가시오가피의 첨가는 영지버섯 균사체를 이용한 분비 단백질 라카아제 및 셀룰라아제의 생산량을 효율적으로 증가시켰다. 영지버섯 균사체의 생장률 측정에서 가시오가피의 첨가는 영지버섯 균사의 생장률이 톱밥에 비해 절반 수준에 밖에 못미치는 결과를 나타내었다. 반면에 영지버섯 균사의 분비 단백질인 라카아제와 셀룰라아제의 양을 증가시키는데 톱밥에 비해 큰 효과가 있음을 확인할 수 있었다. 가시오가피가 첨가된 배양액에서의 라카아제 활성은 톱밥이 첨가된 배양액에서보다 영지 1호, 영지 2호 및 녹각 영지에서 0.61~2.37배 증가하였고, 이 중에서도 영지 2호는 0.947 U/min으로 라카아제 활성이 가장 높게 평가되었다. 셀룰라아제 활성 평가에서는 가시오가피가 첨가된 배양액에서의 셀룰라아제 활성은 톱밥이 첨가된 배양액에서보다 영지 1호, 영지 2호 및 녹각 영지에서 1.77~2.81배 증가하였고, 이 중에서도 영지 1호 및 영지 2호에 비해 녹각 영지에서 0.172 U/min으로 셀룰라아제 활성이 가장 높게 평가되었다. 따라서 가시오가피의 첨가가 영지버섯 균사의 분비 단백질 생산을 증가시키는데 유용한 소재로 사료된다.

Cellulose is the most abundant organic polymer constituent of the cell wall of green plants and of various forms of algae. The complexity of lignocellulosic biomass is a major challenge in industrial research. Most mushroom species that naturally grow on soil or wood possess cellulases and the corresponding enzymatic system and, potential candidates for the direct bioconversion of softwood polysaccharides into fermentable sugars. However, there have been fewer studies on mushroom cellulases than on fungi such as Trichoderma spp., exploit the full potential of mushroom cellulases. This review will focus on the current status ofmushroom cellulase research and applications and will provide insight into promising future prospects.

The objective of this study was to increase starch extraction efficiency from domestic potato by five kinds of foodgrade cellulases (mixture of β-glucanases, pectinase, cellulase, hemicelullase, and β-glucosdiase). Cellulase-treated potato had a maximum of 40% higher starch extraction yield than non-enzyme treated potato. It turned out that the shape and structure of cellulose-treated and nonenzyme-treated potatoes were the same. The average particle size of cellulose-treated potato starch was smaller than non-enzyme treated potato. Interestingly, the small starch granular (<10 μm particle) was shown in extracted starch from cellulose-treated potato. Rapid viscosity analysis showed that starch from cellulase treated potato had lower pasting temperature than starch from nonenzyme-treated potato. The range of the gelatinization temperature (49-62oC) of starches from cellulose-treated potato was broader than that of starches from nonenzyme-treated potato. Therefore, the results of this study confirm that cellulase plays an important role in the extraction of starch from the potato and physicochemical characteristics of potato starch.

The aim of this study is to analyze the functional activity of an endo-β-1, 4-glucanase from the wood dwelling lower termite Coptotermes gestroi. Full length cDNA sequences of the endo-β-1,4-glucanase were obtained by primer walking in conjunction with Rapid Amplification cDNA Ends. With the obtained full length sequences, primers for amplifying open reading frame (ORF) excluding the signal peptide and glycophosphatidylinositol anchor were designed. Amplified endo-β-1,4-glucanase fragment was cloned and expressed using pET30(+) expression vector in BL21 E.coli strain. Expression of endo-β-1,4-glucanase was confirmed by Western blotting and the result revealed that only full ORF was expressed. The cellulase activity of protein preparations from the induced and non-induced cells was analyzed with Congo Red assay with the cellulase from Aspergillus niger (Sigma Aldrich) as a positive control. The activity of C. gestroi endo-β-1,4-glucanase was significantly higher than those observed in the positive control and the enzyme preparation from non-induced cells. Therefore, this study confirmed that C. gestroi endo-β-1,4-glucanase had a function of cellulose hydrolysis.

To search for a variety of cellulase genes from termites with different habitats consuming different foods, we collected three species (Neotermes spp., Odontotermes spp., Macrotermes spp.) from the wood and one species (Nasutitermes spp.) from the cow dung. Total RNA was isolated both from alimentary track tissues containing paunch and from other tissues, and used for the suppression subtractive hybridization (SSH). The resulting EST libraries were sequenced and searched by BLAST to identify cellulase genes. A total of 16 cellulase genes were found from the wood-dwelling termites whereas 4 cellulase genes from the cow dung-dwelling termites. Endo-beta-1,4-glucanase and beta-glucosidase were identified as the most abundant cellulase from the wood-dwelling termites and cow dung-dwelling termites, respectively. This finding suggests that cellulase profiles are significantly different depending on the termite’s habitat and food. In addition, we analyzed phylogenetic relationships among the cellulase genes along with other cellulase genes reported to date. All cellulase genes appeared to be originated from endosymbioants without any hint of horizontal gene transfer. Functional expression of endo-beta-1,4-glucanase using a baculovirus expression system is in progress to characterize its enzymatic properties.

