阅读说明：本技术 一种褐藻胶裂解酶系及其应用 (A kind of algin catenase system and its application ) 是由 李福川 路丹荣 关靖雯 王淑敏 张庆冬 焦润苗 于 2019-08-01 设计创作，主要内容包括：本发明属于酶工程技术领域,涉及一种褐藻胶裂解酶系及其应用。一种褐藻胶裂解酶系,包含内切酶AlyPB1和外切酶AlyPB2；所述内切酶AlyPB1氨基酸序列如SEQ ID NO.3所示；外切酶AlyPB2氨基酸序列如SEQ ID NO.4所示。该酶系可应用于生产生物乙醇。两种褐藻胶裂解酶具有良好的协同作用,当两种酶以1:1的酶活比混合时,单糖的生成量是单独的外切酶降解褐藻胶时的6-8倍,大大提高了褐藻胶的糖化效率,为生物乙醇的生产提供了高效新颖的候选酶系。(The invention belongs to technical field of enzyme engineering, it is related to a kind of algin catenase system and its application.A kind of algin catenase system includes restriction endonuclease AlyPB1 and excision enzyme AlyPB2；The restriction endonuclease AlyPB1 amino acid sequence is as shown in SEQ ID NO.3；Excision enzyme AlyPB2 amino acid sequence is as shown in SEQ ID NO.4.The enzyme system can be applied to production bio-ethanol.Two kinds of algin catenases have good synergistic effect, when enzyme activity ratio mixing of two kinds of enzymes with 1:1,6-8 times when the production quantity of monosaccharide is individual excision enzyme degradation algin, the saccharification efficiency of algin is substantially increased, provides efficiently novel candidate enzyme system for the production of bio-ethanol.)

1. a kind of algin catenase system, it is characterised in that: include restriction endonuclease AlyPB1 and excision enzyme AlyPB2；The restriction endonuclease AlyPB1 amino acid sequence is as shown in SEQ ID NO.3；Excision enzyme AlyPB2 amino acid sequence is as shown in SEQ ID NO.4.

2. algin catenase system according to claim 1, it is characterised in that: the coding base of the restriction endonuclease AlyPB1 Because nucleotide sequence is as shown in SEQ ID NO.1.

3. algin catenase system according to claim 1, it is characterised in that: the coding base of the excision enzyme AlyPB2 Because nucleotide sequence is as shown in SEQ ID NO.2.

4. the application of algin catenase system according to claim 1, it is characterised in that: restriction endonuclease AlyPB1 is to algin Rate activity with guluronic acid is respectively 185U/mg and 1295U/mg.

5. the application of algin catenase system according to claim 1, it is characterised in that: excision enzyme AlyPB2 is to brown alga Glue, guluronic acid, polymannuronate Rate activity be respectively 10.93U/mg, 8.5U/mg and 14.63U/mg.

6. a kind of application of algin catenase system, it is characterised in that: application of the enzyme system in production bio-ethanol.

7. the application of algin catenase system according to claim 6, it is characterised in that: in enzyme system restriction endonuclease AlyPB1 and Excision enzyme AlyPB2 enzyme activity ratio is 1:1.

8. a kind of method for algin of degrading, it is characterised in that: the described in any item enzyme systems of claim 1-5 are used, by 3mg/ The NaH of the algin of mL, 150mM pH8.0₂PO₄-Na₂HPO₄Buffer is mixed according to the ratio of volume ratio 1:1, restriction endonuclease After AlyPB1 and excision enzyme AlyPB2 is with the enzyme activity ratio premix of 1:1, it is added in reaction system, and add deionized water to 300 Microlitre, 10min is reacted under the conditions of 20 DEG C, reaction terminates.

Technical field

The invention belongs to technical field of enzyme engineering, it is related to a kind of algin catenase system and its application.

