Chemo-enzymatic production of bioactive chito-oligosaccahrides

This 3-years post doc project started October 1st 2010 and was prolonged due to a leave in the period October 1s 2012 until June 1s 2014. The report period spans from October 2010 ? June 2015. The project concerns production of chitosan oligomers and their building blocks with enzymatic and chemical methods, in addition to testing of their biological activities. The work was performed at the Norwegian University of Life Sciences (NMBU), Institute for Chemistry, Biotechnology and Food Science in Professor Vincent Eijsink?s group, and at Professor Steve Withers carbohydrate chemistry laboratory at the University of British Columbia (UBC), Vancouver, Canada. The primary objective of this project was chemo-enzymatic production of chitooligosaccharides (CHOS) with documented bioactivities. One of the main goals was to produce small CHOS with a specific sequence to be used as chitinase inhibitors, from smaller glucosamine / GlcNAc building blocks, with chemical and enzymatic methods. Methods for chemo-enzymatic synthesis of small CHOS building blocks and specific oligomers was successfully developed the first two years of the project. The chemical methods was established at UBC, and further optimized at NMBU the last year of the project. We are now able to effectively, in a laboratory scale, produce short CHOS with a specific sequence (alternating GlcN and GlcNAC, DADADA, tetramers, hexamers, octamers etc). We are currently testing these specific CHOS as inhibitors of chitinases. Testing of bioactivities of specific mixtures of CHOS produced by enzymatic methods was an important part of this project. A collaboration with the Chinese Academy of Science resulted in a publication in Carbohydrate Polymers in 2012. (Wu H., Aam B.B., Wang, W., Norberg A.L., Sørlie M., Eijsink V., Du Y. (2012). Inhibition of angiogenesis by chitooligosaccharides with specific degrees of acetylation and polymerization. Carbohydrate Polymers 89(2): 511-518.) We tested the ability of chitooligosaccharides produced in this project to inhibit the development of blood veins, a process called angiogenesis. The chitooligosaccharides was significantly inhibiting this process. This finding might be used in the treatment of cancer, to inhibit the growth of tumors that is depended on new blood veins to form. In 2013, we published a work concerning chitooligosaccharides ability to bind to pathogens in the intestine, to avoid the pathogens from binding and invading epithelial cells and cause disease. This was a collaboration with the University of Nebraska. (Quintero-Villegas M.I., Aam B.B., Rupnow J., Sørlie M., Eijsink V.G., Hutkins R.W (2013) Adherence inhibition of enteropathogenic Escherichia coli by chitooligosaccharides with specific degrees of acetylation and polymerization. J Agric Food Chem. 61 (11) 2748-2754) Another important bioactivity of CHOS is their anti-fungal effect. We have been testing for growth inhibiting activities on plant pathogenic fungi. This work have resulted in a patent application and two publications. (Rahman M.H., Shovan L.R., Hjeljord L.G., Aam B.B., Eijsink V.G., Sørlie M., Tronsmo A. (2014) Inhibition of Fungal Plant Pathogens by Synergistic Action of Chito-Oligosaccharides and Commercially Available Fungicides. PLoS One 25;9(4):e93192 and Rahman M.H., Hjeljord L.G., Aam B.B., Sørlie M., Tronsmo A. (2015) Antifungal effect of chito-oligosaccharides with different degrees of polymerization, European Journal of Plant Pathology 141:147-158.) In addition, we have started a company, BioCHOS (www.biochos.com) which is developing a CHOS- fungicide for plants in agriculture. This anti-fungal effect has also been tested in further applications.

Chitosan, a linear heteropolymer consisting of beta(1-4) linked N-acetylglucosamine (A) and glucosamine (D), can be converted to chito-oligosaccharides (CHOS) with different lengts and sequences by hydrolyzing certain types of chitosan with tailored chiti nases or chitosanases. The resulting CHOS have a variety of interesting bioactivities, including imunomodulary, anti-inflammatory (in f.ex. asthma), and anti-fungal activities. CHOS may also act as elicitors in plant cell defence responses and stimulate b one cell growth. Application of CHOS is hampered by several interrelated factors: (1) It is difficult to produce specific, oligomeric CHOS; (2) CHOS may be too unstable in e.g. the human body and/or bioactivity may be too low; (3) the molecular basis for CHOS functionality has not been sufficiently explored. Building on technology for controlled enzymatic conversion of chitosan that has recently been established in the applicant's laboratory, the present project aims at alleviating all these three limitin g factors by tackling some of the fundamental scientific challenges underlying them. We will: (1) Use innovative enzyme technology to generate specific oligomeric CHOS out of shorter CHOS building blocks that we can produce in-house; this will include the use of enzymes that are tailored to increased transglycosilation efficiency, as well as glycosyntase technology; (2) Chemically modify CHOS to increase their stability and/or bioactivity; (3) Set-up and use a series of biological tests to analyze bioacti vity of highly-defined CHOS preparations. Much of the advanced carbohydrate chemistry and the work on glycosyntase technology are going to be carried out during a one year stay of the project post-doc in the group of Professor Stephen G. Withers at the Un iversity of Brithish Columbia, Vancouver, Canada. Applied studies will be conducted through several collaborations with UMB, Norwegian and foreign laboratories.