CN109852601B - N-glycosylation alginate lyase mutant capable of being efficiently applied and construction method of genetic engineering bacteria - Google Patents

N-glycosylation alginate lyase mutant capable of being efficiently applied and construction method of genetic engineering bacteria Download PDF

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CN109852601B
CN109852601B CN201910159781.1A CN201910159781A CN109852601B CN 109852601 B CN109852601 B CN 109852601B CN 201910159781 A CN201910159781 A CN 201910159781A CN 109852601 B CN109852601 B CN 109852601B
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李恒
史劲松
许正宏
龚劲松
郝瑶
李星霖
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Jiangnan University
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Abstract

The invention discloses an N-glycosylation alginate lyase mutant capable of being efficiently applied and a construction method of a genetic engineering bacterium, belonging to the technical field of biology. On the basis of alginate lyase Aly-Cob, DQNAT repeated sequence fragment is introduced at the N end of the alginate lyase to be used as an N-glycosylation site, and simultaneously, a carrier protein YebF is fused and expressed to carry out extracellular secretion, so that a glycosylation system pgl from Campylobacter jejuni uses a sugar chain GalNAc 2 (Glc)GalNAc 3 Bac is glycosylated. The obtained mutant enzyme NACob has improved enzyme activity and stability compared with those before glycosylation. The N-glycosylation modified alginate lyase mutant provided by the invention can be widely applied to the fields of food, medicine, agriculture and the like related to seaweed processing and utilization.