Background technique

With the continuous growth of world energy sources demand, the rapid exhausted and its generated environmental problem of fossil fuel resource Gradually it attract attention.Macro is greatly paid close attention to recently as the desirable feedstock of production bio-fuel.By In it, containing cellulose, production process are not take up arable land and consumption freshwater resources, do not use fertilizer, can control the dioxy in atmosphere The advantages that changing carbon and capable of evading the contradiction between staple food supply, makes the important candidate of third generation renewable resource (MacDonald LC, Weiler EB, Berger BW. Engineering broad-spectrum digestionof polyuronides from an exolytic polysaccharidelyase. Biotechnology for Biofuels. 2016;9(1):43. Medipally SR, Yusoff FM, Banerjee S, Shariff M. Microalgae as sustainable renewable energy feedstock for biofuel production. BioMed Res Int. 2015;2015:519513. Wargacki AJ, Leonard E, Win MN, Regitsky DD, Santos CN, Kim PB, CooperSR, Raisner RM, Herman A, Sivitz AB, et al. An engineered microbial platform for direct biofuel production from brown macroalgae. Science (NewYork, NY). 2012;335 (6066): 308-13.).Algin (alginate) is The most abundant carbohydrate in brown algae plant cell wall and matrix accounts for about the 40%(Peng CE, Wang of brown alga dry weight QB, Lu DR, Han WJ, Li FC. A Novel Bifunctional Endolytic Alginate Lyase with Variable Alginate-Degrading Modes and Versatile Monosaccharide-Producing Properties. Frontiers in Microbiology. 2018;9 (167): 1-14.).Algin be byβ- D- sweet dew Uronic acid (β-D-Mannuronate, M) and its C5 epimerα- L- guluronic acid (α-L-Guluronate, G) passes through The linear anionic acidic polysaccharose that β-Isosorbide-5-Nitrae glucosides key connection is formed, according to the difference that two kinds of uronic acids put in order, be divided into gather it is sweet Reveal uronic acid section (PolyM), guluronic acid section (PolyG) and mannuronic acid and guluronic acid alternating block (PolyMG/GM) (Cheng YY, Wang DD, Gu JY, Li JG, Liu HH, Li FC, Han WJ. Biochemical Characteristics and Variable Alginate-Degrading Modes of a Novel Bifunctional Endolytic Alginate Lyase. Applied and Environmental Microbiology. 2017;83 (23): e01608-17.).The basic component units of the algin of plant origin and bacterial origin It is similar, but C2 or C3 hydroxyls of the algin of bacterial origin are easy by different degrees of acetylation.Algin is nontoxic, because having The features such as gelation, high viscosity, biocompatibility and chelated metal ions, be widely used in food, medicine, chemical industry, (Han WJ, Gu JY, Cheng YY, Liu HH, Li YZ, Li FC. in the industries such as weaving, production bio-ethanol Novel alginate lyase (Aly5) from a polysaccharide-degrading marine bacterium,Flammeovirga sp. strain MY04: effects of module truncation on biochemical characteristics, alginate degradation patterns, and oligosaccharide-yielding properties. Applied and Environmental Microbiology. 2015;82 (1): 364-74.).

