WO2022257391A1 - 一株能够降解胶原蛋白的菌株及其应用 - Google Patents

一株能够降解胶原蛋白的菌株及其应用 Download PDF

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WO2022257391A1
WO2022257391A1 PCT/CN2021/136035 CN2021136035W WO2022257391A1 WO 2022257391 A1 WO2022257391 A1 WO 2022257391A1 CN 2021136035 W CN2021136035 W CN 2021136035W WO 2022257391 A1 WO2022257391 A1 WO 2022257391A1
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collagen
collagenase
recombinant
recombinant expression
rheinheimera
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French (fr)
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张玉忠
陈秀兰
王珍
张晓玉
程俊慧
宋晓妍
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山东大学
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
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    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/24—Metalloendopeptidases (3.4.24)
    • C12Y304/24003—Microbial collagenase (3.4.24.3)
    • C—CHEMISTRY; METALLURGY
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    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

  • the invention relates to a bacterial strain capable of degrading collagen and its application, belonging to the technical field of biotechnology.
  • Collagen is the most abundant protein in animals and a major structural protein in the extracellular matrix. Collagen is widely distributed and is the main component of mammalian connective tissue. It is also rich in collagen in the skin, bone, scale and other parts of aquatic and marine animals, mainly in the form of insoluble fibrous protein. These collagens are often treated as leftovers in the food processing process, which not only causes waste of biological resources, but also causes adverse effects on the environment. The full and reasonable use of these wastes in food processing can not only promote the development of animal husbandry and aquatic product processing industries, but also reduce environmental pollution.
  • collagen raw materials to produce collagen oligopeptides is an important way for high-value utilization of collagen waste produced in food processing.
  • Collagen oligopeptides have unique biological activities, such as antibacterial, hypotensive, antioxidative, immune regulation, etc.
  • collagen peptides from aquatic and marine animals have higher biological safety, lower immunogenicity and other unique physical and chemical properties, and have good health effects.
  • the health function has great application potential in the fields of biopharmaceuticals, beauty and skin care, hygiene and health, food and health care.
  • Collagen has a large molecular weight and due to the triple helical supramolecular structure and the cross-linking aggregation in all layers, the molecular stability of collagen is very high and it is not easy to be degraded.
  • proteases used in the enzymatic extraction of collagen in industrial production are mainly divided into three categories: microbial proteases, plant proteases, and animal proteases. These enzymes can hydrolyze and remove the terminal peptides of collagen fibers to make them soluble in organic acids. , but cannot destroy the triple helix properties of collagen. Therefore, it is particularly important to screen for new specific high-efficiency collagenases.
  • the mature microbial fermentation process and product extraction and purification process make collagenase from microbial sources particularly attractive.
  • the present invention provides a bacterial strain capable of degrading collagen and its application.
  • the bacterial strain belongs to a new species of the genus Rhineheimer, can secrete collagenase, can be used for degradation of collagen, and can increase the added value of collagen leftovers in animal production and aquatic product processing.
  • the 16S rDNA gene sequence of the Rheinheimera indica SM2107 is shown in SEQ ID NO.1.
  • the growth temperature range of the Rheinheimera indica SM2107 is 4-40°C, and the optimum growth temperature is 25-30°C; the growth NaCl concentration range is 0-10.0% (w /v), the optimum growth NaCl concentration is 3.0-4.0% (w/v); the growth pH range is 6.5-10.5, and the optimum growth pH value is 7.5.
  • Collagenase preparation inoculate Rheinheimera indica SM2107 into the fermentation medium for fermentation culture, and culture it for 4-6 days at a shaking table with a rotation speed of 150-200 rpm and a culture temperature of 23-27°C for 4-6 days to obtain a fermented liquid; then the supernatant obtained by centrifuging the fermented liquid is the collagenase liquid;
  • the fermentation culture conditions are as follows: shaker speed 180rpm, culture temperature 25°C, culture time 5d; described centrifugation speed is 6000 ⁇ 8000r/min, centrifugation time is 8 ⁇ 12min.
  • the centrifugal speed is 6000-8000r/min, and the centrifugal time is 8-12min; the collagen is bovine bone collagen.
  • the addition amount of the collagenase solution is 1300-1700U/g enzyme-material ratio (E/S).
  • a recombinant expression vector which comprises the coding gene shown in SEQ ID NO.2.
  • the recombinant expression vector is constructed with plasmid pET-22b.
  • the recombinant expression vector is transformed into a host cell to generate a recombinant cell.
  • the host cell is Escherichia coli.
  • the Escherichia coli is Escherichia coli BL21(DE3).
  • a recombinant cell comprising the coding gene shown in SEQ ID NO.2.
  • a recombinant expression strain is obtained by culturing recombinant cells.
  • the conditions for culturing the recombinant cells are:
  • LB liquid medium is used, the culture temperature is 37°C, cultured on a shaker, cultured for 5-7 hours, and centrifuged to obtain the recombinant expression strain.
  • a collagenase which is expressed by the above-mentioned recombinant expression strain.
  • the above-mentioned collagenase is used in collagen degradation.
  • a method for degrading collagen comprising:
  • the nucleotide sequence shown in SEQ ID NO.2 is cloned into a plasmid as a recombinant vector, the recombinant vector is transformed into a host cell, and the recombinant cell is constructed; cultivated in a fermentation medium
  • the recombinant cells are obtained to obtain recombinant expression strains; the recombinant expression strains are expressed to obtain collagenase, and the collagenase degrades collagen.
  • the plasmid is plasmid pET-22b.
  • the host cell is Escherichia coli, more preferably Escherichia coli BL21(DE3).