Description

N-glycosylation alginate lyase mutant capable of being efficiently applied and construction method of genetic engineering bacteria
Technical Field
The invention relates to a glycosylation modified marine alginate lyase and application thereof, belonging to the technical field of biology. The algin lyase provided by the invention can be widely applied to the fields of food, medicine, agriculture and the like of seaweed processing and utilization.
Background
The alginate oligosaccharide is a high value-added oligosaccharide which is generated by degrading algin and has multiple biological activities of resisting tumor, regulating immunity, reducing blood sugar and blood fat and the like, and has important application in the fields of food, medicine, feed and the like. The process of preparing the alginate oligosaccharide by enzymolysis by taking alginate lyase as a tool enzyme has the advantages of mild reaction conditions, controllable process, high yield and the like, and is gradually the main mode for preparing the alginate oligosaccharide.
At present, various microorganisms for producing alginate lyase are separated from marine animals and plants and environmental samples, and are rich in types and diverse in performance. Most of the enzyme-producing microorganisms have the characteristics of special growth conditions, low enzyme production amount and the like because of being obtained from marine samples, and the development and application of the enzyme are greatly limited. In order to overcome the defects of the existing alginate lyase, a plurality of enzyme production genes are constructed through gene engineering bacteria, so that exogenous expression is realized, the culture conditions are simplified, and the enzyme production amount is improved. But the problems of low expression level, low enzyme activity and the like still exist generally. Further selection or improvement of the methods of engineering of enzymes is needed to further increase the potential of the enzymes for use.
Glycosylation is a post-translational modification process of proteins which is widely existed and occurs in organisms, and comprises N-position glycosylation, O-position glycosylation, C-position mannosylation and GPI (glyco phosphono diacylglycerol) anchoring connection, wherein the N-position glycosylation is most common. Researches show that N-glycosylation has obvious influence on the aspects of the structure, the function, the stability, the interaction and the like of the enzyme, is an effective means for artificially carrying out enzyme post-modification, and has wide application in various fields of enzyme modification, protein medicines, antibody engineering and the like. In recent years, with the continuous and deep research on the N-glycosylation modification of Campylobacter jejuni (Campylobacter jejuni) protein, the reconstruction of prokaryotic glycosylation system by using Escherichia coli provides conditions for glycosylation modification of enzyme and controllable preparation of glycoprotein. The Escherichia coli has the characteristics of clear genetic background, fast growth, simple operation and the like, and is an ideal host for expression and modification of the enzyme. The Escherichia coli system is used for glycosylation modification of the enzyme, so that the catalytic performance of the enzyme is improved, the stability of the enzyme is improved, and the method has important theoretical research value and application prospect for modification and application of the enzyme.
Disclosure of Invention
The invention aims to provide an N-glycosylation alginate lyase mutant which can be efficiently applied and a construction method of a genetic engineering bacterium.
The mutant is characterized in that on the basis of alginate lyase Aly-Cob, a carrier protein YebF is introduced at the N end of the mutant for fusion expression, and a N multiplied by DQNAT repeated sequence segment (N is 1-10) is introduced as N-glycosylThe glycosylation site is co-expressed with pgl gene cluster of the source campylobacter jejuni glycosylation modification system to form a sugar chain GalNAc 2 (Glc)GalNAc 3 Bac-modified mutant NACob.
The mutant, in one embodiment of the invention, introduces carrier protein YebF and 1 xDQNAT sequence at the N-terminal of Aly-Cob, and the modified algin lyase is named NACob1 through mutation, and the amino acid sequence of the modified algin lyase is shown in SEQ ID No. 1.
SEQ ID No.1：
Figure BDA0001984191550000011
Figure BDA0001984191550000021
In one embodiment of the invention, the mutant is characterized in that a carrier protein YebF and a 4 xDQNAT sequence are introduced into the N end of Aly-Cob, the modified alginate lyase is named NACob4 through mutation, and the amino acid sequence of the modified alginate lyase is shown in SEQ ID No. 2.
SEQ ID No.2：
Figure BDA0001984191550000022
Figure BDA0001984191550000031
The amino acid sequence of the Cobeta sp-source alginate lyase before N-glycosylation mutation is shown in SEQ ID No.2, and the alginate lyase sequences from different Cobeta sp strains have only individual amino acid differences, so that the method is not only suitable for the SEQ ID No.2 sequence, but also suitable for alginate lyase sequences with the amino acid sequence similarity of more than 80 percent.
The expression vector is one of pET28a (+), pET24b, pACYC, pMAF10 and pSP 73.
The genetic engineering bacteria are constructed by taking escherichia coli as a host, and can be any one of E.coli BL21 (DE 3), CLM24 and CLM 37.
In one embodiment of the present invention, the construction method of the genetically engineered bacterium is: constructing recombinant plasmids by the plasmid pTrc, the vector protein YebF and the glycosylated DQNAT repetitive sequence segment, connecting and coding the nucleotide sequence of the alginate lyase after enzyme digestion, and jointly electrotransfering the plasmid and the pACYCpgl plasmid containing a glycosylation system into E.coli BL21 (DE 3) competent cells for expression and glycosylation.
The invention provides a method for improving stability of alginate lyase, which is to perform N-glycosylation mutation and modification on the N end of the alginate lyase.
The invention has the beneficial effects that:
the algin lyase modified by N-glycosylation mutation has the advantage that the expression of the enzyme and the growth of thalli are not influenced by N-glycosylation. After fermentation culture is carried out for 24 hours, the enzyme activity is improved by more than 7 percent, and the temperature stability at 40 ℃ is improved by more than 15 percent.
The algin lyase of the invention can be widely used in the fields of foods, medicines, chemical industry, agriculture and the like related to seaweed processing.
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FIG. 1: comparison of temperature stability of alginate lyase before and after glycosylation
Detailed Description
Example 1: n-glycosylation site-introduced mutant enzyme NACob1 and construction of engineering bacteria
The invention introduces N-glycosylation site 1 XDQNAT at the N-end design of alginate lyase Aly-Cob, and the N-glycosylation site and carrier protein YebF are fused and expressed, and after the fusion expression and glycosylation pglB plasmid are co-expressed, the alginate lyase NACoOB 1 (the amino acid sequence is shown as SEQ ID NO. 1) modified by N-glycosylation is generated.
The specific method comprises the following steps:
(1) Plasmid pTrc99A is subjected to double enzyme digestion by XbaI and HindIII, coding glycosylation sites DQNAT and a 6 XHis gene sequence are introduced, a recombinant plasmid is constructed, the recombinant plasmid is subjected to double enzyme digestion by XbaI and SalI, a YebF (NCBI: B1847) gene sequence is inserted to form a pTrc-YebF-gly-His plasmid, and after double enzyme digestion by XbaI and EcoR I, a Aly-Cob coding gene is inserted to form a recombinant vector pTrc-YebF-gly-Aly-Cob-His;
(2) The constructed expression plasmid and pACYCpgl plasmid carrying campylobacter jejuni N-glycosylation gene cluster are transformed to escherichia coli E.coli CLM24 for co-expression by electric shock, then a transformant is inoculated into LB culture medium containing chloramphenicol (20 mu g/mL) and trimethoprim (100 mu g/mL), after being cultured for 16-20h, isopropyl thiogalactoside (IPTG, 0.01 mM) is added for induction expression for 24h;
(3) The fermentation supernatant was collected by centrifugation, and recombinase NACob1 was purified by a nickel column.
Example 2: n-glycosylation site-introduced mutant enzyme NACob4 and construction of engineering bacteria
The procedure was similar to example 1, except that the glycosylation site was 4 × DQNAT, and the resulting mutant enzyme was named NACob4 (amino acid sequence shown in SEQ ID NO. 2).
Example 3: comparison of enzyme Activity before and after glycosylation modification
The alginate lyase activity in the fermentation supernatant was determined by the DNS method (Table 1). After glycosylation modification, the activity of the alginate lyase is slightly improved, the enzyme activity of NACob4 is NACob Aly-Cob, and the enzyme activity of NACob4 is improved by 12.6%.
TABLE 1 alginate lyase enzymatic Activity before and after glycosylation modification
Figure BDA0001984191550000051
Example 4: comparison of enzyme stability before and after glycosylation modification
And (3) respectively preserving the heat of the enzyme solution before and after glycosylation modification at 40 ℃ for 30min, measuring the residual enzyme activity, and defining the highest enzyme activity as 100%. Sampling every 10min to determine the residual enzyme activity of the sample. Finally, it was confirmed that the stability of alginate lyase, especially NACob4, was improved by 26% compared to Aly-Cob at 40 ℃ after glycosylation modification (FIG. 1).
Sequence listing
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Claims (2)

