Characterization, mechanism, and application of aldolase: A Patulin-degrading enzyme from Kluyveromyces Marxianus YG-4

Abstract

Patulin (PAT) is a mycotoxin produced by Penicillium species, which mainly contaminates fruit and fruit-derived products, posing a serious threat to food safety. A novel heat-resistant PAT degrading enzyme, aldolase (PATY, 16.9 kDa), was isolated and identified from Kluyveromyces marxianus YG-4 (K. marxianus YG-4) isolated from Kefir grain. The whole genome sequencing of K. marxianus YG-4, heterologous expression, purification, and bioinformatics were utilized to study the properties of PATY. PATY showed the highest catalytic efficiency (22.8 U/mg) towards PAT at pH8.0 and 35 °C, and maintained over 80 % relative activity at 70 °C. Bioinformatics analysis revealed that the catalytic reaction is achieved through the hydrogen bonding between the amino acids His19, Arg22, Tyr57, Lys60, and His61 of PATY and PAT. PAT was degraded by PATY into desoxypatulinic acid (DPA), which has no cytotoxicity. PATY can effectively degrade PAT in apple juice, significantly increasing the content of total phenolic and improving the quality of apple juice. In addition, PATY can also degrade Ochratoxin A (OTA, 7.20 %), Zearalenone (ZEN, 31.46 %) and Aflatoxin B₁ (AFB₁, 32.57 %) to a certain extent. In summary, PATY can effectively degrade PAT in contaminated apple juice. PATY provides a feasible and promising solution for achieving PAT detoxification of fruit juice and improving its quality and safety, and is also a potential candidate for simultaneously degrading multiple fungal toxins.