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Breeding of high-yield low-temperature lipase fungi, optimization of fermentation conditions and isolation and purification of lipase

This study successfully bred a high-yield *Aspergillus niger* mutant via mutagenesis and fermentation optimization to produce a purified low-temperature lipase with superior catalytic activity at 15°C, offering a cost-effective solution for energy-efficient industrial applications.

Original authors: Lutong Yang, Mengai Wang, Hongyan Fu, Yun Zhang, Yu Zhang, Mengni Cui, Bowen Jiang, Hong Chen, Yanwei Cui

Published 2026-06-29

📖 5 min read🧠 Deep dive

CC BY 4.0

Original authors: Lutong Yang, Mengai Wang, Hongyan Fu, Yun Zhang, Yu Zhang, Mengni Cui, Bowen Jiang, Hong Chen, Yanwei Cui

Original paper licensed under CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/). ⚕️ This is an AI-generated explanation of a preprint that has not been peer-reviewed. It is not medical advice. Do not make health decisions based on this content. Read full disclaimer

The Big Picture: Finding a "Cold-Weather Worker"

Imagine you have a factory that needs to work, but the building is freezing cold. Most workers (enzymes) are like humans: if it's too cold, they slow down, shiver, and stop working efficiently. They need a heater to get going.

This research paper is about finding and training a special kind of "worker" (a fungus) that loves the cold. The team wanted to create a low-temperature lipase. Think of lipase as a pair of molecular scissors that cuts up fats (like grease or oil). Usually, these scissors only work well in hot water. The scientists wanted scissors that could snip away grease even in a cold sink, saving energy because you wouldn't need to heat the water.

Step 1: The Treasure Hunt (Screening)

The researchers went to the Daxing'anling forest in Heilongjiang, China. This is a place where the winter is brutal, dropping to -52°C. They dug up soil samples, hoping to find a fungus that had already adapted to this extreme cold.

The Analogy: Imagine looking for a fish that can survive in an ice cube. They took soil, mixed it with food (olive oil), and shined a special UV light on it.
The Result: Most fungi didn't glow. But one fungus, which they named Y-A02, glowed brightly with an orange halo. This glow meant it was successfully "cutting" the oil. They identified this fungus as a type of Black Mold (Aspergillus niger).

Step 2: Super-Charging the Worker (Mutagenesis)

The original fungus was good, but the scientists wanted it to be great. They used a technique called mutagenesis, which is like giving the fungus a controlled "vaccination" of stress to force it to evolve.

The Analogy: Think of the fungus as a student. First, they gave the student a little bit of "sunburn" (Ultraviolet light) to wake up their brain. Then, they gave them a specific chemical "quiz" (Nitrosoguanidine) to force them to study harder.
The Result: They created thousands of mutant versions of the fungus. They looked for the one that glowed the brightest. They found a "super-star" mutant named Y-B04. This new version produced 127% more fat-cutting scissors than the original. It was like finding a worker who could do double the job in the same amount of time.

Step 3: Perfecting the Environment (Fermentation)

Even a super-worker needs the right office to perform well. The scientists tweaked the "office conditions" (the liquid the fungus grows in) to get the maximum amount of scissors.

The Analogy: They tried different temperatures, pH levels (acidity), and amounts of "food" (sugar and protein).
The Sweet Spot: They found that the fungus worked best at 21°C (a cool room temperature), with a slightly acidic environment (pH 6.0), and a specific amount of "seeds" (spores) to start the party.
The Outcome: Under these perfect conditions, the fungus produced a massive amount of lipase: 57.26 units per milliliter.

Step 4: Cleaning Up the Product (Purification)

The liquid coming out of the fermentation tank was like a smoothie full of fruit, but also full of seeds, skins, and pulp. They needed just the "fruit" (the pure lipase).

The Analogy: They used a three-step cleaning process:
1. Salting Out: They added salt to the mix. This made the "bad stuff" clump together and sink, while the good lipase stayed floating.
2. The Magnet (Chromatography): They ran the liquid through a special column that acted like a magnet, grabbing the lipase and letting the rest pass through.
3. The Sieve (Gel Filtration): Finally, they ran it through a tiny sieve to separate molecules by size.
The Result: They ended up with a very pure, high-quality product. The "scissors" were now 14 times more powerful per gram than when they started.

What Makes This Lipase Special?

The scientists tested their new "Cold-Worker" scissors and found some amazing traits:

Cold Adaptation: While most scissors need to be heated to 40°C or higher to work, these work best at 30°C and are still very active at 15°C. They are happy in the cold.
Fragile but Fast: Because they are built for the cold, they are a bit delicate. If you heat them up too much (above 40°C), they break down quickly. But in the cold, they are incredibly efficient.
Favorite Foods: They prefer cutting up "medium-length" fat chains (like C8 to C12), which are common in many natural oils.
Metal Reactions: Some metals (like Calcium) made them work even faster, while heavy metals (like Copper or Zinc) acted like poison and stopped them.

The Bottom Line

The paper claims that by finding a fungus in a freezing forest, giving it a genetic "push," and feeding it the perfect diet, the team created a highly efficient, cold-loving enzyme.

What the paper says it can be used for:
The authors specifically mention that this enzyme could be used in:

Food processing: Handling heat-sensitive foods without cooking them.
Low-temperature washing: Detergents that work in cold water (saving energy).
Environmental governance: Cleaning up oil spills or waste in cold environments.

The study provides a blueprint for how to make these enzymes in large quantities, solving the problem of high costs and low activity that usually comes with cold-weather enzymes.

