Description

Abstract

Chitosan and chitosan oligosaccharides have aroused research interest due to their excellent biological activities in recent years. The excellent biological activities of chitosan and chitosan oligosaccharides mainly include antioxidant, anti-tumor, anti-bacterial, anti-fungal, anti-obesity, lowering blood pressure, lowering blood lipid, and anti-inflammatory.

The main ways of obtaining chitosan oligosaccharides are enzymatic, chemical and physical methods. The disadvantages of single enzymatic hydrolysis are that the separation and purification of the enzyme is complicated, the enzyme is easily inactivated, and its hydrolysis efficiency is very low. Physical hydrolysis can make the product impure. The chemical method leads to pollution about the environment and products more mixed, and the chitosan oligosaccharides with a certain degree of polymerization cannot be obtained. The enzymes that degrade chitosan mainly include chitosanase, cellulase, lysozyme, protease, and lipase. Chitosanase is derived from bacteria and fungi. Therefore, in the present study, a novel method was proposed to utilize Escherichia coli cell-displayed chitosanase (CHI-1) (The fusion expression of ice crystal nucleoprotein and chitinase) to degrade the co-fermentation of Bacillus subtilis and Acetobacter sp. from shrimp shell waste.

(https://www.collinsdictionary.com/dictionary/english/pill)

Background

1.Waste of Shrimp and crab’s shell

Nowadays, more and more people’s income increases so that they tend to get a higher quality of life. One of the most important improvements is people’s daily diet. Since the better food material they consume, the better meal they receive, and plays more positive roles in their body. Then shrimps and fish become more and more popular in inland cities, which is tough to get seafood in before. This basic seafood has excellent high-quality protein, low calories and contain zero carbs, which really benefit humans. Furthermore, the price of the most shrimp is acceptable for most people. As a result, the requirement for shrimps has become higher and higher in recent years. According to business wire’s report, “In 2018, China's shrimp production reached 1.9569 million tons, with a compound annual growth rate of 1.6% during the forecast period”. The Southern Shrimp Alliance (SSA) also indicated that “The quantity of shrimp imported in 2017 grew to 1.5 billion pounds, a significant 133.6 million pounds more than 2016 imports. Total shrimp import value increased 15 percent.”

(https://farmingaquaponics.com/can-you-eat-shrimp-shells/)

2.Chitin

Chitin is a polysaccharide extracted from the shells of Marine crustaceans with the chemical formula (C8H13O5N) N. Chitosan is pale beige to white, soluble in concentrated hydrochloric acid, phosphoric acid, sulfuric acid and acetic acid, insoluble in alkali and other organic solvents, also insoluble in water. Chitosan, a deacetyl derivative of chitosan, is insoluble in water and soluble in some dilute acids.

(https://en.wikipedia.org/wiki/Chitin)

Chitin can be used in a wide range of industrial fabrics, clothing, dyes, paper and water treatment. It can be used as insecticide and plant antiviral agent in agriculture. Cosmetic agent, hair protection, moisturizer, etc. Medical supplies can be used for contact lenses, artificial skin, sutures, artificial dialysis membranes and artificial blood vessels.

3.Chitosan Oligosaccharide

Chitosan is a kind of oligosaccharide product with polymerization degree between 2 and 20 obtained by special technology degradation. It is a low molecular weight product with good water solubility, great function and high biological activity. It has many unique functions, such as high solubility, complete solubility in water, easy to be absorbed and utilized by organisms, which is not found in chitosan. Chito-oligosaccharides can also regulate intestinal microecology, cover intestinal tissue morphology, enhance immune function, and alleviate hypertension, hyperlipidemia, cancer and other diseases.

Production

IV.The polymer compound obtained by chitin deacetylation is chitosan, and the small molecular oligosaccharides formed by its re-enzymatic hydrolysis are chitosan oligosaccharides, so the method of making chitosan oligosaccharides is how to degrade chitin.

Chemical method	a lot of pollution and the purification process is very complicated
Physical method	guarantee the degree of product polymerization
Enzyme	the cost of enzymatic degradation is high, and the separation of products is problematic.

Project Design

1.Chassis microorganism

E.coli BL21 - This is a common E. coli in the laboratory, good at gene expression. Therefore, the modified gene can be rapidly expressed in the experiment to degrade chitin.

2.Chitinase

II.Chitinase are hydrolytic enzymes that break down glycosidic bonds in chitin. They catalyse the following reaction:
Random endo-hydrolysis of N-acetyl-β-D-glucosaminide (1→4)-β-linkages in chitin and chitodextrins
As chitin is a component of the cell walls of fungi and exoskeletal elements of some animals (including mollusks and arthropods), chitinases are generally found in organisms that either need to reshape their own chitin or dissolve and digest the chitin of fungi or animals.

(https://en.wikipedia.org/wiki/Chitinase)

3.INP and Cell Surface Display

Surface display technology uses genetic engineering technology to link the gene sequence of the target protein or target polypeptide with the gene sequence of the target protein. The target protein or polypeptide sequence is located on the surface of the host by using the localization ability of the targeting protein. The host range mainly covers viruses, Gram-negative bacteria, Gram-positive bacteria and eukaryotic yeast cells.

In our system, ice crystal nucleoprotein and chitinase are fused and expressed on the surface of Escherichia coli. The ice crystal nucleoprotein is anchored to the cell surface, and the other segment is linked to the chitin chain with a peptide chain.

4.How it works

IV.E. coli expressed chitinase and ice crystal nucleoprotein genes (fusion expression). These two proteins will appear on the cell surface, and the ice crystal ribozyme will make the chitinase better fixed on the cell surface. Then the chitinase will degrade chitin to form chitosan oligosaccharides., thereby accelerating its reaction with the cell surface chitinase.
This method has many advantages. First, there are not many pollutants when it is implemented. Second, the cost is controllable and the response is stable, because after we obtain the gene-edited E. coli, it can be put into production only by storage and propagation. Third, this degradation mode allows us to easily separate the products.

Expectation

We believe that this mode of production is more suitable for the trend of modern industrial production, more environmentally friendly and more stable. The product we're looking for should come in the form of tablet sugar. I hope it can be really used in the future, so that more people can be helped.

(https://www.deccanchronicle.com/lifestyle/health-and-wellbeing/211016/
glucose-tablets-likely-better-for-easing-low-blood-sugar-symptoms.html)