The Case for Developing Recombinant Aflatoxin-Detoxifizyme (rADTZ)

Recombinant aflatoxin-detoxifizyme (rADTZ) is an innovative product developed by China Medicine Corporation. The compound is a derivation of an extracellular enzyme, aflatoxin-detoxifizyme (ADTZ), which exhibits the transformation of aflatoxin (AFT). Hence, rADTZ has the potential to be used for detoxifying AFT in food and feed. In addition, rADTZ has shown promising results in treating liver and stomach cancer. This paper will describe the origin, development and history of rADTZ, its scientific basis, and its market potential in the feed, food and health care industries.

I. Introduction to rADTZ

A. The History of rADTZ rADTZ is a recombinant enzyme. The full-length ADTZ gene was cloned from fungi. The development of rADTZ involves several steps, including fungi cultivation, protein purification, gene cloning, gene recombination, and fermentation. China Medicine was able to successfully engineer the rADTZ-producing gene in yeast. The development of rADTZ has taken 10 years and the project was selected as one of the “863” projects that are considered to be key science projects of China. The financing for the project is fully sponsored by the National Science Foundation. The Company has applied for 23 international patents, which are pending approval.

rADTZ is being developed by Co-win Bioengineering in collaboration with Jinan University. Co-win Bioengineering is a majority-owned subsidiary of China Medicine. Dr. Yao from Co-win Bioengineering is spearheading the development of rADTZ. He has more than 13 years of experience in bioengineering. Dr. Yao’s previous positions included Professor of Jinan University and President of the Institute of Microbial Biotechnology, Jinan University. He also served as a member of the Committee of Enzyme Engineering China.

B. The Scientific Basis for rADTZ
ADTZ is an intracellular enzyme that exhibits detoxification activity on Aflatoxin B1 (AFB1). ADTZ is derived from an edible fungus, which is non-toxic to humans and animals. The molecular mass is 51.8 kDa as determined by SDS–PAGE, a technique used to separate proteins according to their electrophoretic mobility. The isoelectric point was estimated to be 5.4 and optimum activity for the aflatoxin-detoxifizyme was found at pH 6.8 and 35°C. The activity of the purified enzyme was confirmed by the Ames test. Under mild conditions, ADTZ could interact with AFB1 to break down the toxic bifuran structure and function as antidote against AFT.

C. Development Process for rADTZ
The development process for rADTZ involves first isolating and purifying ADTZ from fungi. The primers specific to the ADTZ gene are obtained through purification and sequencing. The gene encoding ADTZ is cloned from the total RNA of Armillariella tabescens. The recombinant protein is then expressed and purified through various expression systems using genetic engineering. ADTZ has bioactivity of transforming AFB1, thus reducing the mutagenic effects of AFB1.

D. Potential Applications of rADTZ
rADTZ is the first enzyme to show efficacy in separating and detoxifying AFT in food and feed. It has also shown promising results in treating liver and stomach cancer. Additionally, the enzymatic sensors based on rADTZ can be used by customs, health care professionals, and food and feed production industries for inspection and quality control applications. It is also believed that rADTZ can be used in transgenic technology, such as the development of genetic modified crops with AFT-resistant properties.

1. Feed Industry: rADTZ can be used as an additive to remove the AFT toxin from feed as well as other food-related products, such as dairy products, flavorings, and peanut oil, among others. Because of the Company’s patent protection, rADTZ has many potential applications in the feed industry as well as in the industrial enzyme market.

2. Food Industry: Currently, it is not possible to detoxify AFT-infected milk. Alkali treatment and UV radiation are used to detoxify peanut oil, but these methods may affect the quality of the peanut oil. ADTZ can detoxify AFB1 under specific conditions, and it has no effect on peanut oil quality and storage.

3. Health Care Industry: rADTZ has shown promising signs in the prevention and the treatment of gastric carcinoma, primary hepatocarcinoma (PHC) and lung cancer. The Company intends to develop rADTZ into therapeutic products that target liver, stomach and other cancers.

4. Detection and Genetically Modified Crops: The Company intends to develop rADTZ into an enzyme biosensor for detecting AFT in food and feed as well as for use in custom and sanitation inspection. Additionally, transferring the ADTZ gene to the genome of crops will enable them to develop AFT-resistant properties by expressing the ADTZ gene.

II. Market Characteristics

A. What is Aflatoxin (AFT)?
Aflatoxins are mycotoxins produced by Aspergillus flavus and Aspergillus parasiticus Speare; including Aflatoxin B1, B2, G1, G2, M1 and M2. AFB1 is the most toxic mycotoxin. The toxicity of AFT is 10 times that of hydrocyanic acid and 68 times that of arsenic. In the animal study, an animal can die from acute toxicity when injected with only 0.294mg/kg of AFB1. AFT is not only toxic, but it is also a potent cancer inducing agent. AFT had been classified by the International Agency for Research on Cancer (IARC) as a Group A carcinogen. It could cause gastric carcinoma, liver cancer and lung cancer.