  • the conditions for cultivating recombinant cells in the fermentation medium are:
  • LB liquid medium is used, the culture temperature is 37°C, cultured on a shaker, cultured for 5-7 hours, and centrifuged to obtain the recombinant expression strain.
  • the collagenase degrades the collagen into peptides with a molecular weight of oligopeptides less than 5000 Da.
  • the collagen is derived from bovine bone or fish skin.
  • the steps of collagenase degrading collagen are as follows:
  • the rotational speed of the centrifugation is 6000-8000 r/min, and the centrifugation time is 8-12 minutes.
  • the added amount of the collagen enzyme solution is 1300-1700 U/g enzyme-material ratio (E/S).
  • the novel bacterium Rheinheimera indica SM2107 provided by the invention is a new species of the genus Rheinheimer and has collagen degradation ability.
  • the collagenase prepared by the invention can be applied to enzymatically hydrolyzing collagen to produce collagen oligopeptides.
  • it has a strong degradation ability for collagen raw materials such as bovine bone, and can further enzymolyze the by-products of meat processing and fishery processing such as bovine bone and fish skin.
  • bovine bone as raw material
  • the prepared collagen oligopeptides have a molecular weight of less than 5000Da reaching more than 77%. The molecular weight is small and the uniformity is high, which is easy to be absorbed and utilized by the human body. Good economic benefits.
  • Fig. 1 is the transmission electron microscope (TEM) figure of Rheinheimera indica SM2107, a new bacterium of the genus Rhineheimer;
  • Figure 2 is a phylogenetic tree diagram constructed based on the 16S rDNA sequences of Rheinheimera indica SM2107 and other bacteria.
  • Figure 3 is a two-dimensional chromatogram of polar lipids of Rheinheimera indica SM2107.
  • Fig. 4 is the enzymolysis effect diagram of the new bacteria Rheinheimera indica SM2107 to bovine bone collagen;
  • Fig. 5 is a line graph of the hydrolysis rate of bovine bone collagen with different enzyme-material ratios in Example 6.
  • Fig. 6 is the molecular weight distribution diagram of the hydrolyzate of bovine bone collagen in Example 6.
  • Fig. 7 is the SDS-PAGE profile of the collagenase of Example 7.
  • Fig. 8 is a graph showing the molecular weight distribution of the product of bovine bone hydrolyzed by collagenase in Example 7.
  • the preparation raw materials of the culture medium in the examples are common raw materials in this field; bovine bone collagen can be purchased in the market.
  • Sediments were collected from hydrothermal vents (E 49.34627°, S 37.89947°) in the polymetallic sulphide area of the Southwest Indian Ocean, placed in an ice box and brought back to the laboratory.
  • the bacteria solution enriched in step (2) was serially diluted according to the multiples of 10 1 , 10 2 , 10 3 , 10 4 , and 10 5 , and then inoculated on screening medium plates respectively, and cultured at 25°C for 48 hours. And further use the plate streaking method to purify and inoculate on the gelatin medium; then pick out the strains that form the degradation circle on the gelatin medium to obtain the strains that have the ability to degrade collagen efficiently, and preserve them by the glycerol tube method.
  • Sole carbon and nitrogen source liquid medium 30 parts of NaCl, 0.5 parts of NH 4 Cl, 3 parts of MgCl 2 6H 2 O, 2 parts of K 2 SO 4 , 0.2 parts of K 2 HPO 4 , 0.01 parts of CaCl 2 , FeCl 3 6H 0.006 parts of 2 O, 0.005 parts of Na 2 MoO 4 ⁇ 7H 2 O, 0.004 parts of CuCl 2 ⁇ 2H 2 O, 6 parts of Tris, 10 parts of chitin, pH 7.8-8.0, all are parts by weight.
  • This medium uses chitin as the sole carbon source.
  • Screening medium add 15g of agar powder on the basis of each liter of the only carbon and nitrogen source liquid medium, pH 7.8-8.0.
  • Gelatin medium 5 parts of peptone, 20 parts of gelatin, 15 parts of agar powder, 30 parts of sea salt, and 1000 parts of distilled water, all in parts by weight.
  • Example 1 The bacterial strain screened and isolated in Example 1 was streaked onto the TYS solid medium plate, and the temperature was cultivated for 24h under the condition of 25°C, and the growth situation of the bacterial colony on the plate was observed and recorded, and the transmission electron microscope (TEM) figure of the bacterial colony morphology ,As shown in Figure 1.
  • TEM transmission electron microscope
  • the bacterial strain is on the TYS solid medium plate, and the colony is transparent (yellow in the later stage), round, and the edges are neat, indicating that it is smooth and moist; Gram staining is red, indicating that it is a Gram-negative bacterium;
  • TEM transmission electron microscopy
  • the cells are rod-shaped, with a length and width of 1.2-2.4 ⁇ 0.5-1.1 ⁇ m, unit cells, and can secrete a large amount of extracellular polymers.
  • TYS solid medium 5 parts of peptone, 1 part of yeast powder, 30 parts of sea salt, 1000 parts of water, pH 7.0-7.4, all in parts by weight.
  • Example 1 The physiological and biochemical characteristics of the bacterial strain screened and isolated in Example 1 were identified by routine physiological and biochemical experiments and API 20NE, ZYM reagent strips.
  • 1 is the isolated bacterial strain of embodiment 1; 2 is bacterial classification Rheinheimera tuosuensis CGMCC 1.12461 T ; 3 is bacterial classification Rheinheimera perlucida BA131 T ; 4 is bacterial classification Rheinheimera longhuensis LH2-2 T ; 5 is bacterial classification Rheinheimera baltica OSBAC1 T 6 is strain Rheinheimera nanhaiensis E407-8 T.
  • + means positive; w means weak positive; -, negative reaction; A, aerobic; FA, facultative aerobic; ND, no data available.