1. A method for constructing an N-glycosylation alginate lyase mutant comprises the following steps:
(1) Plasmid pTrc99A is subjected to double enzyme digestion by XbaI and HindIII, a coding glycosylation site DQNAT and a 6 XHis gene sequence are introduced, a recombinant plasmid is constructed, the recombinant plasmid is subjected to double enzyme digestion by XbaI and SalI, a YebF gene sequence with NCBI accession number of B1847 is inserted to form a pTrc-YebF-gly-His plasmid, the recombinant plasmid is subjected to double enzyme digestion by XbaI and EcoR I, and a Aly-Cob coding gene is inserted to form a recombinant vector pTrc-YebF-gly-Aly-Cob-His;
(2) The constructed expression plasmid containing the alginate lyase gene sequence and the pACYCpgl plasmid carrying the campylobacter jejuni N-glycosylation gene cluster are converted into escherichia coli by electric shock for co-expression, the transformant is inoculated into an LB culture medium containing 20 mu g/mL chloramphenicol and 100 mu g/mL trimethoprim for culture for 16-20h, and 0.01mM isopropyl thiogalactoside is added for induced expression for 24h;
(3) Centrifugally collecting fermentation supernatant, and purifying the recombinase through a nickel column;
the recombinant vector pTrc-YebF-gly-Aly-Cob-His expresses a fusion protein with an amino acid sequence shown as SEQ ID No.1 or SEQ ID No. 2.
2. Use of the alginate lyase produced by the method of claim 1 in the preparation of alginate oligosaccharides.
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CN108285900B (en) * 2018-04-12 2020-06-09 江南大学 Recombinant alginate lyase and construction method and application thereof
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WO2016023018A2 (en) * 2014-08-08 2016-02-11 Glycobia, Inc. Engineered oligosaccharyltransferases
CN108285900A (en) * 2018-04-12 2018-07-17 江南大学 A kind of recombination algin catenase and its construction method and application

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