Algin catenase is a kind of polysaccharide degradation that the fracture of algin intramolecular glycosidic bond is catalyzed by β-elimination reaction Enzyme, and C4=C5 unsaturated double-bond, the unsaturated uronic acid of non-reducing end are formed in the non-reducing end of newly generated algin oligosaccharide It is different from the structure of M and G sugar unit, therefore referred to as Δ monosaccharide, unsaturated product oligosaccharides have characteristic absorption (Peng at 232nm CE, Wang QB, Lu DR, Han WJ, Li FC. A Novel Bifunctional Endolytic Alginate Lyase with Variable Alginate-Degrading Modes and Versatile Monosaccharide- Producing Properties. Frontiers in Microbiology. 2018;9(167):1-14. Cheng YY, Wang DD, Gu JY, Li JG, Liu HH, Li FC, Han WJ. Biochemical Characteristics and Variable Alginate-Degrading Modes of a Novel Bifunctional Endolytic Alginate Lyase. Applied and Environmental Microbiology. 2017;83(23):e01608-17. Han WJ, Gu JY, Cheng YY, Liu HH, Li YZ, Li FC. Novel alginate lyase (Aly5) from a polysaccharide-degrading marine bacterium, Flammeovirga sp. strain MY04: effects of module truncation on biochemical characteristics, alginate degradation patterns, and oligosaccharide-yielding properties. Applied and Environmental Microbiology. 2015;82 (1): 364-74.).Algin catenase is from a wealth of sources, including animal Source (sea mollusk, echinoderm), plant source (marine algae) and microbial source (bacterium, fungi, virus).According to brown alga Glue lyases can be divided into M specificity lyases, G specificity lyases and difunctional lyases to the Preference of substrate；According to brown The difference of the primary structure of phycocolloid lyases, CAZy database are incorporated into 7 polysaceharide lyase families, respectively PL-5, PL-6, PL-7, PL-14, PL-15, PL-17, PL-18 family (Xu F, Dong F, Wang P, Cao HY, Li CY, Li PY, Pang XH, Zhang YZ, Chen XL. Novel Molecular Insights into the Catalytic Mechanism of Marine Bacterial Alginate Lyase AlyGC from Polysaccharide Lyase Family 6. Journal of Biological Chemistry. 2017;292(11):4457-68. Uchimura K, Miyazaki M, Nogi Y, Kobayashi T, Horikoshi K. Cloning and Sequencing of Alginate Lyase Genes from Deep-Sea Strains of Vibrio and Agarivorans and Characterization of a New Vibrio Enzyme. Mar Biotechnol. 2010;12(5):526-33. Zhu B, Yin H. Alginate lyase: Review of major sources and classification, properties, structure-function analysis and applications. Bioengineered Bugs. 2015;6(3):125-31. Lee SI, Choi SH, Lee EY, Kim HS. Molecular cloning, purification, and characterization of a novel polyMG-specific alginate lyase responsible for alginate MG block degradation in Stenotrophomas maltophilia KJ-2. Appl Microbiol Biotechnol. 2012;95(6):1643-53. Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, Henrissat B. The Carbohydrate-Active EnZymes database (CAZy): An expert resource for glycogenomics. Nucleic Acids Res. 2009;37:D233-D238.)；It is different according to the degradation model of algin catenase, it is divided into endo-type lyases and outer Cut type lyases, algin catenase majority identified so far are restriction endonuclease, endo-type algin catenase enzyme activity Height, stability is good, and a series of different unsaturated oligosaccharides of molecular dimensions can quickly be generated by acting on algin, and minimum product is more For unsaturated disaccharides (Badur AH, Jagtap SS, Yalamanchili G, Lee JK, Zhao HM, Rao CV. Alginate Lyases from Alginate-Degrading Vibrio splendidus 12B01 Are Endolytic. Applied and Environmental Microbiology. 2015;81(5):1865-73. Li SY, Wang LN, Chen XH, Zhao WW, Sun M, Han YT. Cloning, Expression, and Biochemical Characterization of Two New Oligoalginate Lyases with Synergistic Degradation Capability. Marine Biotechnology. 2018;20 (1): 75-86.), these are unsaturated few Sugar cannot be converted into bio-fuel.However, circumscribed-type algin catenase is successively degraded from one end of brown alga xanthan molecule, Product is unsaturated monosaccharide, and unsaturated monosaccharide can be automatically changeb to 4-deoxy-L-erythro-5- under non-enzymatic catalysis Hexoseulose uronic acid (DEH) or 2,4,5,6-tetrahydroxytetrahydro-2H-pyran-2- Carboxylic acid (TPC), DEH and TPC are reduced into 2- ketone -3- deoxyglucose under the action of reductase (DehR) Hydrochlorate (KDG), KDG can enter bacterium ED approach to by bacterium utilize or be converted into bio-ethanol (Preiss J, Ashwell G. Alginic acid metabolism in bacteria. I. Enzymatic formation of unsaturated oligosaccharides and 4-deoxy-L-erythro-5-hexoseulose uronic acid. Journal of Biochemistry. 1962;237:309-16. Li SY, Wang LN, Han F, Gong QH, Yu WG. Cloning and characterization of the first polysaccharide lyase family 6 oligoalginate lyase from marine Shewanellasp. Kz7. Journal of Biochemistry. 2016;159 (1): 77-86.).