  • the growth temperature range of the strains screened and isolated in Example 1 is 4-40°C, and the optimum growth temperature is 25-30°C; the NaCl concentration range for growth is 0-10% (w/v), the optimum The NaCl concentration for growth is 3.0-4.0%, the pH range for growth is 6.5-10.5, and the optimum growth pH value is 7.5.
  • the strain screened and isolated in Example 1 can reduce nitrate to nitrite, and its oxidase and catalase reactions are positive. At the same time, the strain can hydrolyze casein, gelatin, Tween 20, Tween 40, Tween 60, Tween 80, starch and aescin.
  • Example 1 The DNA in the bacterial strain screened and isolated in Example 1 was extracted using the BioTeke Bacterial Genome Extraction Kit, and the specific extraction method was referred to the instruction manual of the kit.
  • PCR primers use universal primers:
  • the PCR reaction conditions were: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s; annealing at 55°C for 30 s; extension at 72°C for 90 s; 30 cycles, extension at 72°C for 5 min, and storage at 4°C.
  • the PCR product was sequenced by Beijing Qingke Biotechnology Co., Ltd., and the sequencing result is shown in the sequence table SEQ ID NO.1, with a length of 1523bp.
  • the amplified 16S rDNA sequence was analyzed for homology with the 16S rDNA sequence of all standard strains in the EZBioCloud database. The results showed that the sequence with higher homology belonged to the genus Rhineheimer, and the close relatives in the genus Rhineheimer were selected Phylogenetic analysis was carried out between the related bacterial strains and the bacterial strains isolated in Example 1. A phylogenetic tree was constructed using the Neighbor-joining method using MEGA-X software, as shown in Figure 2.
  • the strain was identified as a new strain of the genus Rheinheimeria and named Rheinheimera indica SM2107.
  • Sherolock automatic bacterial identification system of MIDI Microbial Identification
  • the specific analysis method refers to the manual of the automatic bacterial identification system.
  • the analysis results are shown in Table 2 .
  • composition of the polar lipids of the Rheinheimera indica SM2107 screened and isolated in Example 1 was analyzed by thin-layer two-dimensional chromatography (TLC), and the results are shown in FIG. 3 .
  • the respiratory quinone components of Rheinheimera indica SM2107 were analyzed with an octadecylsilane column (ODS 5mm, 150 ⁇ 4.6mm) by reversed-phase high-performance liquid chromatography.
  • TR Trace ( ⁇ 0.5%); –, not detected or reported.
  • Major fatty acids (>10%) in each strain are shown in bold.
  • the main fatty acids of Rheinheimera indica SM2107 are: C 18:1 ⁇ 7c and/or C 18:1 ⁇ 6c,C 16:0 ,C 17:1 ⁇ 8 locker,and C 16:1 ⁇ 7c and/or C 16:1 ⁇ 6c;
  • the main respiratory quinone is Q-8
  • the main polar lipids are: phosphatidylethanolamine (PE), phosphatidylglycerol (PG).
  • PE phosphatidylethanolamine
  • PG phosphatidylglycerol
  • the application of the new bacteria Rheinheimera indica SM2107 of the genus Rheinheimeria screened and isolated in embodiment 1 in collagen degradation includes the following steps:
  • the shaker speed is 180rpm
  • the culture temperature is 25°C
  • the culture time is 5 days
  • the fermentation liquid is centrifuged at 7000r/min for 10min
  • the obtained supernatant It is the collagenase solution; then add the collagenase solution to the bovine bone collagen according to the enzyme-material ratio of 1700U/g, and react at 50°C for 2 hours; after the completion of the reaction, the precipitate is removed by centrifugation at 7000r/min for 10min, and the obtained The supernatant is the collagen enzymatic hydrolysis solution.
  • the components of the fermentation medium are as follows: 3 parts of bovine bone collagen, 2 parts of yeast powder, 30 parts of sea salt, 1000 parts of water, pH 7.0-7.4, all in parts by weight.
  • the collagenase secreted by the new bacterium Rheinheimera indica SM2107 provided by the present invention is used to degrade bovine bone, and collagen peptides with different molecular weights in the collagen enzymatic hydrolyzate of the obtained collagen have a molecular weight of less than 5000Da. Protein peptides accounted for more than 77% of the total peptides. Therefore, Rheinheimera indica SM2107 and its secreted collagenase can be used to degrade bovine bone collagen to prepare collagen peptides with different molecular weights.
  • the genomic DNA of Rheinheimera indica SM2107 extracted in Example 4 was sequenced (Shanghai Lingen Biotechnology Co., Ltd.).
  • the proteases of the S8 family were found from the protease library MEROPS, and then compared with the proteases of the S8 family according to the sequencing results, the analysis results showed that a collagenase gene was carried on the genome of Rheinheimera indica SM2107.
  • the nucleotide sequence is shown in SEQ ID NO.2, the length is 1539bp, the amino acid sequence is shown in SEQ ID NO.3, consisting of 512 amino acids, named collagenase 48.
  • the Rheinheimera indica SM2107 genome DNA that is extracted with embodiment 4 is template, carries out PCR amplification, and primer is as follows:
  • the PCR reaction conditions were: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s; annealing at 55°C for 30 s; extension at 72°C for 90 s; 30 cycles, extension at 72°C for 5 min, and storage at 4°C.
