• Journal of Microbiology and Biotechnology
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• v.17 no.12
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• pp.1949-1954
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• 2007

A simple way to prevent protein hyperglycosylation in Hansenula polymorpha was found. When glucose oxidase from Aspergillus niger and carboxymethyl cellulase from Bacillus subtilis were expressed under the control of an inducible methanol oxidase (MOX) promoter using methanol as a carbon source, hyperglycosylated forms occurred. In contrast, MOX-repressing carbon sources (e.g., glucose, sorbitol, and glycerol) greatly reduced the extent of hyperglycosylation. Carbon source starvation of the cells also reduced the level of glycosylation, which was reversed to hyperglycosylation by the resumption of cell growth. It was concluded that the proteins expressed under actively growing conditions are produced as hyperglycosylated forms, whereas those under slow or nongrowing conditions are as short-glycosylated forms. The prevention of hyperglycosylation in the Hansenula polymorpha expression system constitutes an additional advantage over the traditional Saccharomyces cerevisiae system in recombinant production of glycosylated proteins.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 1986.12a
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• pp.525.3-526
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• 1986

For the analysis of fermentation characteristics and productivity of plasmid coded product, car-boxymethylcellulase in a recombinant DNA cell fermentation system, batch and continuous fermentations were carried out using a Bacillus megaterium ATCC 14945 transformed with a plasmid, pCK 108 haboring carboxymethyl cellulase gene. The effects of carbon and nitrogen sources and of temperature and pH on cell growth, product yield, plasmid stability, specific plasmid contents of cell, and gene expression efficiency were carefully studied. These experimental results will be discussed in some details.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 1986.12a
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• pp.524.3-524
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• 1986

Carboxymethyl cellulase (CMCase) produced by cloned B. megaterium was found to contain 5.2% carbohydrate but no metal ion. The enzyme was isoelectric at pH 7.23 and was high is basic amino acids. The N-terminal of the enzyme was glutamic acid. The cellulolytic activity of this enzyme was extended to the small molecular substrates such as from cellotriose to cellopentaose. In additon, the enzyme showed transglycoslation activity. The pK values of the enzyme we estimated to be 4.4 and 6.7, andthat of the enzyme-substrate complex were 4.2 and 7.2, respectively. The enzyme was not affected by the treatment with iodoacetic acid, but the modification of enzyme with carbodiimide and diethyl pyrocarbonate resulted in a marked loss of the enzyme activity. These results suggest that the active site of enzyme essentially contains carboxylic and imidazole group of amino acid residues.

• Microbiology and Biotechnology Letters
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• v.21 no.1
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• pp.13-17
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• 1993

When mandarin orange peel was used for a substrate of citric aCid fermentation by Aspergillus niger, principal enzyme activities were investigated. Not only the activity of polygalacturonase and pectin esterase being capable of digesting pectin and crude fiber of mandarin orange peel. but also that of carboxymethyl cellulase, xylanase and amylase was high. In carbohydrate metabolism, the activity of enzymes related in HMP pathway was higher than that in EMP pathway at the orange peel medium designed hereby rather than synthetic medium. Productivity of citric acid was significantly increased when the activity of citrate synthetase was high and 5imultaneously those of aconitase and NADP-dependent dehydrogenase were low.

• Journal of Life Science
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• v.18 no.8
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• pp.1049-1052
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• 2008

Microbial production of cellulolytic enzymes by Aspergillus niger in solid state fermentation (SSF) and submerged state fermentation (SF) in laboratory scale was compared. Czapek Dox liquid broth amended with cellulose (0.5%) was used for cultivation in SF, whereas rice bran was used as a solid support, moistened with cellulose, amended Czapek Dox broth for growth in SSF. The production of Carboxymethyl cellulase, Filter paperase and ${\beta}$-Glucosidase was monitored at regular intervals. The peak production of the enzymes occurred within 3 days of incubation in SSF as against $\geq$ 7 days in SF. SSF gave higher yields of enzymes in comparison to SF. Maximum titres of 0.40, 0.62 and 0.013 U/ml in respect of FPase, CMCase and ${\beta}$-glucosidase in SSF were recovered as against 0.13, 0.06 and 0.0013 U/ml in SF respectively, at their respective peak time intervals. Hence, SSF appeared to be a better choice for production of cellulolytic enzymes by Aspergillus niger.

• Korean Journal of Agricultural Science
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• v.22 no.2
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• pp.151-155
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• 1995

Two types of modified celluloses which contain trinitrophenyl groups as chromophore were synthesized from carboxymethyl cellulose Whatman CM 70 and CM 32. Diaminoethyl groups were added to the CM 70 and CM 32 to make DAE-CM celluloses and then the DAE-CM groups were substituted by 2,4,6-trinitrophenyl groups to produce TNP-celluloses. Average particle size of the TNP-cellulose from CM 32 was $44.6{\pm}9.6{\mu}m$ in diameter and $127.9{\pm}22.5{\mu}m$ in length, which was much smaller than those from CM 70, however its TNP-moiety per gram determined by using the molar extinction coefficient $1.33{\times}10^4$ of ${\varepsilon}$-TNP-lysine at 345 nm, was 0.68 millimoles, which was 5.6-fold greater than those from CM 70. The absorption spectrum of TNP-oligosaccharides which were the soluble products of TNP-celluloses by a cellulase preparation Onozuka R-10, showed a maximal peak at 344 nm. Increases in the absorbance during hydrolysis were linear with the enzyme concentration, and the differences of slope values between two types of TNP-celluloses that the more semsitive assay could be achieved by using those from CM 32 as substrate at the low range of the enzyme concentration.

