Abstract Lichenan, 1,3-1,4-β-Glucan, a linear polysaccharide exists in the cell walls of various cereals, has garnered attention for its industrial applications due to its enzymatic breakdown by lichenase enzymes. In this study, Bacillus licheniformis strain RB16, isolated from cattle faeces, was identified as a robust lichenase producer. The lichenase gene, licA, was successfully cloned and characterized. The cloned RB16 lichenase (LicA) demonstrated its highest activity level at pH 7.5. It also retained over 50% of its activity within the pH range of 6.0–8.5 but experienced a decline to 40% at pH 9.0. LicA was active at temperatures ranging from 25 to 65 °C with an optimum at 45 °C. LicA exhibited more than 60% of its activity at the temperature range of 35–55 °C. Zymogram analysis confirmed LicA’s lichenan-degrading ability and structural analysis revealed a stable enzyme structure primarily composed of random coils and extended strands. Although LicA exhibited low thermostability, consistent with its relatively low α-helix content, it demonstrated promising industrial potential. Evolutionary analysis placed LicA within a cluster of closely related Bacillus lichenases, particularly B. halotolerans, B. atrophaeus, and B. spizizenii. These findings expand our understanding of lichenases of Bacillus and underscore its potential for various industrial applications. Lichenan 1,31,4βGlucan, 1314βGlucan βGlucan 1,3 1,4 β Glucan, 1 3 4 Glucan 1,3-1,4-β-Glucan cereals enzymes study RB faeces producer gene licA characterized RB1 (LicA 75 7 5 7.5 50 6085 6 0 8 6.0–8. 40 90 9 9.0 2 C 60 3555 35 55 35–5 LicAs s lichenandegrading lichenan degrading strands thermostability αhelix α helix content B halotolerans atrophaeus spizizenii 31 4βGlucan 1,31,4βGlucan 13 1, 14 7. 608 6.0–8 9. 355 35– 6.0– 6.0 6.

Abstract This study was undertaken to evaluate the health-promoting potentials of Isatis aucherii, Isatis buschiana, Isatis candolleana, Isatis tinctoria subsp. corymbosa and Isatis tinctoria. I. aucherii and I. candolleana are endemic, I. buschiana, I. tinctoria subsp. corymbosa are native in Turkey. While I. tinctoria is a well studied species, there is insufficient information about other endemic and native species. Therefore, this study is focused to reveal the bioactive compounds of poorly studied endemic and native species. In this context, protein, ash, glucosinolates, fatty acids, total phenolic and flavonoid content, and antioxidant activities were determined in leaf extracts. The highest protein and fatty oil contents were observed in I. tinctoria and I. buschiana. Arachidic acid was predominant inI. tinctoria subsp. corymbosa, I. buschiana and I. aucherii, while predominant fatty acids were arachidonic and oleic acids inI. candolleana and I. tinctoria. Glucobrassicin was the main glucosinolate in I. tinctoria, while the others contained gluconapin as the main glucosinolate. Antioxidant activities were correlated with phenolic and flavonoid content, the highest and lowest antioxidant activities were observed in I. buschiana and I. aucherii, respectively. According to results, I. buschiana leaves were high in contents of bioactive compounds; it could be a promising plant with its health- promoting effects.

Fresh leaves of four Isatis species culture form of I. tinctoria L and wild forms of I. buschiana Schischkin, I. candolleana Boiss. (endemic) and I. tinctoria L. subsp. corymbosa. (Boiss.) were used for indigo production. Dyes were extracted by fermentation and hot water application. The extracted dyes were optimized with different pH and reducing agents. Results showed that the dye from hot water application produced the desired dying quality at pH 11. Reducing agent concentrations had no significant effect on color quality. Dark blue and blue colors were obtained from I. tinctoria and I. candolleana extracts although I. tinctoria subsp. corymbosa and I. buschiana produced mostly yellow-gray colors. Light, dry and wet rubbing fastness values varied between 3 and 3/4 while washing fastness was between 2 and 4/5. The highest indigo amounts were determined spectrophotometrically as 4.19 mg/g and 2.53 mg/g in I. tinctoria and I. candolleana, respectively. Results also showed that harvesting season was important for indigo production and the highest indigo amount was observed in mid-June.