  • the PCR product was double-digested with Nde I and Xho I, and the digested PCR product was recovered by agarose gel electrophoresis, and then the digested PCR product was ligated with the pET-22b vector plasmid that had also been double-digested, and the ligated product was transformed into the large intestine
  • the Bacillus DH5 ⁇ strain was spread on the solid plate of LB medium containing 100 ⁇ g/mL ampicillin sodium, cultured at 37°C for 14 hours, and a single clone was picked; the single clone was inserted into the liquid LB medium containing 100 ⁇ g/mL ampicillin sodium Cultivate and extract plasmids;
  • Collagenase 48 was a single band on the electrophoresis gel, with a molecular weight between 35-45kDa, and the position coincided with the predicted molecular weight .
  • the collagenase 48 provided by the present invention is used to degrade bovine bone, and the collagen peptides with different molecular weights in the obtained collagen hydrolyzed solution account for more than 76% of the total peptides. Collagen peptides with a molecular weight below 1000Da accounted for more than 40% of the total peptides. Therefore, collagenase 48 can be used to degrade bovine bone collagen to prepare collagen peptides with different molecular weights, especially collagen peptides with a molecular weight below 1000 Da.

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Abstract

提供了一株莱茵海默氏属新菌（Rheinheimera indica）SM2107及其应用。该菌株的保藏编号为CCTCC M 2021506。所述菌株具有胶原蛋白降解能力，可以制备胶原蛋白酶，该菌株所产的胶原蛋白酶能够应用于酶解胶原蛋白，生产胶原蛋白寡肽。

Description

一株能够降解胶原蛋白的菌株及其应用 技术领域
本发明涉及一株能够降解胶原蛋白的菌株及其应用，属于生物技术技术领域。
背景技术
胶原蛋白是动物体内含量最丰富的蛋白质，是细胞外基质内的一种主要结构蛋白。胶原蛋白分布广泛，是哺乳动物结缔组织的主要成分，在水产及海洋动物的皮、骨、鳞等部位也含有丰富的胶原蛋白，主要以不溶性纤维状蛋白的形式存在。这些胶原蛋白在食品加工过程中往往作为下脚料处理，不仅造成生物资源的浪费，还对环境造成不良的影响。充分合理的利用这些食品加工过程中的废弃物，不仅能够推动畜产、水产加工业的发展，而且能够减少环境污染。
利用胶原蛋白原料生产胶原蛋白寡肽是食品加工过程中产生的胶原蛋白下脚料高值化利用的重要途径。胶原蛋白寡肽具有独特的生物活性，如抗菌、降血压、抗氧化、免疫调节等。另外，相较于陆源动物为原料来源制备的胶原蛋白肽，水产及海洋动物胶原蛋白肽具有更高的生物安全性、更低的免疫原性和其它独特的理化性质，且对机体健康具有良好保健功能，在生物制药、美容护肤、卫生健康、食品保健等领域均有着巨大的应用潜力。
胶原蛋白分子量大且由于三股螺旋超分子的结构以及存在于各级层中的交联聚集作用，胶原蛋白的分子稳定性很高，不易被降解。目前工业生产上酶法提取胶原蛋白这一工艺中使用的蛋白酶主要分为微生物蛋白酶、植物蛋白酶、动物蛋白酶这三类，这些酶可以水解除去胶原纤维的末端肽，使之可溶于有机酸，但无法破坏胶原蛋白的三股螺旋特性。因此筛选获得新的特异性的高效胶原蛋白酶尤为重要。成熟的微生物发酵工艺和产物提取纯化工艺使得微生物来源的胶原蛋白酶尤为引人关注。
目前，人们已经从枯草杆菌、蜡状芽孢杆菌、放线菌、假交替单胞菌等微生物中获得了胶原蛋白酶，但大多数菌株产酶活力较低或需要特殊的诱导条件，能够分泌高效胶原蛋白酶的进而降解胶原蛋白的菌株还很缺乏。
发明内容
针对现有技术的不足，本发明提供一株能够降解胶原蛋白的菌株及其应用。该菌株属于莱茵海默氏属的一个新种，能够分泌的胶原蛋白酶，可用于胶原蛋白的降解，提高畜产、水产加工中胶原蛋白下脚料的附加值。
本发明的技术方案如下：
