• Journal of Microbiology and Biotechnology
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• 제27권9호
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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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• 제27권3호
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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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• 제31권10호
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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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• 제28권12호
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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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• 제32권1호
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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.

• 한국식품과학회지
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• 제48권2호
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• pp.147-152
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• 2016

전통 감주 제조 중 쌀 콜렛 분말 사용량과 당화시간에 따른 이화학적 특성과 관능검사를 조사하였다. pH는 동일한 당화시간과 동일 시료의 경우 불규칙하게 미미한 변화가 있었으나, 당화에 특별한 지장은 없었다. 점도는 콜렛 분말 비율의 증가 및 당화시간이 경과할수록 미세하게 높아졌으며, 쌀 콜렛 분말:엿기름 추출물에서 쌀 콜렛 분말의 비율이 25% 이상일 때는 그 값이 낮아졌다(p<0.05). 색도는 콜렛 분말이 증가할수록 L값, a값 및 b값은 동일시간에서 높아졌고, ${\Delta}E$값은 낮아졌다. 당화시간이 경과할수록 L값은 5:95는 불규칙하게 약간 높아졌으나, 그 외는 시료와 무관하게 시간이 경과할수록 낮아졌다. a값, b값 및 ${\Delta}E$값은 당화시간이 경과할수록 불규칙하지만 높아지는 경향이었다(p<0.05). 당도는 콜렛 분말의 양이 증가하거나 당화시간이 경과할수록 모든 시료에서 당도가 높아졌다. 5:95-15:85는 14.8-27.6으로 급격히 증가하였고, 15:85 이상에는 완만하게 증가되었다. 환원당은 콜렛분말의 증가 및 당화시간이 경과할수록 모든 시료에서 17.92-20.71 mg/mL로 매우 높게 나타났으며(p<0.05), 지금까지 보고된 어떠한 식혜보다 점도, 당도 및 환원당 함량은 높은 값을 보였다. 유리아미노산은 시료 중 콜렛 분말의 비율이 증가할수록 함량이 증가되는 경향이었으며, 생균수는 실활 시키지 않은 모든 시료에서 균이 검출되었으므로(p<0.05) 반드시 멸균이 필요하였다. 관능검사에서는 시료 25:75, 당화는 5시간일 때 패널의 기호도를 충족하는 것으로 평가되었다(p<0.05).

• 한국식품과학회지
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• 제44권5호
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• pp.553-558
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• 2012

팽화미분 첨가량[0, 25, 50, 75 및 100%(w/w)]에 따른 식혜의 당화 과정 중 pH, 탁도, 색도, 당도, 환원당, 총당, ketose, 아미노산, 단백질 및 관능 성질 변화를 조사하였다. 팽화미분 첨가량이 증가할수록 그리고 시간이 경과할수록 pH는 낮아지고(p<0.05) 탁도는 높아지는 경향을 보였다. L값은 25% 0시간을 제외하고 팽화미분 첨가량이 증가할수록 높았고 모든 시료에서 1시간에 최고값을 나타낸 후 감소하였지만(p<0.05) 0%는 1시간 후 비교적 일정한 값을 나타내었다. 당화 초기를 제외하고 팽화미분 첨가량이 증가할수록 그리고 시간이 경과할수록 적색도는 높아지는 경향을 보였고 황색도(p<0.05)는 높았다. 팽화미분 첨가량이 증가할수록 그리고 시간이 경과할수록 당도, 환원당, 총당 및 sucrose는 높았다(p<0.05). 모든 시료에서 당화 6시간에 sucrose는 0.79-0.86%로 시판 식혜 보다 매우 낮은 값을 보였다. 동일한 시간에 아미노산은 시료 사이에 큰 차이가 없었지만 단백질은 100%가 다른 시료보다 항상 높았다. 동일한 시료에서 아미노산과 단백질은 시간이 경과 할수록 높았다(p<0.05). 팽화미분 0과 75% 및 0과 100%는 0.1%내에서 유의적인 차이가 있었고 차이정도는 있음(moderate)에 가까웠다. 팽화미분 75와 100%는 0%보다 선호도가 높았다(p<0.05).

• KSBB Journal
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• 제29권5호
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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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• 제21권6호
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• pp.474-478
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• 2006

본 연구에서는 에탄올의 생산단가를 낮추기 위해, 음식물쓰레기 당화액을 이용하여 효소당화비용을 줄이고 환원당의 기질저해를 감소시키기 위해 회분식의 반연속식 동시당화발효 시스템을 개발하였다. 음식물쓰레기 200 g와 최종효소액 (amylase 기준으로 $3.0\;U/m{\ell}$) $40\;m{\ell}$가 반응하였을 때 생산되는 환원당의 속도는 $35^{\circ}C$에서 $5.84\;g/{\ell}{\cdot}h$로서, strain KJ가 소비하는 환원당 속도는 $-3.88\;g/{\ell}{\cdot}h$와 비슷하여 동시당화발효의 최적온도는 $35^{\circ}C$로 결정되었다. 그리고 음식물쓰레기 당화를 위한 최적 효소농도는 $2.0\;U/m{\ell}$로서 생산되는 환원당의 속도는 4.80 g/L h이었다. 이는 에탄올 생산균주가 $35^{\circ}C$에서 소비하는 환원당의 속도인 $-3.88\;g/{\ell}{\cdot}hr$와 비슷하므로, 효소의 최적농도는 $2.0\;U/m{\ell}$로 결정하였다. Fed-batch식 동시당화발효에서 생산된 환원당을 다 소모하고 나서 12시간 단위로 음식물쓰레기를 공급하여 배양한 결과, 배양 120시간째 에탄올발효 후의 잔존 환원당 농도는 $18.3\;g/{\ell}$, 생성된 에탄올 농도는 $64\;g/{\ell}$, 에탄올의 수율은 0.45 g-ethanol/g-reducing sugar이었다. 그리하여 음식물쓰레기의 Fed-batch식 동시당화발효기술을 개발하여 효소당화비용을 줄이고 환원당의 기질저해를 감소시킴으로써 에탄올 수율을 향상시키는데 성공하였다.

• 한국미생물·생명공학회지
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• 제43권1호
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• pp.9-15
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• 2015

본 연구는 해조류, 꼬시래기를 발효하여 에탄올을 생산하였다. 최적 전처리 조건은 12% (w/v) 해조류 슬러리, 270 mM 황산, 121도 60분동안 실시하였다. 열산가수분해 후에, 꼬시래기 가수분해산물에 16 U/ml의 혼합효소 Viscozyme L과 Celluclast 1.5 L를 이용하여 효소당화를 수행하였다. 50.4 g/l의총단당류의농도는, 120 g dw/l 꼬시래기 슬러리로부터 열산가수분해와효소당화에 의해 총 탄수화물 60 g/l의 전환율 84.2%를 나타내었다. 꼬시래기 가수분해산물은 분리당화발효(SHF)로 에탄올 생산을 위한 기질로 사용하였다. 고농도 galactose로 순치한 Candida lusitaniae ATCC42720에 의한 에탄올 생산은 0.43의 에탄올 수율(Y_EtOH)인 22.0 g/l를 생산하였다. 특정 당에 순치한 효모는 혼합당의 흡수에 유용하며, 그 결과 해조류가수분해산물배지로부터 높은 에탄올 수율을 나타내었다.