Recipient Organization
NORTH CAROLINA STATE UNIV
(N/A)
RALEIGH,NC 27695
Performing Department
Plant & Microbial Biology
Non Technical Summary
There is a strategic opportunity with regard to health and bioremediation to convert commodity sugars such as lactose and others into commercially viable high value added products. Reducing the overall process cost to improve the overall food processing economy will be the key factor to consider. Example of this are the multiple independent uses of lactose in nutrition and medicine, as well as the generation of its derivatives galacto-oligosaccharides (GOS), highly valued functional food ingredients able to mimic Human Milk Oligosaccharides (HMO) properties.The economic value of these functional products is easily demonstrated by comparing the current market price of lactose at $3/kg to the $5.20-8.50/kg market price of GOS. Thanks to the possibility of industrial production of GOS these compounds are a part of a trend in digestive health food ingredients valued at $265.9 million in 2010 with an annual growth rate of 18.3% and expected to grow at a compound annual growth rate of 13.2% from 2010 to 2015 (Affertsholt-Allen, 2007; Markets & Markets, 2010). In addition, the global GOS market is expected to reach $1.69 billion by 2025 (Report, 2016).Lactose, a major component of milk of mammals, is also a residue from cheese manufacturing process that generates two products: cheese and whey. For every pound of cheese made, nine pounds of whey are generated, creating a growing surplus of whey (186 million tons in 2008), which contains ~5% lactose. This lactose fraction has a BOD that is approximately 175-fold greater than typical sewage effluent (Smithers, 2008), therefore the untreated waste cannot be directly disposed of into bodies of water (Affertsholt-Allen, 2007; Ganzle et al., 2008; Markets & Markets, 2010; Ubic-consulting, 2010).The traditional solution to this problem has been to bioremediate lactose-rich effluents by applying expensive processes to extract the lactose, which can then be sold as a commodity product at a ceiling value of $3/kg. Only 50% of the cheese whey produced annually is recycled into useful products such as food ingredients and animal feed. The rest is considered waste either because critical volumes needed for economical recycling are not reached or due to the high degree of technical difficulty involved in biotransformation.On the other hand, components of Chitin are the second most abundant polysaccharide in nature after cellulose. Its annual biotic availability is in the range of several gigatons (Khoushab & Yamabhai, 2010; Synowiecki & Al-Khateeb, 2003) and unlike cellulose the 6-7% nitrogen content of chitin could provide an additional nitrogen source when assimilated. Its abundance and applications have gained interest in different biotechnological applications including immunology and nutrition.Previously, we designed and patented and licensed a method by which lactose can be enzymatically converted into galactosyl-lactose derivatives called galacto-oligosaccharides (GOS) (Dagher et al., 2013). GOS are one of the most extensively evaluated prebiotics, and systematic evidence shows either GOS or HMO are able to stimulate the growth and activity of beneficial bacteria in the digestive system, and are widely used in food products such as, nutritional supplements, yogurts, baked goods, and animal feed. Additionally, GOS are the most inexpensive alternative and often added to infant and follow-on formulas to mimic the beneficial effects of the human milk oligosaccharides (HMOs). Due to the lack of cost-effective industrial production methods currently infant and follow-on formulas do not contain nitrogen-containing HMOs, which are considered essential factors, able to increase the prevalence of Lactobacillus bifidus of the intestinal flora of the normal breast-fed infants, in contrast to the mixed flora of infants fed cow's milk formulas.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Goals / Objectives
Here, we propose to further evaluate new approaches to simultaneously biotransform/bioremediate the commodity chemicals lactose and Chitin derivatives to obtain complex human milk oligosaccharides (Han et al., 2012). We consider that an opportunity exists for using the oligosaccharide structure to develop a new industrial method of biological synthesis of HMOs sugars; which could be one of the first examples leading to low cost industrial production feasibility of high purity HMO sugars containing human milk bifidogenic factors.
Project Methods
Research Plan of Work. Here we propose to obtain and study the protein 3D-structure to further evaluate the catalytic domains of BHT as well as study its catalytic capabilities to simultaneously generate GOS, N-acetyllactosamine and other HMO-like sugars leading to a new production process.To assess the use of lactose-rich dairy products and chitin byproducts as viable precursors for GOS, and alternative HMOsugars production. We have met the milestone for obtaining economically efficient amounts of soluble enzyme to catalyze the reaction. Since we already have an integrated technology platform in place, we are in a position to immediately begin screening and testing raw materials. Therefore, we do not request specific equipment or enhancements for this project.To determine the 3-D structure of the BHT protein generated by Pichia pastoris. We obtained a three-dimensional molecular model by using MUSTER online but a 3-D structure of this protein needs yet to be obtained. We plan to utilize, x-ray crystallography to solve the tridimensional structure of the BHT protein alone and the ES complex with the substrate lactose. Once the protein structure is revealed, it will be useful in comparing this enzyme to other beta-glycosidases and to establish its evolutionary origins. This is a promiscuous enzyme able to use alternative hexoses or pentoses as acceptors. Thus, we anticipate that the deduced structural elements contributing to its beta hexosyl transferase activity and stability will be unveiled, allowing for further site-directed mutagenesis.To further improve the secretion of soluble BHT applying genetic modification methods. Native BHT is expressed and later localized into the cell membrane facing outside the cell. We previously achieved surprisingly cost-effective levels of soluble BHT generating a chimeric gene by replacing the native leader for an alternative secretory sequence. Furthermore, BHT still contains structural suitable features candidates for enhancement of soluble protein secretion. However, there are additional protein features (transmembrane domains and N-glycosylation sequences) with functional significance requiring further examination. Therefore, we plan to use site-directed mutagenesis of the transmembrane domain and substitutions of the N-glycosylation sequences Asn-Xaa-Ser for Asn-Xaa-Thr (Thr is more efficiently glycosylated than Ser).