一株莱茵海默氏属新菌Rheinheimera indica SM2107，该菌株2021年5月8日保存于中国典型培养物保藏中心，保藏编号CCTCC M2021506，地址：中国，武汉，武汉大学。
上述菌株自西南印度洋多金属硫化物区热液口(E 49.34627°,S 37.89947°)的沉积物样品分离得到。
根据本发明优选的，所述莱茵海默氏属新菌Rheinheimera indica SM2107的16S rDNA基因序列如SEQ ID NO.1所示。
根据本发明优选的，所述莱茵海默氏属新菌Rheinheimera indica SM2107的生长温度范围为4～40℃，最适生长温度为25～30℃；生长的NaCl浓度范围为0～10.0％(w/v)，最适生长的NaCl浓度为3.0～4.0％(w/v)；生长的pH范围为6.5～10.5，最适生长的pH值为7.5。
本发明所述莱茵海默氏属新菌Rheinheimera indica SM2107在制备胶原蛋白酶以及降解胶原蛋白中的应用。
上述应用，包括步骤如下：
(1)制备胶原蛋白酶：将莱茵海默氏属新菌Rheinheimera indica SM2107接种至发酵培养基中进行发酵培养，在摇床转速150～200rpm，培养温度23～27℃下培养4～6d，得到发酵液；再将发酵液经离心得到的上清液即为胶原蛋白酶液；
(2)降解胶原蛋白：向胶原蛋白中加入步骤(1)得到的胶原蛋白酶液，在45～55℃的条件下反应2～3h，经离心后去除沉淀，得到的上清液即为胶原蛋白酶解液。
根据本发明优选的，步骤(1)中，所述的发酵培养基组分如下：牛骨胶原蛋白3份，酵母粉2份，海盐30份，水1000份，pH＝7.0～7.4，均为重量份。
根据本发明优选的，步骤(1)中，所述的发酵培养条件为：摇床转速180rpm，培养温度25℃，培养时间5d；所述的离心转速为6000～8000r/min，离心时间为8～12min。
根据本发明优选的，步骤(2)中，所述的离心转速为6000～8000r/min，离心时间为8～12min；所述的胶原蛋白为牛骨胶原蛋白。
根据本发明优选的，步骤(2)中，所述的胶原蛋白酶液的加入量为1300～1700U/g的酶料比(E/S)。
一种重组表达载体，该重组表达载体包含如SEQ ID NO.2所示的编码基因。
根据本发明，优选的，所述的重组表达载体用质粒pET-22b构建。
根据本发明，优选的，所述重组表达载体转化到宿主细胞中产生重组细胞。
根据本发明，优选的，所述的宿主细胞为大肠杆菌。
根据本发明，优选的，所述的大肠杆菌为大肠杆菌BL21(DE3)。
一种重组细胞，该重组细胞包含如SEQ ID NO.2所示的编码基因。
一种重组表达菌株，该菌株是对重组细胞进行培养得到。
根据本发明，优选的，对重组细胞进行培养的条件为：
采用LB液体培养基，培养温度37℃，摇床培养，培养5-7h，离心分离得到重组表达菌株。
一种胶原蛋白酶，该胶原蛋白酶由上述重组表达菌株表达得到。
根据本发明，上述胶原蛋白酶，在胶原蛋白降解中的应用。
一种胶原蛋白的降解方法，包括：
使用如SEQ ID NO.2所示的核苷酸序列，将所述核苷酸序列克隆到质粒中作为重组载体，该重组载体转化到宿主细胞中，构建得到重组细胞；在发酵培养基中培养重组细胞，得到重组表达菌株；重组表达菌株表达得到胶原蛋白酶，胶原蛋白酶对胶原蛋白进行降解。
根据本发明，优选的，所述的质粒为质粒pET-22b。
根据本发明，优选的，所述的宿主细胞为大肠杆菌，进一步优选为大肠杆菌BL21(DE3)。
根据本发明，优选的，在发酵培养基中培养重组细胞的条件为：
采用LB液体培养基，培养温度37℃，摇床培养，培养5-7h，离心分离得到重组表达菌株。
根据本发明，优选的，胶原蛋白酶对胶原蛋白进行降解至寡肽分子量小于5000Da的肽段。
根据本发明，优选的，所述的胶原蛋白来源于牛骨或鱼皮。
根据本发明，优选的，胶原蛋白酶对胶原蛋白进行降解的步骤如下：
向胶原蛋白中加入胶原蛋白酶液，在45～55℃的条件下反应2～3h，经离心后去除沉淀，得到的上清液即为胶原蛋白酶解液。
根据本发明，优选的，离心的转速为6000～8000r/min，离心时间为8～12min。
根据本发明，优选的，胶原蛋白酶液的加入量为1300～1700U/g的酶料比(E/S)。
本发明的有益效果：
本发明提供的莱茵海默氏属新菌Rheinheimera indica SM2107是莱茵海默氏属的一个新种，具有胶原蛋白降解能力。本发明制备的胶原蛋白酶能够应用于酶解胶原蛋白，生产胶原蛋白 寡肽。特别是对牛骨等胶原蛋白原料具有很强的降解能力，可以对牛骨、鱼皮等肉类加工和渔业加工的副产品做进一步的酶解处理。采用牛骨作为原料，制备的胶原蛋白寡肽分子量小于5000Da的肽段达到了77％以上，分子量小且均一度高，易于被人体吸收利用，有效的提高了胶原蛋白副产品的附加值，具有很好的经济效益。
附图说明
图1为莱茵海默氏属新菌Rheinheimera indica SM2107的透射电子显微镜(TEM)图；
图2为根据莱茵海默氏属新菌Rheinheimera indica SM2107以及其它细菌的16S rDNA序列构建的系统发育树图。
图3为莱茵海默氏属新菌Rheinheimera indica SM2107的极性脂双向层析图。
图中：图a、磷钼酸染色，图b、茚三酮染色
图4为莱茵海默氏属新菌Rheinheimera indica SM2107对牛骨胶原蛋白的酶解效果图；
图中：1、降解前，2、降解后。
图5为实施例6不同酶料比对牛骨胶原蛋白的水解率折线图。
图6为实施例6牛骨胶原蛋白水解物的分子量分布图。
图7为实施例7胶原蛋白酶的SDS-PAGE图谱。
图8为实施例7牛骨经胶原蛋白酶酶解后产物的分子量分布图。
具体实施方式
下面结合实施例对本发明的技术方案作进一步说明，但本发明所保护范围不限于此。