• Journal of Microbiology and Biotechnology
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• v.16 no.8
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• pp.1269-1275
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• 2006

The selection of multienzyme complex-producing bacteria under aerobic condition was conducted for improving the degradation of lignocellulosic substances. The criteria for selection were cellulase and xylanase enzyme production, the presence of cellulose-binding domains and/or xylan-binding domains in enzymes to bind to insoluble substances, the adhesion of bacterial cells to insoluble substances, and the production of multiple cellulases and xylanases in a form of a high molecular weight complex. Among the six Bacillus strains, isolated from various sources and deposited in our laboratory, Paenibacillus curdlanolyticus B-6 strain was the best producer of cellulase and xylanase enzymes, which have both cellulose-binding factors (CBFs) and xylan-binding factors (XBFs). Moreover, multiple carboxymethyl cellulases (CMCases) and xylanases were produced by the strain B-6. The zymograms analysis showed at least 9 types of xylanases and 6 types of CMCases associated in a protein band of xylanase and cellulase with high molecular weight. These cells also enabled to adhere to both avicel and insoluble xylan, which were analyzed by scanning electron microscopy. The results indicated that the strain B-6 produced the multienzyme complex, which may be cellulosome or xylanosome. Thus, P. curdlanolyticus B-6 was selected to study the role and interaction between the enzymes and their substrates and the cooperation of multiple enzymes to enhance the hydrolysis due to the complex structure for efficient cellulases and xylanases degradation of insoluble polysaccharides.

• Korean Journal of Agricultural Science
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• v.25 no.1
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• pp.131-137
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• 1998

The cellulase components were partially purified from the culture filtrate of the alkalophilic bacterium Pseudomonas sp. AC-711 and its enzymatic properties were characterized. The specific activity of the purified major enzyme component was 3.5 units/mg protein as carboxymethyl cellulase and the yield was 23% of the total activity of the culture broth. The molecular weight of the component was 46,000 and the Km and Vmax on CMC were determined as $15.4mg\;mL^{-1}$ and $4.17{\mu}moles\;mL^{-1}\;min^{-1}$, respectively. The enzyme was stable at the temperatures below $60^{\circ}C$ and at the pH range of 4.0~11.0, and the optimal temperature and pH were $60^{\circ}C$ and pH 8.0, respectively. The enzyme activity was not significantly affected by the common surfactants (concentration: 0.05%) such as ${\alpha}$-olefin sulfonate, linear alkylbenzene sulfonate, sodium dodecyl sulfonate, hexadecyltrimethylammonium bromide and Tween 80. The enzyme was activated by the metal ions such as $Ca^{2+}$, $Cu^{2+}$, $Co^{2+}$, whereas inhibited by $Hg^{2+}$ and $Zn^{2+}$. The enzyme exhibited relatively high activity toward amorphous CMC as compared with crystalline substrates such as filter paper and avicel.

• Journal of Microbiology and Biotechnology
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• v.17 no.11
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• pp.1782-1788
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• 2007

Cellulosomes in Clostridium cellulovorans are assembled by the interaction between the repeated cohesin domains of a scaffolding protein (CbpA) and the dockerin domain of enzyme components. In this study, we determined the synergistic effects on cellulosic and hemicellulosic substrates by three different recombinant mini-cellulosomes containing either endoglucanase EngB or endoxylanase XynA bound to mini-CbpA with one cohesin domain (mini-CbpAl), two cohesins (mini-CbpA12), or four cohesins (mini-CbpAl234). The assembly of EngB or XynA with mini-CbpA increased the activity against carboxymethyl cellulose, acid-swollen cellulose, Avicel, xylan, and com fiber 1.1-1.8-fold compared with that for the corresponding enzyme alone. A most distinct improvement was shown with com fiber, a natural substrate containing xylan, arabinan, and cellulose. However, there was little difference in activity between the three different mini-cellulosomes when the cellulosomal enzyme concentration was held constant regardless of the copy number of cohesins in the cellulosome. A synergistic effect was observed when the enzyme concentration was increased to be proportional to the number of cohesins in the mini-cellulosome. The highest degree of synergy was observed with mini-CbpAl234 (1.8-fold) and then mini-CbpAl2 (1.3-fold), and the lowest synergy was observed with mini-CbpAl (1.2-fold) when Avicel was used as the substrate. As the copy number of cohesin was increased, there was more synergy. These results indicate that the clustering effect (physical enzyme proximity) of the enzyme within the mini-cellulosome is one of the important factors for efficient degradation of plant cell walls.

• Korean Journal of Agricultural Science
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• v.25 no.1
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• pp.124-130
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• 1998

An alkalophilic bacterium, the strain AC-711 as a potent producer of alkaline cellulase, was selected among many isolates from soil environments. Morphological, physiological and chemical characteristics of the strain AC-711 suggested that it belongs to the genus Pseudomonas according to the Bergey's Manual of Systematic Bacteriology, however the G+C mol% (54.43) of its chromosomal DNA is lower than the normal values of the genus. The major cell wall fatty acids were determined as 15:0 and 17:0 anteiso. The production of alkaline CMCase by the strain was maximal when grown on the mediun containing 1% carboxymethyl cellulose, 0.1% $KH_2PO_4$, 0.02% $CoCl_2$, 0.02% Tween 80, 0.5% $Na_2CO_3$, 0.8% yeast extract, pH 10.3 at $30^{\circ}C$ for 3 days, and the most of enzyme was excreted into culture mediun.