D-Tagatose, a functional sweetener, is converted from d-galactose by l-arabinose isomerase, which catalyzes the conversion of l-arabinose to l-ribulose. In this study, the araA gene encoding l-arabinose isomerase from Klebsiella pneumoniae was cloned and expressed in Escherichia coli, and the expressed enzyme was purified and characterized. The purified l-arabinose isomerase, a soluble protein with 11.6-fold purification and a 22% final yield, displayed a specific activity of 1.8 U/mg for d-galactose and existed as a homohexamer of 336 kDa. The enzyme exhibited maximum activity at pH 8.0 and 40 °C in the presence of Mn²⁺ and relative activity for pentoses and hexoses in the order l-arabinose > d-galactose > l-ribulose > d-xylulose > d-xylose > d-tagatose > d-glucose. The thermal stability of recombinant E. coli cells expressing l-arabinose isomerase from K. pneumoniae was higher than that of the enzyme. Thus, the reaction conditions of the recombinant cells were optimized to pH 8.0, 50 °C, and 4 g/L cell concentration using 100 g/L d-galactose with 1 mM Mn²⁺. Under these conditions, 33.5 g/L d-tagatose was produced from d-galactose with 33.5% molar yield and 67 g/L/h productivity. Our findings will help produce d-tagatose using whole-cell reactions, extending its industrial application.

An isolated bacterium from soil that highly hydrolyzes cellulose was identified as Paenibacillus peoriae and named P. peoriae MK1. The cellulase from P. peoriae MK1 was cloned and expressed in Escherichia coli. The purified recombinant cellulase, a soluble protein with 13.2-fold purification and 19% final yield, displayed a specific activity of 77 U/mg for CM-cellulose and existed as a metal-independent monomer of 65 kDa. The enzyme exhibited maximum activity at pH 5.0 and 40 °C with a half-life of 9.5 h in the presence of Ca²⁺ ion. The highest activity was observed toward CM-cellulose as an amorphous substrate, followed by swollen cellulose, and sigmacell cellulose and α-cellulose as crystalline substrates. The enzyme and substrate concentrations for the hydrolysis of CM-cellulose were optimized to 133 U/mL and 20 g/L CM-cellulose, respectively. Under these conditions, CM-cellulose was hydrolyzed to reducing sugars composed mostly of oligosaccharides by cellulase from P. peoriae MK1 as an endo-type cellulase with a productivity of 11.1 g/L/h for 10 min. Our findings will contribute to the industrial usability of cellulase and the research for securing cellulase sources.

A commercial immobilized d-glucose isomerase from Streptomyces murines (Sweetzyme) was used to produce l-rhamnulose from l-rhamnose in a packed-bed reactor. The optimal conditions for l-rhamnulose production from l-rhamnose were determined as pH 8.0, 60 °C, 300 g L⁻¹ l-rhamnose as a substrate, and 0.6 h⁻¹ dilution rate. The half-life of the immobilized enzyme at 60 °C was 809 h. Under the optimal conditions, the immobilized enzyme produced an average of 135 g L⁻¹ l-rhamnulose from 300 g L⁻¹ l-rhamnose after 16 days at pH 8.0, 60 °C, and 0.6 h⁻¹ dilution rate, with a productivity of 81 g/L/h and a conversion yield of 45% in a packed-bed reactor.

Commercial β-galactosidase from Aspergillus oryzae (SUMILACT L^TM) was used for the bioconversion of the ginsenosides Rb1, Rb2, Rc, Rd, and Rg3 to gypenoside-XVII, compound-O, compound-MC1, F2, and Rh2, respectively. The optimal conditions were pH 4.5, 50 °C, 60 U·mL⁻¹ enzyme, and 8.0 mM substrate. Interestingly, the enzyme hydrolyzed only the outer β-(1,2)-d-glucose linkage at the C-3 position of ginsenosides. Under optimum conditions, the enzyme completely converted Rb1, Rb2, Rc, Rd, and Rg3 to gypenoside-XVII, compound-O, compound-MC1, F2, and Rh2, respectively, with the highest productivity.

This study aimed to prepare a valuable minor ginsenoside Compound K, using ginsenosides from ginseng leaf, by-products of ginseng, with a food-grade commercial enzyme Cytolase PCL 5. The total content of major ginsenosides in ginseng leaf extract was 146.9 mg/g and that of the PPD-type ginsenosides such as Rb1, Rb2, Rc, Rd and F2 was 5.7, 11.5, 19.8, 16.4, and 9.8 mg/g, respectively. Cytolase PCL5 showed very efficient enzymatic activity to produce Compound K from the PPD-type gisenosides of ginseng leaf extract. The Cytolase PCL5 exhibited the highest activity at pH 4.5 and 65℃, and the half-lives of the enzyme at 50, 55, 60, 65, 70, and 75℃ were 118, 94, 87, 60, 47, and 8 h, respectively. During Compound K production, the optimal concentrations of PPD-type ginsenosides and enzyme were 10 mg/g and 100 U/ml, respectively. Under optimum conditions, Cytolase PCL5 produced Compound K from PPD-type ginsenosides with a molar yield of over 97% and productivity of 1 mmole/L/h within 10 h. These results suggest that the ginseng leaf is a potential producer of Compound K and Cytolase PCL5 is a very useful enzyme for Compound K production.