一株莱茵海默氏属新菌Rheinheimera indica SM2107，2021年5月8日保存于中国典型培养物保藏中心，保藏编号CCTCC M 2021506，地址:中国，武汉，武汉大学。
实施例中培养基的配制原料均为本领域常用原料；牛骨胶原蛋白可在市场购得。
实施例1 菌株的筛选与分离
(1)样品采集
从西南印度洋多金属硫化物区热液口(E 49.34627°,S 37.89947°)采集沉积物，放置在冰盒中带回实验室。
(2)富集驯化
吸取沉积物样品500μL加入到50mL唯一碳源液体培养基中，在摇床中以25℃，180rpm的条件，培养一周，得到富集的菌液。
(3)菌株的筛选和分离
将步骤(2)中富集的菌液按照10 1，10 2，10 3，10 4，10 5的倍数进行梯度稀释，然后分别 接种于筛选培养基平板上，在25℃条件下培养48h，并进一步采用平板划线法纯化接种至明胶培养基上；接着在明胶培养基上将形成降解圈的菌株挑出，得到具有高效降解胶原蛋白能力的菌株，通过甘油管方法保藏。
上述培养基组分如下：
唯一碳氮源液体培养基：NaCl 30份，NH 4Cl 0.5份，MgCl 2·6H 2O 3份，K 2SO 4 2份，K 2HPO 4 0.2份，CaCl 2 0.01份，FeCl 3·6H 2O 0.006份，Na 2MoO 4·7H 2O 0.005份，CuCl 2·2H 2O 0.004份，Tris 6份，几丁质10份，pH 7.8～8.0，均为重量份。本培养基以几丁质为唯一碳源。
筛选培养基：在每升唯一碳氮源液体培养基基础上添加15g琼脂粉，pH 7.8～8.0。
明胶培养基：蛋白胨5份，明胶20份，琼脂粉15份，海盐30份，蒸馏水1000份，均为重量份。
实施例2 菌株形态学的鉴定
将实施例1筛选和分离的菌株划线到TYS固体培养基平板上，在温度为25℃的条件下培养24h，观察并记录平板上菌落的生长情况，菌落形态的透射电子显微镜(TEM)图，如图1所示。
由图1可知，该菌株在TYS固体培养基平板上，菌落呈透明(后期发黄)，圆形，边缘整齐，表明光滑较湿润；革兰氏染色呈红色，表明为革兰氏阴性菌；透射电子显微镜(TEM)图中细胞呈杆状，长宽为1.2～2.4×0.5～1.1μm，单胞，可分泌大量的胞外聚合物。
TYS固体培养基：蛋白胨5份，酵母粉1份，海盐30份，水1000份，pH 7.0～7.4，均为重量份。
实施例3 菌株生理生化的鉴定
通过常规生理生化实验和API 20NE、ZYM试剂条对实施例1筛选和分离的菌株的生理生化特征进行鉴定。
鉴定分析结果见表1。
表1 实施例1分离的菌株和莱茵海默氏属亲缘关系相近菌株的生理生化特征比较
Figure PCTCN2021136035-appb-000001
Figure PCTCN2021136035-appb-000002
表中：1为实施例1分离的菌株；2为菌种Rheinheimera tuosuensis CGMCC 1.12461 T；3为菌种Rheinheimera perlucida BA131 T；4为菌种Rheinheimera longhuensis LH2-2 T；5为菌种Rheinheimera baltica OSBAC1 T；6为菌种Rheinheimera nanhaiensis E407-8 T。+代表阳性；w代表弱阳性；-,negative reaction；A,好氧；FA,兼性好氧；ND，代表没有可用数据。
由表1可知，实施例1筛选和分离的菌株生长温度范围为4～40℃，最适生长温度为25～30℃；生长的NaCl浓度范围为0～10％(w/v)，最适生长的NaCl浓度为3.0～4.0％；生长的pH范围为6.5～10.5，最适生长的pH值为7.5。实施例1筛选和分离的菌株能够还原硝酸盐为亚硝酸盐，其氧化酶和触酶反应为阳性。同时该菌株能够水解酪蛋白、明胶、Tween 20、Tween 40、Tween 60、Tween 80、淀粉和七叶苷。
实施例4 菌株的16S rDNA序列扩增与鉴定
使用BioTeke细菌基因组提取试剂盒提取实施例1筛选和分离的菌株中的DNA，具体提取方法参照该试剂盒的说明书。
PCR引物采用通用引物：
27F：5'-AGAGTTTGATCCTGGCTCAG-3'，
1492R：5'-GGTTACCTTGTTACGACTTC-3'。
PCR反应条件为：温度为95℃预变性5min；95℃变性30s；55℃退火30s；72℃延伸90s；30个循环，72℃延伸5min，4℃保存。
PCR产物由北京擎科生物科技有限公司进行测序，测序结果如序列表SEQ ID NO.1所示，长度为1523bp。
将扩增出的16S rDNA序列在EZBioCloud数据库与所有标准菌株16S rDNA序列进行同源性分析，结果显示同源性较高的序列属于莱茵海默氏属，选取莱茵海默氏属内的相近亲缘关系的菌株与实施例1分离的菌株进行系统发育分析。采用MEGA-X软件运用邻接法(Neighbor-joining)构建系统发育树，如图2所示。
根据16S rDNA序列比对结果并结合该菌株的生物学特性，将该菌株鉴定为莱茵海默氏属新菌，命名为Rheinheimera indica SM2107。
实施例5 菌株的脂肪酸、细胞醌和极性脂成分分析
使用MIDI(Microbial Identification)公司的Sherolock全自动细菌鉴定系统对莱茵海默氏属新菌Rheinheimera indica SM2107进行脂肪酸成分分析，具体分析方法参照该全自动细菌鉴定系统的说明书，分析结果如表2所示。
通过薄层双向层析方法(TLC)分析实施例1筛选和分离的莱茵海默氏属新菌Rheinheimera indica SM2107极性脂组成，结果如图3所示。
使用反相高效液相色谱分析法，用十八烷基硅烷色谱柱(ODS 5mm，150×4.6mm)分析莱茵海默氏属新菌Rheinheimera indica SM2107呼吸醌组分。