During algin is converted into bio-ethanol, circumscribed-type algin catenase plays vital work With.However it is less about the research of excision enzyme at present, and to show enzyme activity low for identified circumscribed-type algin catenase, The disadvantages of stability is poor (Wang LN, Li SY, Yu WG, Gong QH. Cloning, overexpression and characterization of a new oligoalginate lyase from a marine bacterium,Shewanellasp. Biotechnol Lett.2015;37 (3): 665-71.).In addition, in a usual bacterial genomes The gene for often existing simultaneously endo-type algin catenase and circumscribed-type algin catenase shows that bacterium is common in two kinds of enzymes It can be degradable by algin and be absorbed and utilized under effect.Therefore, the synergistic effect of restriction endonuclease and excision enzyme can be very big The saccharification efficiency of ground promotion algin.However past research is concentrated mainly on classification, the zymologic property, degradation of substrates of single enzyme It is less to the research of the algin catenase system from same bacterial strain in the structure and function research of mode and enzyme.It is raw with single enzyme Object catalytic phase ratio, multienzyme synergism catalysis can be improved the utilization rate of algin and reduces production cost, but internal, circumscribed brown at present Phycocolloid lyases concerted catalysis research seldom, lacks synergistic effect mechanism and understands, the algin cracking of energy efficient degradation algin Therefore enzyme system finds new and effective algin catenase system and is of great significance for the low cost production of bio-ethanol.

Summary of the invention

In order to make up deficiency in the prior art, the present invention provides a kind of algin catenase system, which includes inscribe Type algin catenase AlyPB1 and circumscribed-type algin catenase AlyPB2 has the enzymatic activity of efficient degradation algin.

The technical solution used to solve the technical problems of the present invention is that: a kind of algin catenase system includes restriction endonuclease AlyPB1 and excision enzyme AlyPB2；The restriction endonuclease AlyPB1 amino acid sequence is as shown in SEQ ID NO.3；Excision enzyme AlyPB2 Amino acid sequence is as shown in SEQ ID NO.4.

As further improvement of the invention, the encoding gene nucleotide sequence of the restriction endonuclease AlyPB1 such as SEQ ID Shown in NO.1.

As a further improvement of the present invention, the encoding gene nucleotide sequence of the excision enzyme AlyPB2 such as SEQ ID Shown in NO.2.

The present invention also provides a kind of application of algin catenase system, application of the enzyme system in production bio-ethanol.

As a further improvement of the present invention, in the application, restriction endonuclease AlyPB1 and excision enzyme AlyPB2 enzyme in enzyme system Ratio living is 1:1.

Further, restriction endonuclease AlyPB1 to the Rate activity of algin and guluronic acid be respectively 185U/mg and 1295U/mg, it is lower to the Rate activity of polymannuronate (< 1 U/mg).

Further, excision enzyme AlyPB2 is respectively to the Rate activity of algin, guluronic acid, polymannuronate 10.93U/mg, 8.5U/mg and 14.63U/mg.

The present invention also provides a kind of method using enzyme system degradation algin of the invention, this method are as follows: by 3mg/mL Algin, 150mM pH8.0 NaH₂PO₄-Na₂HPO₄Buffer is mixed according to the ratio of volume ratio 1:1, restriction endonuclease After AlyPB1 and excision enzyme AlyPB2 is with the enzyme activity ratio premix of 1:1, it is added in reaction system, and add deionized water to 300 Microlitre, 10min is reacted under the conditions of 20 DEG C, reaction terminates.

Algin catenase system of the invention, has the advantages that

1, endo-type algin catenase AlyPB1, stable in physicochemical property, degrading activity is high, and final principal product is unsaturation two Sugar, unsaturated trisaccharide and unsaturated tetrose, are a kind of tendentious endo-type algin catenases of G-, have the latent of industrial application Matter.

