G C Pradeep

A multi-tolerant low molecular weight mannanase from Bacillus sp. CSB39 and its compatibility as an industrial biocatalyst.

Bacillus sp. CSB39, isolated from popular traditional Korean food (Kimchi), produced a low molecular weight, thermostable mannanase (MnCSB39); 571.14U/mL using locust bean gum galactomannan as a major substrate. It was purified to homogeneity using a simple and effective two-step purification strategy, Sepharose CL-6B and DEAE Sepharose Fast Flow, which resulted in 25.47% yield and 19.32-fold purity. The surfactant-, NaCl-, urea-, and protease-tolerant monomeric protein had a mass of ∼30kDa as analyzed by SDS-PAGE and galactomannan zymography. MnCSB39 was found to have optimal activity at pH 7.5 and temperature of 70°C. The enzyme showed ˃55% activity at 5.0-15% (w/v) NaCl, and ˃93% of the initial activity after incubation at 37°C for 60min. Trypsin and proteinase K had no effect on MnCBS39. The enzyme showed ˃80% activity in up to 3M urea. The N-terminal amino acid sequence, ALKGDGX, did not show identity with reported mannanases, which suggests the novelty of our enzyme. Activation energy for galactomannan hydrolysis was 26.85kJmol(-1) with a Kcat of 142.58×10(4)s(-1). MnCSB39 had Km and Vmax values of 0.082mg/mL and 1099±1.0Umg(-1), respectively. Thermodynamic parameters such as ΔH, ΔG, ΔS, Q10, ΔGE-S, and ΔGE-T supported the spontaneous formation of products and the high hydrolytic efficiency and feasibility of the enzymatic reaction, which strengthen its novelty. MnCSB39 activity was affected by metal ions, modulators, chelators, and detergents. Mannobiose was the principal end-product of hydrolysis. Bacillus subtilis CSB39 produced a maximum of 1524.44U mannanase from solid state fermentation of 1g wheat bran. MnCSB39 was simple to purify, was active at a wide pH and temperature range, multi-stress tolerant and catalyzes a thermodynamically possible reaction, characteristics that suggests its suitability for application as an industrial biocatalyst.

A novel low-molecular weight alkaline mannanase from Streptomyces tendae

A novel, low-molecular weight, alkaline mannanase from Streptomyces tendae (MnSt) was purified to homogeneity and biochemically characterized. The extracellular mannanase was purified with 26.3% yield using a Sepharose Cl-6B column. The molecular mass of MnSt was approximately 24 kDa. MnSt was stable over a broad pH range (5 ∼ 12.5), was thermally stable at 60°C, and functioned optimally at 50°C and a pH of 12.0. MnSt had Km and Vmax values of 0.05 ± 1 mg/mL and 439 ± 0.5mmol/min, respectively, using bean gum galactomannan as a substrate. The N-terminal sequence of MnSt was GWSVDAPYIAXQPFS. Thin layer chromatography (TLC) analysis of the MnSt hydrolysis products suggested that the major oligosaccharide produced was mannobiose. MnSt activity was remarkably affected by metal ions, modulators, chelators, and detergents. MnSt was simple to purify, had high thermal stability, was stable over a broad pH range, and produced mannooligosaccharides. MnSt has high potential for use as an industrial biocatalyst, particularly as a bio-bleaching agent or for oligosaccharide production.

Understanding β-mannanase from Streptomyces sp. CS147 and its potential application in lignocellulose based biorefining.

Hydrolytic enzymes such as cellulase and hemicellulase have been attracted in lignocellulose based biorefinery. Especially, mannanase has been a growing interest in industrial applications due to its importance in the bioconversion. In this study, an extracellular endo‐β‐1,4‐D‐mannanase was produced by Streptomyces sp. CS147 (Mn147) and purified 8.5‐fold with a 43.4% yield using Sephadex G‐50 column. The characterization of Mn147 was performed, and the results were as follows: molecular weight of ∼25 kDa with an optimum temperature of 50°C and pH of 11.0. The effect of metal ions and various reagents on Mn147 was strongly activated by Ca+2 but inhibited by Mg+2 , Fe+2 , hydrogen peroxide, EDTA and EGTA. Km and Vmax values of Mn147 were 0.13 mg/mL and 294 μmol/min mg, respectively, when different concentrations (3.1 to 50 mg/mL) of locust bean gum galactomannan were used as substrate. In enzymatic hydrolysis of heterogeneous substrate (spent coffee grounds), Mn147 shows a similar conversion compared to commercial enzymes. In addition, lignocellulosic biomass can be hydrolyzed to oligosaccharides (reducing sugars), which can be further utilized for the production of biomaterials. These results showed that Mn147 is attractive in quest of potential bioindustrial applications.

An alkaline and metallo-protein type endo xylanase from Streptomyces sp. CSWu-1

An alkaline xylanase (XynWu-1) from Streptomyces sp. CSWu-1 was isolated from the Korean soil sample, purified and biochemically characterized. The extracellular xylanase was purified 4.8 fold with a 16% yield using Sephadex G-50 followed by DEAE-Sepharose (fast flow) column chromatography. The molecular mass of the enzyme was approximately 37 kDa estimated by SDS-PAGE and xylan zymography. N-terminal amino acid sequence of XynWu-1 was AINVLVAALX. The enzyme was found to be stable in a broad range of pH (7.0 ∼ 13.0) and to 50°C and have an optimal pH and temperature of 11.0 and 60°C, respectively. XynWu-1 activity was found to be affected by Mn2+ ion with highest activity at 6 mM and produced xylose, xylobiose, and xylotetraose as major hydrolyzed end products. It was found to degrade agro waste materials like corncob and wheat bran by XynWu-1 (2,000 U/g) as shown by electron microscopy. As being stable in extreme alkaline pH, diverse peculiar biochemical characteristics, and ability to produce oligosaccharide shows that XynWu-1 has potential application in various bioindustries like probiotics, ethanol, etc.