Technical Summary: Breeding of High-Yield Low-Temperature Lipase Fungi, Optimization of Fermentation Conditions, and Isolation and Purification of Lipase

Problem Statement
Conventional lipases often possess elevated optimal catalytic temperatures, which can lead to substrate inactivation and product degradation when processing heat-sensitive materials. This limitation results in reduced biotransformation efficiency and compromised yields. While low-temperature lipases (active below 40°C, with optima between 0–30°C) offer a solution for eco-friendly, energy-efficient industrial processes, they frequently suffer from low activity, high production costs, and limited availability. This study addresses the need for an efficient, high-yield production system for low-temperature lipases to support applications in food processing, low-temperature washing, and environmental governance.

Methodology
The research followed a multi-stage approach involving strain screening, mutagenesis breeding, fermentation optimization, and enzyme purification:

Strain Screening: Soil samples were collected from the cold forest regions of the Daxing'anling area in Heilongjiang Province (annual mean temperature -4°C to -2°C). Initial screening utilized Rhodamine B lipid-screening medium to identify fluorescent halos, followed by re-screening using the p-nitrophenyl butyrate method to quantify lipase activity.
Strain Identification: The selected strain, designated Y-A02, was identified via PCR amplification of the ITS region and phylogenetic analysis, confirming it as Aspergillus niger.
Mutagenesis Breeding: To enhance lipase yield, the study employed a compound mutagenesis strategy combining Ultraviolet (UV) radiation and nitrosoguanidine (NTG).
- UV Treatment: Spore suspensions were exposed to 254 nm UV light for varying durations (0–160 s). A lethality rate of ~82.9% at 120 s was selected as optimal.
- NTG Treatment: Spores were treated with varying NTG concentrations (0.05–0.30%). A concentration of 0.15%, yielding a 78.9% lethality rate, was selected.
- Compound Mutagenesis: Strains underwent 120 s UV exposure followed by 0.15% NTG treatment. Mutants with larger fluorescent halos were selected for further evaluation.
Fermentation Optimization: Single-factor experiments determined the effects of temperature, pH, inoculum size, nitrogen sources, and carbon sources. Subsequently, a Box-Behnken response surface methodology (RSM) was used to optimize three critical factors: temperature, pH, and inoculum size.
Purification: The lipase was purified from the fermentation supernatant of the mutant strain (Y-B04) using a sequential protocol: ammonium sulfate fractional precipitation (30% and 60% saturation), dialysis, Q-HP anion exchange chromatography, and Superdex 75 gel filtration chromatography.
Characterization: The purified enzyme's properties were evaluated, including optimal reaction temperature and pH, thermal and pH stability, effects of metal ions and organic solvents, substrate specificity, and kinetic parameters ( $K_m$ and $V_m$ ).

Key Results

Strain Improvement: The mutant strain Aspergillus niger Y-B04, derived from the parent strain Y-A02 via UV-NTG compound mutagenesis, demonstrated a 127.35% increase in lipase production compared to the original strain.
Genetic Stability: Strain Y-B04 exhibited excellent genetic stability over five consecutive generations, with lipase activity fluctuating only within a narrow range (48.45–50.21 U/mL).
Fermentation Optimization: Through RSM, the optimal fermentation conditions were determined to be 21°C, pH 6.0, and an inoculum size of 6.5×10⁶ spores/mL. Under these conditions, the lipase production reached 57.26 U/mL, closely matching the model's predicted value (58.06 U/mL) with a deviation of only 1.4%.
Purification and Specific Activity: The purification process resulted in a 14.15-fold increase in specific activity, reaching 113.52 U/mg. The final preparation showed a single band of approximately 36 kDa on SDS-PAGE.
Enzymatic Properties:
- Temperature: The optimal reaction temperature is 30°C. The enzyme retains over 60% activity at 15°C and over 80% activity between 20–40°C. It is thermally unstable above 30°C, retaining less than 20% activity after 1 hour at 50°C.
- pH: Optimal activity occurs at pH 7.0, with over 80% relative activity maintained between pH 6.0 and 7.0.
- Stability: The enzyme is stable at 4°C and 20°C for 5 hours but loses activity rapidly at higher temperatures.
- Influence of Additives: Ca²⁺, Na⁺, and K⁺ (at 1 mM) activated the enzyme, while heavy metal ions (Fe³⁺, Mn²⁺, Zn²⁺, Cu²⁺) and Mg²⁺ significantly inhibited activity. Most organic solvents inhibited the enzyme, though 10% methanol enhanced activity by 18.41%.
- Specificity: The lipase showed highest activity toward medium-chain substrates (C8-C12), with C8 (p-NPC8) exhibiting 100% relative activity.
- Kinetics: The enzyme displayed a Michaelis constant ( $K_m$ ) of 19.86 mmol/L and a maximum reaction rate ( $V_m$ ) of 0.178 mmol/(L·min).

Significance and Claims
The authors claim that this study provides a scientific basis and feasible solutions for the large-scale production of low-temperature lipases. By successfully isolating a high-yield Aspergillus niger strain and optimizing its production through mutagenesis and fermentation control, the research offers a microbial source capable of efficiently producing enzymes that function effectively at low temperatures.

The study highlights that the resulting lipase can reduce energy consumption and operating costs in industries such as food processing, low-temperature washing, and environmental remediation. Compared to commercial lipases, the developed enzyme offers a shorter reaction time, minimal impact on substrate quality, and a reduced risk of thermal damage to heat-sensitive products. The authors position this work as a contribution to the resource pool of low-temperature lipase-producing bacteria, demonstrating that mutation breeding combined with refined fermentation and purification control can significantly enhance both the yield and performance of these enzymes.

The paper acknowledges limitations, noting that while the strain is stable under laboratory conditions, industrial scalability requires further evaluation regarding production costs, process feasibility, and long-term strain preservation. Future work is proposed to explore multi-omics technologies to understand molecular mechanisms and to conduct protein engineering for further performance enhancement.