# Efficient Production of γ-CD from Starch by γ-CGTase Heterologously Produced in Pichia pastoris, Assisted by β-CGTase Liquefaction and Pullulanase Debranching

## TL;DR

This study improves the production of γ-cyclodextrin (γ-CD) from starch using optimized enzymes in Pichia pastoris, resulting in a more efficient process.

## Contribution

A new process for efficient γ-CD production is developed using codon-optimized enzymes and optimized fermentation.

## Key findings

- Recombinant BFγ-CGTase yield was increased 13.3 times to 463 U/L through codon optimization and fermentation improvements.
- γ-CD yield increased by 17.67% using optimized amounts of BFγ-CGTase and BtPul in the reaction.
- A new process combining Bsβ-CGTase liquefaction and pullulanase debranching was established for efficient γ-CD production.

## Abstract

Cyclodextrins (CDs) are cyclic oligosaccharides composed of α(1 → 4) linked glucose units, which are widely used as solubilizers and stabilizers in the food, pharmaceutical and cosmetic industries. Among the CDs, γ-CD has attracted much attention due to its larger hydrophobic cavity and higher solubility. However, the industrial production of γ-CD is limited by lack of suitable enzymes and production process shortcomings. In this study, various strategies of improving heterologous enzyme production and optimization of the starch conversion process were applied to increase the production of γ-CD. A γ-cyclodextrin glucanotransferase with good product specificity from Bacillus sp. FJAT-44876 (BFγ-CGTase) and a liquefying β-CGTase from Bacillus sp. 1011 (Bsβ-CGTase) were successfully secreted by Pichia pastoris. After codon optimization and using the one-factor-at-a-time (OFAT) principle to improve the fermentation, the yield of recombinant BFγ-CGTase was increased 13.3 times to 463 U/L. Next a process was established involving Bsβ-CGTase-assisted starch liquefaction and simultaneous pullulanase debranching and BFγ-CGTase production of γ-CD. The yield of γ-CD increased by 17.67% via optimizing the amounts of BFγ-CGTase and BtPul used for the reaction. Overall, combination of the various improvements provided a new process for efficient preparation of γ-CD.