AFT can also be potentially hazardous to the environment. Dr Jiujiang Yu, Research Geneticist from the United States Department of Agriculture (USDA), has conducted studies on the presence of AFT in corn feed and its implications in biofuel conversion to ethanol. He has demonstrated that AFT could be potentially hazardous to the environment when ethanol containing AFT is used. As a result, there will be a need to remove or reduce AFT levels in corn for use in ethanol production. The gene encoding for the ADTZ enzyme can be potentially engineered into fermentation organisms used for ethanol production and hence effectively detoxify AFT from the corn. There is currently no legislation in China governing AFT levels in food and feed.

B. Common Methods to Detoxify AFT
There are currently several methods used to eliminate AFT from feed.

1. The Physical Method: eliminating AFT from feed by hand picking the infected feed, or rinsing or heating. Downside to the physical method: It does not eliminate AFT from feed completely and the feed has shorter reserve time as a result. It is also very easy for secondary contamination to occur.

2. The Chemical Method: eliminating AFT by ammoniation, through clay or zeolite; or alkylation.
Downside to chemical method: The process is expensive and it does not eliminate AFT completely in feed.

3. The Biological Method: lactobacillus fermentation. Downside to biological method: It only works for feed with minor contamination. The treated feed can only be used as a supplement.

C. Potential Markets for rADTZ
1. Feed Industry: Feed production in China totaled 107 million tons in 2005. The country’s entire feed industry is valued at approximately RMB 274.2 billion ($39.8 billion, 1 RMB = $0.145). The total cost of industrial enzymes is roughly 1% of the total cost of feed. Thus, the size of the potential domestic feed market for rADTZ is around RMB 2.7 billion ($392.3 million) per year. In the past five years, the market for industrial enzymes in feed has experienced rapid growth of 11% a year, and is projected to reach RMB 5.5 billion ($800 million) by 2010.

2. Food Industry: The annual production for peanut oil was 2.3 million tons in 2005. The size of the peanut oil industry in China is estimated at RMB 22.5 billion ($3.3 billion). The estimated potential for rADTZ in the domestic peanut oil market is projected to be approximately RMB 220 million ($32 million) per year. The seasoning and fermented food industry in China was approximately RMB 54 billion ($7.8 billion) in 2005 and growing 35% annually. The estimated market size for rADTZ in the domestic seasoning market is approximately RMB 540 million ($78.5 million) per year. The total output of dairy products in China was 23 million tons in 2005. The total dairy industry is estimated to be around RMB 230 billion ($33.4 billion). The projected total market for the Company’s product in the food industry is estimated at RMB 2.3 billion ($334.2 million) per year.

3. Health Care Industry: The prevalence for hepatitis B virus (HBV) carriers in the global population is over 10% and it is approximately 15%-20% in certain regions in China. The number of HBV carriers is about 130 million in China and 600 million worldwide. It is estimated that the total domestic market for rADTZ in the health care industry will be over RMB 10 billion ($1.5 billion) per year.

4. Detection: The projected market for enzyme and AFT biosensor applications is approximately RMB 100 million ($14.5 million) per year. It is estimated that the potential size of the market for genetically modified crop is approximately RMB 690 million ($100 million)

D. Regulation of AFT in Advanced Markets
In well-developed countries, AFT contamination rarely occurs at levels that cause acute ‘aflatoxicosis,’ a poisoning that results from ingestion of aflatoxins in contaminated food or feed. The relative frequency of aflatoxicosis in humans in United States is not known since outbreaks have not been reported. Because aflatoxins are proven carcinogens, the U.S. Food and Drug Administration (FDA) does not permit food products containing more than 20 ppb AFT fit for human consumption. In developed countries in Europe, the exposure rate to aflatoxins is also low due to even stricter European Union (EU) regulation.

1. Feed Industry: Outbreaks of AFT vary among grain producing regions in developed countries and depend on growing conditions within a region. There are years during which aflatoxins appear dormant, and periods when outbreaks can occur. For instance, corn grown in the Southeastern U.S. has had the most frequent occurrence. Drought, insect damage and other stresses on crops are associated with greater occurrence of AFT in corn. Delayed or inadequate drying after harvest and poor storage conditions can contribute to incidences of AFT.

2. Food Industry: The mold that produces AFT can be found in peanuts and peanut butter; tree nuts such as pecans; corn; wheat; and oil seeds such as cotton seed. In the U.S., peanuts and peanut butter are some of the most rigorously tested products by the FDA since they are susceptible to AFT and are widely consumed. In the EU, the European Commission has imposed conditions on importation from specific countries due to possible risk of contamination. These food Items include and peanut products from China and Egypt; pistachios and pistachio products from Iran; dried figs, hazelnuts, and pistachios from Turkey; and unshelled Brazil nuts from Brazil.

III. Conclusion

In summary, rADTZ is a proprietary product developed by China Medicine in collaboration with Jinan University. It has the potential to be the most effective method in removing, detecting and detoxifying AFT from feed and in food. Other potential applications for rADTZ include preventing and treating various cancers, detecting AFT in feed and food as an enzyme biosensor, and developing genetic modified crops.

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[i] Aflatoxin-detoxifizyme Application Development by Guangzhou Co-win Bioengineering Co., Ltd

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