Preparation of arginase and arabinoxylanase

Posted by Lisa Clara on November 15th, 2019

Arginase (Arg) is a protein that decomposes arginine. Arginine is one of the indispensable amino acids for cell growth. Normal cells can absorb citrulline by intracellular pathway and synthesize arginine under the catalysis of argininosuccinate synthase (ASS).

Arginase catalyzes the conversion of arginine to urea and ornithine. The rat liver extract rich in arginase was added to the medium to inhibit the in vitro proliferation of tumor cells, and its anti-tumor effect was discovered. It was later confirmed that its anti-tumor mechanism is arginine depletion. Since arginine is an essential amino acid in the medium, when the concentration is lower than 8, the tumor cells undergo irreversible cell death. Wheatley et al. further confirmed the in vitro antitumor activity of arginine depletion. In the 24 different tumor cell lines tested, all cells died within 5 days of arginine depletion. These cells include breast cancer, colon cancer, rectal cancer, Lung cancer, prostate cancer and ovarian cancer.

In order to promote the development of arginase, Ma Xiaoli et al. cloned the human arginase gene into yeast expression vector, transfected Pichia pastoris, screened highly expressed strains, and carried out fermentation, purification process and activity. The research laid the foundation for further research and evaluation of the substance. These studies constitute the basis for the treatment of tumors by arginine deprivation. The preparation of human arginase is still rare in the literature. Ikemoto et al. used human E. coli to express human arginase and literature reports on human arginase expression using Bacillus subtilis. The activity of the target substance obtained by Ma Xiaoli et al. was similar to that reported in the literature. A Pichia pastoris strain with high expression of recombinant human arginase was established. Because it is secreted and expressed, it does not form inclusion bodies. The purification process is simple and the target substance activity is good. . This has laid a solid foundation for further research on its anti-tumor effect and is conducive to industrialization.

Wheat bran, corn bran, and bagasse are all by-products of the plant. The common feature is that the fiber content is high but not fully utilized. Due to the high content of arabinoxylan in wheat bran and other scraps, there have been some reports on the preparation of xylooligosaccharides by enzymatic hydrolysis. Compared with other oligosaccharides, xylooligosaccharides have the following advantages: First, the proliferative ability of bifidobacteria is strong, the amount of addition is small, and the effect is remarkable; second, it can promote the absorption of calcium; and third, the stability is high. At present, the production of xylooligosaccharides generally uses arabinoxylanase, but since the polysaccharide is cross-linked by ferulic acid, the efficiency of enzymatic hydrolysis using a single enzyme is very low. Therefore, the current process is to treat the raw materials with alkali to break the cross-linking of ferulic acid before enzymatic hydrolysis, and then use arabinoxylanase to degrade the polysaccharide. The disadvantage of this process is that the process is more complicated, polluting the environment, the product color is dark (browning after xylan alkali treatment), and ferulic acid is discarded.

Ferulic acid has many health-care functions and is currently approved for use as an antioxidant and preservative in foods in the United States, and is also permitted as a food additive in the United States. A new method for preparing xylooligosaccharides and trans-ferulic acid, a double enzyme method, eliminates the alkali treatment step, and simultaneously prepares xylooligosaccharides and trans-ferulic acid. The method uses Aspergillus niger to ferment production of arabinoxylanase and ferulic acid esterase, and uses ferulic acid esterase to break the cross-linking of ferulic acid and sugar, and arabinoxylanase breaks the glycosidic bond to make the two synergistic acting on the scrap to achieve efficient degradation.