Effect of proteolytic enzyme and fiber of papaya fruit on human digestive health

The World Health Organization (WHO, 2005) recommends consumption of fruits and vegetables as part of a healthy diet with daily recommendation of 5 servings or at least 400 g per day. Fruits and vegetables are good sources of vitamins, minerals, antioxidants, and fiber. Papaya fruit is known for his high nutrient and fiber content, and with few exceptions, it is generally consumed ripe due to its characteristic flavor and aroma. Digestion improvement has been attributed to consumption of papaya; this we speculate is attributed to the fiber content and proteolytic enzymes associated with this highly nutritious fruit. However, research is lacking that evaluates the impact of papaya fruit on human digestion. Papain is a proteolytic enzyme generally extracted from the latex of unripe papaya. Previous research has focused on evaluating papain activity from the latex of different parts of the plant; however there are no reports about papain activity in papaya pulp through fruit maturation. The activity of papain through different stages of ripeness of papaya and its capacity of dislodging meat bolus in an in vitro model was addressed. The objective of this study was to investigate whether papain activity and fiber content are responsible for the digestive properties attributed to papaya and to find a processing method that preserves papaya health properties with minimal impact on flavor. Our results indicated that papain was active at all maturation stages of the fruit. Ripe papaya pulp displayed the highest enzyme activity and also presented the largest meat bolus displacement. The in vitro digestion study indicated that ripe papaya displayed the highest protein digestibility; this is associated with proteolytic enzymes still active at the acidity of the stomach. Results from the in vitro fermentation study indicated that ripe papaya produced the highest amount of Short Chain Fatty Acids SCFA of the three papaya substrates (unripe, ripe, and processed). SCFA are the most important product of fermentation and are used as indicators of the amount of substrate fermented by microorganisms in the colon. The combination of proteolytic enzymes and fiber content found in papaya make of this fruit not only a potential digestive aid, but also a good source of SCFA and their associated potential health benefits. Irradiation processing had minimal impact on flavor compounds of papaya nectar. However, processed papaya experienced the lowest protein digestibility and SCFA production among the papaya substrates. Future research needs to explore new processing methods for papaya that minimize the detrimental impact on enzyme activity and SCFA production.

Biosynthesis and mode of action of ribosomally synthesized and post-translationally modified antimicrobial peptides

One of the greatest sources of biologically active compounds is natural products. Often these compounds serve as platforms for the design and development of novel drugs and therapeutics. The overwhelming amount of genomic information acquired in recent years has revealed that ribosomally synthesized and post-translationally modified natural products are much more widespread than originally anticipated. Identified in nearly all forms of life, these natural products display incredible structural diversity and possess a wide range of biological functions that include antimicrobial, antiviral, anti-inflammatory, antitumor, and antiallodynic activities. The unique pathways taken to biosynthesize these compounds offer exciting opportunities for the bioengineering of these complex molecules. The studies described herein focus on both the mode of action and biosynthesis of antimicrobial peptides. In Chapter 2, it is demonstrated that haloduracin, a recently discovered two-peptide lantibiotic, possesses nanomolar antimicrobial activity against a panel of bacteria strains. The potency of haloduracin rivals that of nisin, an economically and therapeutically relevant lantibiotic, which can be attributed to a similar dual mode of action. Moreover, it was demonstrated that this lantibiotic of alkaliphile origin has better stability at physiological pH than nisin. The molecular target of haloduracin was identified as the cell wall peptidoglycan precursor lipid II. Through the in vitro biosynthesis of haloduracin, several analogues of Halα were prepared and evaluated for their ability to inhibit peptidoglycan biosynthesis as well as bacterial cell growth. In an effort to overcome the limitations of in vitro biosynthesis strategies, a novel strategy was developed resulting in a constitutively active lantibiotic synthetase enzyme. This methodology, described in Chapter 3, enabled the production of fully-modified lacticin 481 products with proteinogenic and non-proteinogenic amino acid substitutions. A number of lacticin 481 analogues were prepared and their antimicrobial activity and ability to bind lipid II was assessed. Moreover, site-directed mutagenesis of the constitutively active synthetase resulted in a kinase-like enzyme with the ability to phosphorylate a number of peptide substrates. The hunt for a lantibiotic synthetase enzyme responsible for installing the presumed dehydro amino acids and a thioether ring in the natural product sublancin, led to the identification and characterization of a unique post-translational modification. The studies described in Chapter 4, demonstrate that sublancin is not a lantibiotic, but rather an unusual S-linked glycopeptide. Its structure was revised based on extensive chemical, biochemical, and spectroscopic characterization. In addition to structural investigation, bioinformatic analysis of the sublancin gene cluster led to the identification of an S-glycosyltransferase predicted to be responsible for the post-translational modification of the sublancin precursor peptide. The unprecedented glycosyltransferase was reconstituted in vitro and demonstrated remarkable substrate promiscuity for both the NDP-sugar co-substrate as well as the precursor peptide itself. An in vitro method was developed for the production of sublancin and analogues which were subsequently evaluated in bioactivity assays. Finally, a number of putative biosynthetic gene clusters were identified that appear to harbor the necessary genes for production of an S-glycopeptide. An additional S-glycosyltransferase with more favorable intrinsic properties including better expression, stability, and solubility was reconstituted in vitro and demonstrated robust catalytic abilities.