본 연구는 도축장에서 폐기되는 도축 반추위 내용물을 사일리지 혹은 TMR (total mixed ration) 사료 첨가용 효소제로 개발하기 위한 목적으로 수행되었다. 도축 반추위 내용물에는 상당한 수분이 함유되어 있어, 적절한 건조과정을 거치지 않고서는 그 활용이 원활하지 않다. 그러나 효소는 열에 민감한 단백질로 구성되어 있기 때문에 적절한건조 조건의 개발이 필요하다. 본 연구에서는 통계적 방법을 이용하여 가열온도 (60, 75, 90°C), 가열시간 (12, 30, 48시간) 및 부형제의 비율 (12, 22.5, 33%)이 도축 반추위 내용물에 함유된 다양한 효소활성에 미치는 효과를 분석하였다. 총 3가지 효소, xylan 분해효소, 셀룰로오스 분해효소및 전분 분해효소를 검토하였고, 각 효소활성들에 대한 각요인들의 효과가 매우 다양하게 나타났다 (p<0.05). 셀룰로오스 분해효소와 전분 분해효소의 활성은 가열온도가 증가함에 따라 감소하였으며 (p<0.05), xylan 분해해소는 열에대한 안전성이 다른 효소들에 비하여 우수하였다. 자일란,셀룰로오스, 전분 분해효소를 증가시키는 적정 부형제의비율은 22.5, 12 그리고 33%로 나타났다. 비록 3가지 효소들의 제형화에 있어 공통적으로 적용될 수 있는 최적점을도출하지는 못하였으나, 본 연구결과에서 얻어진 효소들의반응 값들을 이용하여 목적하는 효소에 따라서 다양한 방법으로 적용이 가능할 것으로 판단된다.

A carboxymethyl cellulase gene, cel5B, was cloned, sequenced, and expressed in Escherichia coli. pRCS20 in E. coli was identified from metagenomic cosmid library of cow rumen for cellulase activity on a carboxymethyl cellulose agar plates. Cosmid clone (RCS20) was partially digested with Sau3AI, ligated into BamHI site of pBluescript II SK+ vector, and transformed into E. coli DH5α. The insert DNA of 1.3 kb was obtained, designated cel5B, which has the activity of hydrolyzation of CMC. The cel5B gene had an open reading frame (ORF) of 1,059 bp encoding 352 amino acids with a signal peptide of 48 amino acids and the conserved region, VIYEIYNEPL, belongs to the glycosyl hydrolase family 5. The molecular mass of Cel5B protein expressed from E. coli DH5α exhibited to be about 34 kDa by CMC-SDS-PAGE. The optimal pH was 8.0, and the optimal temperature was about 50℃ for its enzymatic activity.

From the used sawdust medium of commercial mushroom, Lyophyllum ulmarium and Pleurotus eryngii, cellulase was extracted by distilled water. Optimum temperature of extraction was 25 ℃. Cellulase including this medium could attack cellulose powder, wood tip of a Japan ceder and cypress, and rice hull. Both cellulase from Lyophyllum ulmarium and Pleurotus eryngii showed high activities for rice hull. Now, by affinity chromatography, these cellulase is tried to purify.

In order to obtain the fundamental informations on cellulase of Trichoderma viride QM 9414 for its production and utilization, some physico-chemical properties of the enzyme were reviewed. When T. viride QM 9414 was cultured on wheat bran medium, filter paper-disintegrating and carboxymethyl cellulose(CMC)-saccharifying activity were incresed with the cell growth, and thereafter CMC-saccharifying activity was kept on almost the same leved while filter-paper disintegrating activity was decreased sharply. And β-glucosidase was formed maximally on the late stationary phase of growth. The crude cellulase of cell-free extracts was purified by (NH_4)_2SO, fractionation, Sephadex-G 200 column chromatography and DEAE Sephadex A-50 column chromatography. Filter paper-disintegrating, CMC-saccharifying and β-glucosidase activity were purified 10-fold, 47-fold and 38-fold, respectively. The crude enzyme was proved to be a complex of three different enzyme proteins which were showing filter paper-disintegrating, CMC-saccharifying and β-glucosidase activity. The optimal pH of the three enzyme components was alike pH 4.0, and the optimal temperature for CMC-saccharifying, filter paper-disintegrating and β-glucosidase activity were 40℃, 45℃ and 50℃ respectively. The Km and Vmax values of CMC saccharifying activity for CMC were 0.485% and 3.10(μmole/min·㎖), and the Km and Vmax values of β-glucosidase for ONPG(o-nitrophenol glucopyranoside) were 0.944×10 exp (-3)M and 0.097(μmole/min·㎖), respectively. The Km and Vmax values of filter paper-disintegrating activity for Avicel were determined to be 0.09% and 0.178(μmole/min·㎖), respectively. β-Glucosidase activity was competitively inhibited by glucose, and the Ki value for this enzyme was 3.54×10 exp (-3)M, CMC saccharifying activity was found to be greatly inhibited by cellobiose.

수집한 장류 시료에서 분리한 균주들을 CMC를 함유하는 배지에 접종하여 섬유소 분해 활성이 우수한 4-1 균주를 선발하였다. 4-1 균주의 16S rRNA 염기서열을 분석한 결과, B. subtilis로 동정되었다. B. subtilis 4-1의 효소생산을 위한 최적 배양조건은 탄소원으로 1.0% soluble starch와 질소원으로 0.1% yeast extract를 첨가하여 45℃에서 24시간 배양하였을 때로 나타났다. 최적 배양 pH를 조사한 결과, pH 5.0～9.0에서 cellulase 효소활성이 높았다. B. subtilis 4-1의 조효소 특성은 효소반응의 최적 pH가 pH 9.0, 반응온도는 60℃에서 효소활성이 가장 높았으며, 20～90℃ 온도에서 60분간 열처리시 효소 활성이 80%이상 유지되었다. 따라서 B. subtilis 4-1에 의해 생산되는 cellulase는 내알칼리성 효소로 추정되며, 높은 열에도 안정한 것으로 나타났다. B. subtilis 4-1이 생산하는 cellulase는 CMC에 가장 높은 효소활성을 나타내었으며 avicel과 pNPG에서도 활성을 보여 복합효소로 생각된다.