β-Cyclodextrin Production by Cyclodextrin Glucanotransferase from an Alkaliphile Microbacterium terrae KNR 9 Using...

Area of Science:

• Biochemistry
• Enzymology
• Carbohydrate Chemistry

Background:

• Cyclodextrin glucanotransferase (CGTase) is an alpha-amylase enzyme crucial for starch conversion.
• CGTase catalyzes intramolecular transglycosylation, producing cyclodextrins (CDs) from starch substrates.
• Alkaliphilic Microbacterium terrae KNR 9 produces a potent CGTase enzyme.

Purpose of the Study:

• To evaluate various raw and gelatinized starches as substrates for beta-cyclodextrin (β-CD) production using purified CGTase.
• To optimize the reaction parameters for maximizing β-CD yield.
• To confirm starch degradation by CGTase through microscopic analysis.

Main Methods:

• Screening of six different starch substrates (soluble starch, potato, sago, corn, corn flour, rice flour) for β-CD production.
• Enzymatic assays using purified CGTase from Microbacterium terrae KNR 9.
• Statistical optimization employing Central Composite Design (CCD) for starch concentration, enzyme concentration, and temperature.
• Confirmation of cyclodextrin production via thin-layer chromatography (TLC).
• Microscopic observation to verify starch granule degradation.

Main Results:

• Raw potato starch was the most effective substrate, yielding 13.46 gm/L of CDs within 1 hour at 60°C.
• Raw sago starch was the second-best substrate, producing 12.96 gm/L of CDs.
• Optimized conditions (4.8 U/L CGTase, 150 gm/L potato starch, 55.6°C) yielded a maximum of 28.22 gm/L β-CD.
• The optimized temperature of 55.6°C resulted in a ~15% increase in β-CD production compared to 60°C.

Conclusions:

• Purified CGTase from Microbacterium terrae KNR 9 effectively produces β-cyclodextrins from various starch sources.
• Raw potato starch is a superior substrate for CGTase-mediated β-CD synthesis.
• Statistical optimization significantly enhances β-CD production efficiency, demonstrating the potential for industrial applications.