Brown algae oligosaccharides are functional oligosaccharides obtained by subjecting alginates to specific enzymatic or chemical cleavage reactions. They exhibit a wide range of biological activities, including antitumor, anti-inflammatory, immunomodulatory, and lipid-lowering effects. As a result, they hold great potential for applications in fields such as pharmaceuticals, health supplements, cosmetics, animal feed, and plant growth regulators. Traditionally, brown algae oligosaccharides have been prepared by using alkaline solutions to degrade large brown algae rich in alginates. However, this method not only damages the active components in brown algae but also causes significant environmental pollution. Moreover, brown algae oligosaccharides produced via alkaline hydrolysis have unstable degrees of polymerization, which in turn leads to inconsistent efficacy.

In December 2017, Hanhe Biology collaborated with the Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, to develop a research and development project focused on alginate lyase and the preparation of alginate oligosaccharides. Through this research, a novel bifunctional alginate lyase with alkali tolerance was identified and characterized. This enzyme exhibits high activity against both poly-mannuronic acid (polyM) and poly-guluronic acid (polyG) in alginates, enabling complete degradation of alginates into main products such as alginobiose, alginotriose, and alginotetraose. Furthermore, by modifying the protein’s structural domains, the enzyme’s enzymatic activity and thermal stability were significantly enhanced, endowing it with strong potential for industrial-scale applications.

Today, the author will reveal to you, step by step, how to screen strains that produce alginate lyase, how to screen and transform enzyme genes and construct engineered strains, how to optimize fermentation processes, and how to optimize the enzymatic preparation of alginooligosaccharides.

I. Screening Microorganisms That Produce Alginate Lyase

Brown Alginate lyases are primarily found in marine algae, marine mollusks, echinoderms, and various microorganisms. We have screened a variety of alginate lyases from marine and terrestrial bacteria, mollusks, and algae plants, identifying more than 100 strains capable of producing alginate lyases. Subsequently, we further selected the strain with the highest alginate lyase enzyme activity from among these producer strains for detailed analysis.

✷ Preliminary screening of alginate lyase activity: The larger the clear zone, the stronger the activity.

II. Screening of alginate lyase genes and construction of genetically engineered strains

The highly active strains that have been screened will undergo genome sequencing. By investigating the mechanisms of enzyme translation and transport processing, we will develop a set of molecular components mediated by molecular chaperones and proteases, establishing a control system for the self-assembly and maturation of multi-domain alginate lyase proteins. We will then engineer an efficient tool enzyme—alginate lyase—for the high-yield preparation of alginate oligosaccharides with high specificity, excellent hydrolytic efficiency, and controllable molecular weight and structure. To achieve this, we will ligate the genes encoding the screened alginate lyase into expression vectors (plasmid DNA), construct recombinant expression plasmids, transform Bacillus subtilis host cells, and screen for positive clones, thereby establishing the genetically engineered strain capable of producing alginate lyase.

3. Optimize the fermentation process for alginate lyase to enhance enzyme activity.

Compared with other similar enzymes, the alginate lyase exhibits an enzyme activity more than 40 times higher than that of the currently available sigma-reagent-type alginate lyase. It features high salt tolerance—withstanding 15% NaCl—and exceptional stability, maintaining its full enzyme activity for one year when stored at 4°C without any loss. Additionally, it displays substrate specificity with targeted cleavage. Hanhe Bio has developed a Bacillus subtilis fermentation enzyme preparation technology (genetically engineered strain fermentation technology) with an enzyme activity exceeding 40,000 U/mL. Optimized to reach the global highest level certified by the Ministry of Science and Technology’s novelty search.

✷ On the left is kelp without any bacteria; on the right is kelp after bacterial inoculation, in which the highly active alginate lyase has already degraded the kelp into a slurry.

✷2L Four-fermentation-tank system

Compared to other microbial fermentations, Bacillus subtilis grows rapidly, has a short fermentation cycle for enzyme production, and employs secretory expression, making its post-enzyme processing relatively simple. In contrast, Escherichia coli fermentation for enzyme production requires cell disruption and involves complex operations, while Pichia pastoris fermentation for enzyme production has a long cycle and high costs.

4. Production of brown algae oligosaccharides by enzymatic hydrolysis of algal polysaccharides (alginic acid and alginates) using alginate lyase.

Currently, most commercial processes for preparing alginate oligosaccharides rely on chemical methods. These methods suffer from difficulties in controlling reaction conditions, poor functional activity, low product recovery rates, and environmental unfriendliness. Hanhe Bio, in collaboration with the Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, uses alginate lyase to cleave alginic acid and produce algal oligosaccharides. The main products are algal disaccharides, trisaccharides, and tetrasaccharides, which exhibit stable degree of polymerization and high activity.

✷ A shows the time-course analysis of alginate oligosaccharides produced after enzymatic hydrolysis by alginate lyase; B shows the relative content of alginate oligosaccharides.

Brown algae oligosaccharides prepared by enzymatic hydrolysis using alginate lyase, It features rapid reaction speed, mild reaction conditions, and a short enzymatic hydrolysis cycle. The degree of polymerization of brown algae oligosaccharides can be controlled to ensure that the proportion of dimers to tetramers exceeds 75%, with molecular weights concentrated between 414 and 810. When combined with strong acids or bases (pH: 3–10), calcium, and magnesium, and subjected to high temperatures above 150°C, it exhibits no flocculation or precipitation. It has low addition costs and high activity.

V. Functions of Brown Algae Oligosaccharides

1. Activating the SA (salicylic acid) and JA (jasmonic acid) immune systems can help resist bacterial and viral infections, thereby reducing the need for pesticide use.

2. Induces plants to synthesize ABA (increasing abscisic acid content by 5 times) and JA (jasmonic acid), enhancing plant stress resistance, improving fruit coloration and sweetness, and promoting early maturation and early market availability.

3. Induces plants to synthesize IAA (increasing auxin levels by 8 times), promoting rapid root formation and germination, and accelerating overall growth.

4. Adding alginate oligosaccharides gives the product plant vaccine-like efficacy.

Algal oligosaccharides (AOS) have been shown to... It is an important signaling molecule in plants that promotes plant growth, enhances plant resistance to diseases, strengthens plants' adaptability to environmental conditions, and improves crop yield and quality. Also Bifidus factor, which promotes the growth of human endothelial cells and keratinocytes, possesses unique biological activities, including antitumor, anti-inflammatory, anticoagulant, antioxidant, immunomodulatory effects, as well as the ability to lower blood lipids and blood glucose levels. Therefore, it can be used in both pharmaceuticals and health foods.

✷ The left image shows a pharmaceutical product whose core ingredient is alginate oligosaccharides; the right image shows a research paper titled: “The Promoting Effect of Alginate Oligosaccharides on Rice Root Development Under Auxin Signaling.”