2, circumscribed-type algin catenase AlyPB2, which is the difunctional algin catenase of circumscribed-type, from non-reducing end Degradation of polysaccharide and oligosaccharide substrates, product are unsaturated monosaccharide.

3, two kinds of algin catenases have good synergistic effect, when enzyme activity ratio mixing of two kinds of enzymes with 1:1, monosaccharide 6-8 times when being individual excision enzyme degradation algin of production quantity, the saccharification efficiency of algin is substantially increased, for biology second The production of alcohol provides efficiently novel candidate enzyme system.

Detailed description of the invention

Fig. 1, the protein three-dimensional structure model for recombinating algin catenase AlyPB1 (A) and AlyPB2 (B)；

The polyacrylamide gel electrophoresis of Fig. 2, the expression of algin catenase AlyPB1 (A) and AlyPB2 (B) and purifying situation Map；

Wherein: swimming lane 1, protein molecular weight standard, size is 116 kD, 66.2 kD, 45 kD, 35 to band from top to bottom KD, 25 kD, 18.4 kD, 14.4 kD；Thallus before swimming lane 2, control strain broken wall, 10 μ L of applied sample amount, swimming lane 3, recombinant bacterium are broken Bacterium solution after wall, 10 μ L of applied sample amount, supernatant after swimming lane 4, recombinant bacterium broken wall, 10 μ L of applied sample amount, swimming lane 5, through ni-sepharose purification AlyPB1 or AlyPB2,10 μ L of applied sample amount；

Fig. 3, temperature are to the activity influence curve of algin catenase AlyPB1 (A) and AlyPB2 (B)；

The activity influence curve of Fig. 4, pH to algin catenase AlyPB1 (A) and AlyPB2 (B)；

Fig. 5, temperature are to the stability influence curve of algin catenase AlyPB1 (A) and AlyPB2 (B)；

Fig. 6, metal ion and chemical reagent are to algin catenase AlyPB1 (A) and AlyPB2 (B) active influence curve；

Fig. 7, recombination algin catenase AlyPB1 different time degradation algin catabolite efficient liquid phase (HPLC) point Analysis figure；

In figure: UDP2: unsaturated disaccharides, UDP3: unsaturated trisaccharide；UDP4: unsaturated tetrose, UDP5: unsaturated pentasaccharides, UDP6: six sugar of unsaturation；

Fig. 8, recombination algin catenase AlyPB2 different time degradation algin catabolite efficient liquid phase (HPLC) point Analysis figure；

In figure: UDP1: unsaturated monosaccharide, DEH and TPC: unsaturated monosaccharide conversion product；

Fig. 9, algin catenase AlyPB2 different time degradation fluorescent marker saturation algin pentasaccharides catabolite height Imitate liquid phase (HPLC) analysis chart；

In figure: 2-AB-Alg5: the saturation algin pentasaccharides of fluorescent marker, 2-AB-Alg2-4: the unsaturated brown alga of fluorescent marker Glue disaccharides, trisaccharide and tetrose；

The enzyme activity analysis chart of Figure 10, algin catenase AlyPB2 to different molecular size algin substrate；

The synergistic effect analysis chart of Figure 11, algin catenase AlyPB1 and AlyPB2；

In figure: UDP1: unsaturated monosaccharide, UDP2: unsaturated disaccharides, UDP3: unsaturated trisaccharide；UDP4: unsaturated tetrose.

Specific embodiment

The elaboration of following embodiment is some common technologies how implemented for the comprehensive disclosure present invention, rather than is Limitation application range of the invention.Inventor tried one's best ensure in embodiment the accuracy of parameter (such as measure, Temperature, etc.), but some experimental errors and deviation should also pay attention to.Unless otherwise indicated, middle-molecular-weihydroxyethyl of the present invention is Refer to average molecular weight, temperature refers to degree Celsius.

Biological material source

One plant of luminous bacillus (PhotobacteriumSp.) FC615, on December 10th, 2018 are preserved in Chinese microorganism strain Preservation administration committee common micro-organisms center, preservation address: the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences Institute of microbiology, deposit number CGMCC NO. 16918.