-2 with CBP showed the highest ethanol production and the lowest lactate formation compared with those of other strains, such as the hydrolytic cellobiose-fermenting S. cerevisiae expressing mutant CDT-1 with GH1-1, and the glucose-fermenting S. cerevisiae with extracellular β-glucosidase. These results suggest that the cellobiose-fermenting yeast strain exhibiting low energy consumption can enhance the efficiency of the SSF of cellulosic biomass.

• Korean Journal of Food Science and Technology
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• v.48 no.2
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• pp.147-152
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• 2016

This study was conducted to investigate the physicochemical quality properties and provide basic data for the activation of traditional Kamju of juice type product prepared by mixing malt and extruded rice collet powder. Malt extracts were prepared by extracting the mixture of malt and water at a weight ratio of 25:75 after soaking for 2 h at $45^{\circ}C$. Rice collet powder was prepared by adjusting the barrel temperature to $95^{\circ}C$, screw speed to $3.07{\times}g$, discharge port diameter to 7 mm and a raw material input to 50 kg/h, the powder was then ground to a particle size of 80 mesh. The physicochemical characteristics (pH, color, viscosity, reducing sugars, number of viable cells, free amino acids) and sensory evaluations were conducted at various time points during the saccharification and at different mixing ratios of the extruded rice collet powder to malt extract (5:95, 15:85, 25:75, 35:65, each at $55^{\circ}C$ for 9 h). As a result, with an increase in the proportion of the extruded rice collet powder and saccharification time, the physicochemical properties of traditional Kamju significantly improved (p<0.05). A mixing ratio of 35:65 rice collet powder to malt extract and a saccharification time of 9 h were found to be the most desirable conditions. However, based on the sensory evaluation, a mixing ratio of rice collet powder and malt extract of 25:75 and a saccharification time of 5 h resulted in the most preferable palatability of traditional Kamju (p<0.05). Therefore, the mixing ratio and saccharification time should be determined to provide a better choice with respect to the taste and economic aspects of traditional Kamju.

• Korean Journal of Food Science and Technology
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• v.44 no.5
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• pp.553-558
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• 2012

The pH, turbidity, color, brix, reducing sugar, total sugar, ketose, amino acid, protein and organoleptic characteristics of sikhye prepared with puffed rice powder (0, 25, 50, 75 and 100% (w/w)) were investigated during saccharification. A higher concentration of puffed rice powder and longer saccharification time resulted in pH reduction (p<0.05) and tended to increase turbidity. A higher Hunter "L" value was observed with higher puffed rice powder content, except for the addition of 25% at 0 h. The Hunter "L" value peaked at hour 1 and slowly declined thereafter. Elevations in brix, reducing sugar, total sugar and sucrose were observed with higher puffed rice powder content and longer duration of saccharification (p<0.05). The sucrose content ranged from 0.79-0.86% (w/w) at hour 6, which was markedly lower than that of commercial sikhye. With time, levels of amino acid and protein increased (p<0.05). Sensory evaluation revealed moderate differences between 0% and both 75 and 100% (p<0.001), with 75 and 100% preferred over 0% (p<0.05).

• KSBB Journal
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• v.29 no.5
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• pp.316-322
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• 2014

Xylanase have been used to convert the polymetric xylan into fermentable sugars from the production of ethanol and xylitol from plant biomass. The aim of this study was to isolate and identify xylanolytic bacterium from herbivore feces and was to used the xylanase for enzymatic hydrolysis of biomass. Xylanolytic strains were isolated from 59 different feces of herbivores from Seoul Grand Park located in Gwacheon Gyeonggi-do. The xylanolytic strains were selected by congo red staining and DNS method. Total 67 strains isolated from the herbivores feces were tested for xylanase activity. Among the strains, H10-1, which has the highest xylanase activity, was isolated from feces of Ceratotherium simum. The H10-1 strain was identified as Bacillus pumilus based on its morphological/biochemical characteristics and partial 16S rDNA gene sequences. Culture conditions of B. pumilus H10-1 such as initial medium pH, incubation temperature and incubation time were optimized for maximum xylanase production. And also xylanase produced by B. pumilus H10-1 was applied for the saccharification of Miscanthus sacchariflorus cv. 'Geodae 1', which was pretreated with 1.5M NaOH. The optimized culture conditions of B. pumilus H10-1 were pH 9, $30^{\circ}C$ incubation temperature, and 7 day incubation time, respectively. This xylanase activity under the optimized conditions was $20.4{\pm}3.3IU$. The crude xylanase produced by B. pumilus H10-1 was used for the saccharification of xylan derived from pretreated 'Geodae 1'. The saccharification conditions were $50^{\circ}C$, 200 rpm, and 5 days. Saccharification efficiency of pretreated 'Geodae 1' by B. pumilus H10-1 was 8.2%.

• KSBB Journal
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• v.21 no.6 s.101
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• pp.474-478
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• 2006

For the economically feasible production of ethanol, utilization of SFW (saccharified food wastes) as substrate for synchronous saccharification and fermentation (SSF) process was developed in this study. When 200 g of food wastes and 40 mL of enzyme ($amylase activity,\;3.0\;U/m{\ell}$) were reacted, production rate of reducing sugar was $5.84\;g/{\ell}{\cdot}h$, and consumption rate was $-3.88\;g/{\ell}{\cdot}h\;at\;35^{\circ}C$ So suitable condition of SSF was concluded at temperature of $35^{\circ}C$. Also, optimal enzyme concentration of SSF was concluded in $2.0\;U/m{\ell}$, at this condition, the production rate of reducing sugar was $4.80\;g/{\ell}{\cdot}h$ At SSF process, when 50 g of food wastes was supplied in 12 h interval, $64\;g/{\ell}$ of ethanol and 0.45 g-ethanol/g-reducing sugar in yield were obtained in 120 h fermentation. Thus, the technology of high yield of ethanol production using food wastes was confirmed. And semi-continuos SSF system for cutting off cost of enzymatic saccharification was developed in this study.

• Microbiology and Biotechnology Letters
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• v.43 no.1
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• pp.9-15
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• 2015

The seaweed, Gracilaria verrucosa, was fermented to produce bioethanol. Optimal pretreatment conditions were determined to be 12% (w/v) seaweed slurry and 270 mM sulfuric acid at 121℃ for 60 min. After thermal acid hydrolysis, enzymatic saccharification was carried out with 16 U/ml of mixed enzymes using Viscozyme L and Celluclast 1.5 L to G. verrucosa hydrolysates. A total monosaccharide concentration of 50.4 g/l, representing 84.2% conversion of 60 g/l total carbohydrate from 120 g dw/l G. verrucosa slurry was obtained by thermal acid hydrolysis and enzymatic saccharification. G. verrucosa hydrolysate was used as the substrate for ethanol production by separate hydrolysis and fermentation (SHF). Ethanol production by Candida lusitaniae ATCC 42720 acclimated to high-galactose concentrations was 22.0 g/l with ethanol yield (Y_EtOH) of 0.43. Acclimated yeast to high concentrations of specific sugar could utilize mixed sugars, resulting in higher ethanol yields in the seaweed hydrolysates medium.