Jiangnan University

Wenjing Cui

Li Zhou

Zhemin Zhou

Zhongmei Liu

Junling Guo

Yu Yan

Laichuang Han

Yuanyuan Xia

Chao Wang

Bo Pang

Ting Li

Zhongyi Cheng

Wenliang Hao

Yao Lan

Wenqi Ye

Ting Xie

Long Yu

Yufeng Liu

Dezhi Chen

Sailan Zhang

The present disclosure discloses a thermophilic recombinant type II pullulanase and the application thereof, and belongs to the technical field of genetic engineering. The present disclosure obtains a thermophilic recombinant type II pullulanase by heterologously expressing type II pullulanase in. Its optimum pH is 6.6, it has better pH tolerance under the conditions of pH 5.8-8.0, and its optimum temperature is 95° C. After incubating at 95° C. for 10 h, the remaining enzyme activity is greater than 50%. It can exhibit higher specific enzyme activity under strong reducing conditions. For example, adding DTT to the culture environment can increase the specific enzyme activity of Sumo-Pulby 237.2%. The present disclosure also provides the combined truncation mutant Δ28N+Δ791C of type II pullulanase Sumo-Pul. The specific enzyme activity of the enzyme mutant is 32.18±0.92 U/mg, which is 5.99 times as high as that of the wild-type enzyme, thereby having important industrial application value and potential.

Thermophilic recombinant type II pullulanase and application thereof

The present disclosure discloses a method for constructing an efficientpromoter, and belongs to the technical field of gene engineering. According to the present disclosure, natural promoters identified by different sigma subunits are connected in series to obtain some double-series and triple-series promoters, the lengths of intervening sequences between core areas of the promoters are optimized on the basis of series connection of the promoters to further improve the activity of the promoters, finally, different RBS designs are performed on the promoters, and it is verified that this strategy can not only improve the compatibility between the promoters and other gene expression regulating and controlling elements, but also controllably regulate the expression of exogenous genes. Through the method provided by the present disclosure, people can obtain the promoters with higher activity and stronger designability and compatibility through simple and convenient promoter design and modification methods. The method is simple and easy to implement and has wide application prospects in the construction of an exogenous protein efficient expression system and synthetic biology research.

Method for constructing efficientpromoter

The disclosure discloses a mutant of nitrile hydratase derived from, and belongs to the technical field of enzyme engineering. The nitrile hydratase mutantNhase-A20V provided by the disclosure has a half-life of about 10 min at 70° C., which does not change much compared with the thermal stability of the wild enzyme. The specific enzyme activity of the mutantNhase-A20V is 128% of that of the wild enzyme. At the same time, the mutant also has better tolerance to a substrate and a product, and the final yield of nicotinamide produced by whole-cell catalysis reaches 598 g/L. Therefore, the nitrile hydratase mutantNhase-A20V provided by the disclosure has good enzymatic properties and is beneficial to future industrial production.

Mutant of nitrile hydratase derived from

The present disclosure discloses aefficiently-induced expression system based on an artificial series promoter, and belongs to the technical field of gene engineering. According to the present disclosure, an efficient artificial series constitutive promoter is used, and by compositely designing the promoter and elements (a repressor and a binding site thereof) relevant to an operon, the activity of the constitutive promoter is regulated and controlled by an inducer to finally construct theefficiently-induced expression system induced by the inducer. The result indicates that compared with a Pstrongly constitutive promoter, the activity of the artificial series promoter in the system is about 15 times higher. The activity of the promoter can be accurately controlled by adding different concentrations of inducers. Therefore, the efficient expression system is simple in structure, high in activity and strict in regulating and controlling and has wide application prospects in heterologous protein efficient expression and synthetic biology research.