表2 莱茵海默氏属新菌Rheinheimera indica SM2107和亲缘关系相近菌株的脂肪酸含量比较
Figure PCTCN2021136035-appb-000003
Figure PCTCN2021136035-appb-000004
表中：1为菌种Rheinheimera indicate SM2107 T；2为菌种Rheinheimera tuosuensis CGMCC1.12461 T；3为菌种Rheinheimera perlucida BA131 T；4为菌种Rheinheimera longhuensis LH2-2 T；5为菌种Rheinheimera baltica OSBAC1 T；6为菌种Rheinheimera nanhaiensis E407-8 T。
TR,Trace(<0.5％)；–,not detected or reported.Major fatty acids(>10％)in each strain are shown in bold。
由表2可知，莱茵海默氏属新菌Rheinheimera indica SM2107的主要脂肪酸为：C 18:1ω7c and/or C 18:1ω6c,C 16:0,C 17:1ω8с,and C 16:1ω7c and/or C 16:1ω6c；主要呼吸醌为Q-8，主要的极性脂类为：磷脂酰乙醇胺(phosphatidylethanolamine，PE)，磷脂酰甘油(phosphatidylglycerol，PG)。莱茵海默氏属新菌Rheinheimera indica SM2107的基因组DNA的G+C含量为48.76mol％。
实施例6
实施例1筛选和分离的莱茵海默氏属新菌Rheinheimera indica SM2107在胶原蛋白降解中的应用，包括步骤如下：
将莱茵海默氏属新菌Rheinheimera indica SM2107接种至发酵培养基中进行发酵培养，摇床转速180rpm，培养温度25℃，培养时间5d，发酵液经7000r/min，离心10min，得到的上清液即为胶原蛋白酶液；然后按照1700U/g的酶料比向牛骨胶原蛋白中加入胶原蛋白酶液，在50℃下反应2h；两者反应完成后经7000r/min离心10min后去除沉淀，得到的上清液即为胶原蛋白酶解液。
所述的发酵培养基组分如下：牛骨胶原蛋白3份，酵母粉2份，海盐30份，水1000份，pH7.0～7.4，均为重量份。
本实施例中牛骨胶原蛋白的酶解效果如图4所示，反应后，称量反应剩余的底物量，计算牛骨胶原蛋白底物的酶解率，结果如图5所示。
由图4～5可知，莱茵海默氏属新菌Rheinheimera indica SM2107产生的胶原蛋白酶能够高效地酶解牛骨胶原蛋白，最高酶解率达到97.52％。
本实施例中牛骨胶原蛋白降解得到的胶原蛋白酶解液经冷冻干燥后，用流动相溶液(乙腈：蒸馏水：三氟乙酸＝45：55：0.1)配制成5.0mg/mL的胶原蛋白酶解液，待样品充分溶解后，用0.22μm的滤器过滤后，利用HPLC分析酶解液中胶原蛋白肽的分子量分布，结果如图6和表3所示。
表3 牛骨胶原蛋白酶解液中不同分子量肽段的相对丰度
Figure PCTCN2021136035-appb-000005
由图6和表3可知，采用本发明提供的莱茵海默氏属新菌Rheinheimera indica SM2107分泌的胶原蛋白酶来降解牛骨，所得的胶原蛋白酶解液中不同分子量的胶原蛋白肽分子量5000Da以下的胶原蛋白肽占总肽段77％以上。因此，可将莱茵海默氏属新菌Rheinheimera indica SM2107和其分泌的胶原蛋白酶用于降解牛骨胶原蛋白制备含有不同分子量的胶原蛋白肽。
实施例7
将实施例4提取的莱茵海默氏属新菌Rheinheimera indica SM2107基因组DNA进行测序(上海凌恩生物科技有限公司)。从蛋白酶库MEROPS中找出S8家族的蛋白酶，再根据测序结果与S8家族的蛋白酶进行比对分析，分析结果显示莱茵海默氏属新菌Rheinheimera indica SM2107基因组上携带一个胶原蛋白酶的基因，该基因的核苷酸序列如SEQ ID NO.2所示，长度为1539bp，氨基酸序列如SEQ ID NO.3所示，由512个氨基酸组成，命名为胶原蛋白酶48。
以实施例4提取的莱茵海默氏属新菌Rheinheimera indica SM2107基因组DNA为模板，进行PCR扩增，引物如下：
pET-22b-F:AAGAAGGAGATATACATATGAAAACTACAAAAACCCTCTTA
pET-22b-R:TGGTGGTGGTGGTGCTCGAGGTACTGATAGCTGTATGTCA
PCR反应条件为：温度为95℃预变性5min；95℃变性30s；55℃退火30s；72℃延伸90s；30个循环，72℃延伸5min，4℃保存。
将PCR产物经Nde I和Xho I双酶切，琼脂糖凝胶电泳回收酶切PCR产物，然后将酶切后的PCR产物与同样经过双酶切的pET-22b载体质粒连接，连接产物转化大肠杆菌DH5α菌株后涂布于含有100μg/mL氨苄青霉素钠的LB培养基固体平板上，37℃培养14h，挑取单克隆；将单克隆接入含有100μg/mL氨苄青霉素钠的液体LB培养基中培养，提取质粒；
接着将该重组质粒送去北京擎科生物科技有限公司，结果表明，在pET-22b的Nde I和Xho I酶切位点之间成功插入SEQ ID NO.2所示的胶原蛋白酶基因，且插入方向正确。
胶原蛋白酶基因的异源表达：
(1)将构建好的重组质粒载体按照《分子克隆实验指南》上的热激转化方法转化至大肠杆菌BL21(DE3)(购自南京诺唯赞生物科技股份有限公司)感受态细胞，并涂布于含100μg/mL氨苄青霉素的淡水LB固体平板上，37℃过夜培养；
