Team:NWU-CHINA-B/Description

NWU-CHINA-B

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Ginseng is one of the oldest relict plants on earth. It originated from the "East Asian" and "North American" flora of paleotropical mountain areas in the Tertiary, and the modern distribution center of the genus is mainly located in southwest China.

Ginseng has a long history of application in China. Legend as early as the time of the Ancient Yellow Emperor, the magical effects of ginseng have been known. Shen Nong’s herbal classic (It was written into a book around the Western Han Dynasty 206B.C-A.D24), the earliest Chinese medicine book, records in detail: "Ginseng tastes sweet, mainly nourishes the internal organs, soothes the nerves, calms the soul, stops palpitations, drives away evil spirits, improves eyesight, makes people happy, improves intelligence, long-term use can be light and prolong life" medicinal function.

Yellow Emperor

Shennong Hundred Grass Sutra

During the Eastern Han Dynasty（A.D25-220）, Treatise on Febrile Diseases written by Zhongjing Zhang, a famous doctor, contained 113 prescriptions, 21 of which are related to ginseng, accounting for 18.6 percent of the total. It is also discussed that ginseng has the functions of "warming, nourishing, making man strong, making sperm strong, keeping warm, enhancing vision and stabilizing spirit".

Nowadays, the application of ginseng involves all aspects of our life. Ginseng can be used to dilate skin capillaries, promote skin blood circulation, increase skin nutrition, regulate skin water and oil balance, prevent skin dehydration, hardening, wrinkle, is the perfect for skin care and beauty.
    In addition, ginseng is also suitable for adjusting blood pressure, restoring heart function, neurasthenia and physical weakness and other diseases. Also, it can make expectoration easy, invigorating stomach, facilitate urination, get people excited and so on. It can be used to treat diabetes, cardiovascular disease, neurasthenia and other diseases.

After a full understanding of ginseng, we realized that its pharmacological value is very high, so we have a strong research interest in ginseng, a Chinese herbal medicine with a long history.

After data collection and material analysis of ginseng, we know that the pharmacological activity of ginseng is mainly ginsenosides. Considing this is a triterpenoid saponins linked to sugar chain, ginseng can be divided into the main ginsenosides and rare ginsenosides according to the differences of ginsenoside content in ginseng.
    Compared to the main ginsenosides, rare ginsenosides have the advantages of small size, high bioavailability and strong ability to penetrate cell membranes, so it can be seen that rare ginsenosides show higher biological activity.

Among them, rare ginsenoside CK has excellent biological activities such as treating diabetes, anti-tumor, anti-inflammation and anti-aging. However, CK almost does not exist in natural ginseng, and it is expensive in the market due to its low yield and high production cost.
    Therefore, we considering how to solve the production problems of rare ginsenoside CK, so as to make full use of the pharmacological activity value of ginseng and reduce the market economic cost at the same time.

We have learned that there are still deficiencies in the current production methods of rare ginsenosides. Although physical radiation method and chemical conversion method can improve the yield of rare ginsenosides to a certain extent, it is very bad for the environment, so it is not suitable for large-scale production. However, the microbial transformation method is difficult to strictly control the growth and enzyme production of each batch of microorganisms, so it is difficult to detect its influence on the transformation of ginsenoside. Meanwhile, the experiment is not suitable for us because of its low repeatability. As a result, we chose a new method - enzyme conversion method.

We found that the rare ginsenoside CK can be produced from the main ginsenoside Rb1 by enzymatic transformation, so we successfully expressed β-glucosidase (SS-bgly) derived from leaf sulfosulphide in Pichia pastoris GS115 for the deglycosylation of the Rb1 and CK production.

The target gene SS-bgly optimized by codon was submitted to the company for synthesis, and then we constructed the target gene SS-bgly and E.coli TOP10 into a plasmid containing pPIC9K-SS-bgly.

After the strain had been activated, we extracted the plasmid and used SaI I enzymeto linearize it ,and then transferred to Pichia pastoris GS115. MD plate and colony PCR were used to screen the positive transformants. The successful transformants were screened byshaking flask fermentation toobtain the transformant with high enzyme activity.

The SS-bgly gene sequence was amplified from the previously extracted plasmid pPIC9K-SS-bgly as a template, and was linked to the pPICZαA vector.

E. Coli TOP 10 strain containing pPICZαA-ss-bgly plasmid was successfully constructed. After the strain was activated on the low-salt LB resistance plate, the plasmid was extracted and linearized by restriction endonuclease Sac 1 enzyme.

The plasmid was transferred to the previously constructed GS115-pPIC9K-ss-bgly strain. The transmutants were preliminarily screened by resistance plate and colony PCR with different concentration gradients. Strain GS115/9K-ZαA-ss-bgly was screened by shaking flask fermentation.

After that, the constructed strain was fermented, and the concentrated crude enzyme was obtained through salting out, dialysis and ultrafiltration.

The enzymatic reaction was carried out in sodium acetate buffer, and a series of optimizations were made on the condition temperature, pH, metal ion concentration and substrate concentration of enzymatic reaction. Finally, we verify our experimental hypothesis.