# Construction of pectinase gene engineering microbe and its application in tobacco sheets

## TL;DR

Scientists engineered a microbe to produce pectinase, which reduces pectin in tobacco leaves, improving cigarette quality.

## Contribution

A recombinant Pichia pastoris strain was constructed to produce polygalacturonase for tobacco pulp pectin degradation.

## Key findings

- The recombinant strain produced 2872.91 U/mL of crude enzyme with a 60 kDa protein band.
- Pectin degradation in tobacco pulp reached 17.53% with 0.8% enzyme addition.
- Sensory quality improved when 0.4% enzyme was used in the process.

## Abstract

Pectin has bad effects on the sensory quality of cigarettes. In order to reduce the pectin content in tobacco leaves, polygalacturonase (PG) gene was extracted from Aspergillus niger sw06, and recombinant plasmid pPICZαA was constructed and transformed into Pichia pastoris X33 to build an engineered strain X33/pPICZαA-PG. Transformant genomic fragment was 1,608 bp. The genomic fragment was amplified and recovered, and sequencing indicated that PG gene expression have been successfully inserted into P. pastoris expression vector. Positive clones were detected by SDS protein with a molecular weight of about 60 kDa. The enzyme production cycle of the recombinant strain was 36 h, and crude enzyme activity was 2872.91 U/mL. The fusion protein was purified by nickel Sepharose affinity chromatography. A clear band was detected and the concentration of recombinant protein was 8.1 μg/μL. It showed a good effect on degrading pectin after addition of the PG crude enzyme produced by recombinant yeast on the tobacco pulp. The optimized addition amount on process product line was 0.8%, which could reduce tobacco pulp pectin from 3.65 to 3.01% and achieve a degradation rate of 17.53%. Sensory evaluation showed that the effect was better when the addition amount of pulping was 0.4%.