Papain expression in the cytoplasm by T7-promoter engineering and co-expression with human protein disulfide isomerase (PDI) and thiol peroxidase (GPx7) genes.

UNLABELLED

Difficulties exist in obtaining full-length, correctly folded, and soluble papain or papain-like proteases that necessitate the exploration of alternative strategies. This study describes the development of an strain capable of producing soluble papain without the need for complex and time-consuming refolding steps. To enhance the production of soluble papain, engineered T7 promoters and a recombinant papain translationally fused with varying tags were constructed. The tags investigated include the maltose-binding protein, small ubiquitin modifier protein, and glutathione transferase. An SHuffle strain was engineered to accumulate hydrogen peroxide (HO) by disruption of the redox pathway. This was accomplished by co-expression of the fusion constructs with two human endoplasmic reticulum-resident proteins, thiol peroxidase glutathione peroxidase-7 (GPx7), and protein disulfide isomerase (PDI). The oxidizing capacity of HO was used to improve disulfide bond formation in papain. The GPx7-PDI fusion dyad played a significant role in consuming harmful HO generated by the SHuffle cells. This consumption of HO helped provide the necessary oxidizing conditions for the efficient production of soluble papain. In shake-flask experiments, the recombinant strain produced ~110 mg/L of papain. Moreover, in batch fermentation, the volumetric yield reached ~349 mg/L. This work provides insights into recombinant papain microbial production that can lead to an industrial viable production strain.

IMPORTANCE

Papain, a cysteine-like protease, has extensive applications across various industries including food, chemical, pharmaceutical, drug, and polymer. However, the traditional isolation of papain from plants results in a complex mixture of proteases. Such protease mixtures result in an inability to understand which component enzyme contributed to substrate conversions. Concentrations of constituent enzymes likely differ based on the ripeness of the papaya fruit. Also, constituent enzymes from papaya differ in optimal activity as a function of temperature and pH. Thus, by using papain-like enzymes from papaya fruit, valuable information on component enzyme activity and specificity is lost. Numerous methods have been reported to purify papain and papain-like enzymes from the crude mixture. Often, methods involve at least three steps including column chromatography to separate five cysteine proteases. Such procedures represent tedious processes to manufacture the pure enzymes in extracts. The numerous uses of papain for industrial processes, as well as the probability that certain components of papain crude mixtures will be preferred for specific applications, necessitate alternative methods such as recombinant expression from microbial production systems to meet the high world demand for papain.