• Journal of Microbiology and Biotechnology
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• v.24 no.12
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• pp.1699-1706
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• 2014

A lichenase gene (mt-lic) was identified for the first time through function-based screening of a soil metagenomic library. Its deduced amino acid sequence exhibited a high degree of homology with endo-${\beta}$-1,3-1,4-glucanase (having both lichenase and chitosanase activities), encoded by the bgc gene of Bacillus circulans WL-12. The recombinant lichenase overexpressed and purified from Escherichia coli was able to efficiently hydrolyze both barley ${\beta}$-glucan and lichenan. The enzyme showed maximal activity at a pH of 6.0 at $50^{\circ}C$, with Azo-barley-glucan as the substrate. The metal ions $Mn^{2+}$, $Mg^{2+}$, $Ca^{2+}$, and $Fe^{2+}$ enhanced the enzymatic activity, whereas the $Cu^{2+}$ and $Zn^{2+}$ ions inhibited the enzymatic activity. The $K_m$ and $V_{max}$ values of the purified lichenase were determined to be 0.45 mg/ml and 24.83 U/min/mg of protein, respectively.

• Journal of Microbiology and Biotechnology
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• v.32 no.4
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• pp.484-492
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• 2022

Lichenase is an enzyme mainly implicated in the degradation of polysaccharides in the cell walls of grains. Emerging evidence shows that a highly efficient expression of a thermostable recombinant lichenase holds considerable promise for application in the beer-brewing and animal feed industries. Herein, we cloned a lichenase gene (CelA203) from Bacillus subtilis B110 and expressed it in E. coli. This gene contains an ORF of 729 bp, encoding a protein with 242 amino acids and a calculated molecular mass of 27.3 kDa. According to the zymogram results, purified CelA203 existed in two forms, a monomer, and a tetramer, but only the tetramer had potent enzymatic activity. CelA203 remained stable over a broad pH and temperature range and retained 40% activity at 70℃ for 1 h. The K_m and V_max of CelA203 towards barley β-glucan and lichenan were 3.98 mg/ml, 1017.17 U/mg, and 2.78 mg/ml, 198.24 U/mg, respectively. Furthermore, trisaccharide and tetrasaccharide were the main products obtained from CelA203-mediated hydrolysis of deactivated oat bran. These findings demonstrate a promising role for CelA203 in the production of oligosaccharides in animal feed and brewing industries.

• Journal of Microbiology and Biotechnology
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• v.16 no.3
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• pp.368-373
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• 2006

Bacillus subtilis and related Bacillus species are frequently used as hosts for the mass production of recombinant proteins. Accordingly, this study examined the cellular response of B. subtilis to the overexpression of a soluble secretory protein. As such, the lichenase derived from B. cereus was overexpressed in B. subtilis, initially localized in the cytoplasm as a mature form and then secreted into the medium. Thereafter, the proteome of B. subtilis was analyzed using 2D electrophoresis and MALDI-TOF mass spectrometry. The expression of several heat-shock proteins, such as dnaK and groEL, was increased under this condition. In addition, manganese superoxide dismutase and NADH dehydrogenase were also upregulated in the lichenase-secreting B. subtilis. Therefore, it was concluded that the transient accumulation of a secreted protein in B. subtilis before secretion acted as a stress on the cell, which in turn induced the expression of various protective proteins.

• Korean Journal of Microbiology
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• v.48 no.2
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• pp.73-78
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• 2012

We previously reported that the truncated Cel44C-$Man26A_{P558}$ ${\beta}$-glycosyl hydrolase protein exhibits multifunctional activities, including cellulase, xylanase, and lichenase. DNA shuffling of the truncated Cel44C-$Man26A_{P558}$ enzyme was performed to enhance the enzymatic activity of the multifunctional ${\beta}$-glycosyl hydrolase. Two mutant enzymes, M2Cel44C-$Man26A_{P558}$ that carries one mutation (P438A) and M21Cel44C-$Man26A_{P558}$ that carries two mutations (A273T and P438A) were obtained. The enzymatic activity of the M21Cel44C-$Man26A_{P558}$ double mutant was lower than enzymatic activity of the single mutant (M2Cel44C-$Man26A_{P558}$). However, both mutants displayed the enhancements in their enzyme activities that were ${\approx}1.3$- to 2.2-fold higher than the original enzymatic activity in Cel44C-$Man26A_{P558}$. In particular, the mutant M2Cel44C-$Man26A_{P558}$ exhibited an approximate 1.5- to 2.2-fold increase in the cellulase, xylanase, and lichenase activities in comparison with the control (Cel44C-$Man26A_{P558}$). The optimum cellulase, linchenase, and xylanase activities of ${\beta}$-glycosyl hydrolase were observed at pH 7.0, pH 7.0 and pH 6.0, respectively. These results, therefore, suggest that the amino acid residue Ala438 plays important roles in the enhancement of the activity of multifunctional ${\beta}$-glycosyl hydrolase.

• Preventive Nutrition and Food Science
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• v.3 no.4
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• pp.382-386
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• 1998

A soluble dietary fiber, $\beta$-glucan, contained in oat and barley has nutritional benefits such as hypocholesterolemic effects and influences blood glucose regulation. The contents of $\beta$-glucan in both cereals range from 3 to 7% with the exception of a certain barley genotype which contains up to 16% $\beta$-glucan. $\beta$-Glucan is distributed mainly in the cell walls of endosperm and the distal (bran) portion of kernel. Various procedures have been developed for increasing the extraction yield of $\beta$-glucan. Oat gum prepared with weak alkali extractionand alcohol proecipitation following protein removal usually contains 80% $\beta$-glucan.The most commonly used method for $\beta$-glucan quantitiation is an enzymatic procedure combining lichenase plus $\beta$-glucosidase followed by measuring the amount of glucos released by glucose oxidase-peroxidase treatment. The increase in foam-and emulsion-stabilizing capacity of $\beta$-glucan is due to the increase in viscosity of the aqueous phase. Therefore, $\beta$-glucan shows great potentials as a thickener and a stabilizer.

• Food Science and Biotechnology
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• v.18 no.1
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• pp.271-274
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• 2009

Cereal $\beta$-glucans, linked essentially by mixed $\beta$-(1,4/1,3) glycosidic bonds, were extracted, purified, and structurally identified. Previously chemical structure of barley $\beta$-glucans was characterized from 3 varieties of 'Gang', 'Ohl', and 'Gwangan', and the (1,4)/(1,3) linkage ratio of the $\beta$-glucans was identical. In this study, $\beta$-glucans from 1 barley ('Chal') and 3 oat ('Ohl', 'Samhan', and 'Donghan') varieties were structurally scrutinized, and the linkage pattern of total 7 cereal $\beta$-glucans was compared. The amount of 2 major 3-O-$\beta$-cellobiosyl-D-glucose (DP3) and 3-O-$\beta$-cellotriosyl-D-glucose (DP4) from barley and oat accounted for only 66.6-73.3 and 68.12-81.89% of water-extractable $\beta$-glucan fractions, and the (1,4)/(1,3) linkage ratios of both barley and oat $\beta$-glucans were within very narrow range of 2.27-2.31 and 2.38-2.39, respectively, among the cultivars tested. Structural difference in the cereal $\beta$-glucans was evident when DP3:DP4 ratio in the $\beta$-glucan structure was compared. As a result, this ratio was significantly greater for barley $\beta$-glucan (2.26-2.74) than for oat (1.54-1.66). Chal-B had the greatest DP3 to DP4 ratio among the samples, which in turn reflected the least amount of (1,4)-linkages.