Bacillus subtilis efficiently-induced expression system based on artificial series promoter

Disclosed is a double enzyme tandem preparation method of L-2-aminobutyric acid, and belongs to the field of bioengineering. In the disclosure, recombinantexpressing L-glutamate mutase and recombinantexpressing L-aspartate-β-decarboxylase are separately cultured to obtain L-glutamate mutase and L-aspartate-β-decarboxylase. The two enzymes are added to a reaction system at a certain mass ratio, and L-glutamate is used as a substrate to carry out an enzyme reaction to prepare the L-2-aminobutyric acid. When the dosage of the L-aspartate-β-decarboxylase is 2 mg/mL, and the reaction time is 24 h, 8.5 mmol/L L-2-aminobutyric acid is produced by conversion, with a molar conversion rate of 85.00%. Compared with a chemical production method, the method disclosed by the disclosure has a safe production process and no environmental pollution. Compared with a multi-enzyme synthesis system with threonine as a substrate, the substrate is cheaper and the process is simpler.

Double enzyme tandem preparation method of l-2-aminobutyric acid

The disclosure discloses an L-aspartate &#x3b1;-decarboxylase mutant and application thereof, and belongs to the technical field of enzyme engineering. In the disclosure, lysine at position 221 of L-aspartate &#x3b1;-decarboxylase is mutated to arginine, glycine at position 369 is mutated to alanine, and the obtained new mutant enzymes have better temperature tolerance and are beneficial to industrial production. The K221R and G369A recombinant strains are subjected to high-density fermentation, and with sodium L-aspartate as a substrate, a whole cell catalytic reaction is carried out to prepare &#x3b2;-alanine. Compared with a chemical production method, the method has the advantages that the production process is safe and clean, and has no environmental pollution. Compared with a pure enzyme catalysis method, the method has the advantages that the operation is simple and convenient. The yield of the final product &#x3b2;-alanine reaches 91% and 90% respectively, and the concentration reaches 162.15 g/L and 160.42 g/L respectively.

L-aspartate alpha-decarboxylase mutant and application thereof

The disclosure discloses a nitrile hydratase mutant, a genetically engineered bacterium containing the mutant and applications thereof, and belongs to the technical field of enzyme engineering. In the disclosure, glycine at position 47 of a nitrile hydratase mutant &#x3b1;L6T/A19V/F126Y-&#x3b2;M46K/E108R/S212Y (disclosed in the patent of disclosure CN102216455A) is mutated to asparagine. The obtained new mutant enzyme has better temperature tolerance and tolerance to a product, and is conducive to future industrial production. The recombinant strain containing the nitrile hydratase mutant is fermented at high density, and 3-cyanopyridine is used as a substrate to carry out a whole-cell catalytic reaction to prepare nicotinamide. Compared with a chemical production method, the method has a safe and clean production process and no environmental pollution. Compared with an enzymatic method, the substrate price is cheap and the catalytic efficiency is high. The yield of the final product nicotinamide is over 95%, the concentration reaches 680 g/L, and the separation and purification steps of the product are simplified.

Nitrile hydratase mutant, genetically engineered bacterium containing mutant and applications thereof

The invention relates to the technical field of bioengineering, and discloses a method for synthesizing L-aspartic acid with maleic acid by whole-cell biocatalysis. In the invention, a recombinant strain co-expressing maleate cis-trans isomerase and L-aspartate lyase is constructed, and engineered and optimized to produce L-aspartic acid from maleic acid with a high conversion rate by whole-cell catalyzing. Relatively inexpensive maleic acid is utilized by the recombinant strain to produce L-aspartic acid, where maleic acid is reacted completely in 40-120 min, there is almost no buildup of the intermediate fumaric acid, and the conversion rate is up to 98% or more.

Method for preparing L-aspartic acid with maleic acid by whole-cell biocatalysis

The present invention provides a fused NHase with improved specific activity and stability, which relates to the field of genetic engineering. This invention provides a method of overexpressing a fused NHase inand producing a mutant NHase with improved stability and product tolerance. The invention provides a simple, efficient and safe method of making mutant NHase, and can produce a large amount of soluble NHases in a short period. The present invention makes a contribution to large-scale industrial production and further theoretical study of NHases.

Fused NHase with improved specific activity and stability

The present invention provides a fused NHase with improved specific activity and stability, which relates to the field of genetic engineering. This invention provides a method of overexpressing a fused NHase inand producing a mutant NHase with improved the stability and product tolerance. The invention provides a simple, efficient and safe method of making mutant NHase, and can produce a large amount of soluble NHases in a short period. The present invention makes a contribution to large-scale industrial production and further theoretical study of NHases.

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Wuxi, China

Wenjing Cui