(2)在平板上挑取单菌落，接种至含100μg/mL氨苄青霉素的淡水LB液体培养基中，37℃过夜培养，得到种子液；
(3)将种子液按1％(v/v)的接种量转接至含100μg/mL氨苄青霉素的淡水LB液体培养基中，在摇床转速150～200rpm，培养温度37℃,下培养5-7h，之后调整培养条件为18℃、120rpm，继续培养半小时后加入IPTG至终浓度0.1mM，并于18℃，120rpm诱导表达12-16h，得到发酵液。再将发酵液经离心得到的上清液即为胶原蛋白酶液。
胶原蛋白酶的分离纯化：
(1)粗酶液的制备：将菌体重悬于Lysis buffer(50mM Tris-HCl,100mM NaCl,0.5％甘油，pH＝8.0)中，使用压力破碎仪破碎细胞，于4℃、10000rpm离心60min，所得上清即为粗酶液；
(2)镍亲和层析
镍亲和柱用Lysis buffer预处理后，将粗酶液上样至镍亲和柱，然后再用Lysis buffer平衡一个柱体积，之后用Wash buffer(50mM Tris-HCl,100mM NaCl,0.5％甘油，10mM Imidazole,pH 8.0)冲洗1个柱体积，最后用Elution buffer(50mM Tris-HCl,100mM NaCl,0.5％甘油，250mM Imidazole，pH＝8.0)洗脱目的蛋白；
(3)凝胶过滤层析
用分子筛buffer(10mM Tris-HCl,100mM NaCl,pH＝8.0)将Superdex 200 Increase column凝胶过滤层析柱平衡后，将镍亲和层析纯化后的样品浓缩后上样至层析柱，使用分子筛buffer洗脱；收集目的蛋白峰的峰尖样品，加10％甘油后-80℃保存备用；
(4)蛋白纯度检测
用聚丙烯酰胺凝胶电泳检测胶原蛋白酶48的表达情况，结果如图7所示，胶原蛋白酶48在电泳胶上呈单一条带，分子量大小在35-45kDa之间，位置与预测的分子量相吻合。
实施例8
取1mL实施例7异源诱导表达的胶原蛋白酶48，加入至10mg牛骨中进行酶解反应，酶解反应条件为：摇床转速180rpm，反应温度50℃，反应时间2h。反应产物经7000r/min，离心10min，得到的上清液即为蛋白酶解液；然后蛋白酶解液冷冻干燥后，按照实施例6中的方法处理后经HPLC分析酶解液中的胶原蛋白肽的分子量分布。结果如图8和表4所示。
表4 牛骨胶原蛋白酶解液中不同分子量肽段的相对丰度
Figure PCTCN2021136035-appb-000006
由图8和表4可知，采用本发明提供的胶原蛋白酶48的来降解牛骨，所得的胶原蛋白酶解液中不同分子量的胶原蛋白肽分子量5000Da以下的胶原蛋白肽占总肽段76％以上，分子量在1000Da以下的胶原蛋白肽占总肽段40％以上。因此，可将胶原蛋白酶48用于降解牛骨胶原蛋白制备含有不同分子量的胶原蛋白肽，特别是分子量在1000Da以下的胶原蛋白肽。

Claims (25)

  1. 一株莱茵海默氏属新菌Rheinheimera indica SM2107，该菌株2021年5月8日保存于中国典型培养物保藏中心，保藏编号CCTCC M2021506，地址：中国，武汉，武汉大学。
  2. 根据权利要求1所述的莱茵海默氏属新菌Rheinheimera indica SM2107，其特征在于，所述莱茵海默氏属新菌Rheinheimera indica SM2107的16S rDNA基因序列如SEQ ID NO.1所示。
  3. 根据权利要求1所述的莱茵海默氏属新菌Rheinheimera indica SM2107，其特征在于，所述莱茵海默氏属新菌Rheinheimera indica SM2107的生长温度范围为4～40℃，生长的NaCl浓度范围为0～10.0％(w/v)，生长的pH范围为6.5～10.5。
  4. 权利要求1所述莱茵海默氏属新菌Rheinheimera indica SM2107在制备胶原蛋白酶以及降解胶原蛋白中的应用。
  5. 如权利要求4所述的应用，其特征在于，包括步骤如下：
    (1)制备胶原蛋白酶：将莱茵海默氏属新菌Rheinheimera indica SM2107接种至发酵培养基中进行发酵培养，在摇床转速150～200rpm，培养温度23～27℃下培养4～6d，得到发酵液；再将发酵液经离心得到的上清液即为胶原蛋白酶液；
    (2)降解胶原蛋白：向胶原蛋白中加入步骤(1)得到的胶原蛋白酶液，在45～55℃的条件下反应2～3h，经离心后去除沉淀，得到的上清液即为胶原蛋白酶解液。
  6. 如权利要求5所述的应用，其特征在于，步骤(1)中，所述的发酵培养条件为：摇床转速180rpm，培养温度25℃，培养时间5d；所述的离心转速为6000～8000r/min，离心时间为8～12min。
  7. 如权利要求5所述的应用，其特征在于，步骤(2)中，所述的胶原蛋白为牛骨胶原蛋白；所述的胶原蛋白酶液的加入量为1300～1700U/g的酶料比(E/S)。
  8. 一种重组表达载体，其特征在于，该重组表达载体包含如SEQ ID NO.2所示的编码基因。
  9. 根据权利要求8所述的重组表达载体，其特征在于，所述的重组表达载体用质粒pET-22b构建。
  10. 根据权利要求9所述的重组表达载体，其特征在于，所述重组表达载体转化到宿主细胞中产生重组细胞。
  11. 根据权利要求10所述的重组表达载体，其特征在于，所述的宿主细胞为大肠杆菌；优选的，所述的大肠杆菌为大肠杆菌BL21(DE3)。
  12. 一种重组细胞，其特征在于，该重组细胞包含如SEQ ID NO.2所示的编码基因。
  13. 一种重组表达菌株，其特征在于，该菌株是对包含如SEQ ID NO.2所示编码基因的重组细胞进行培养得到。
  14. 根据权利要求13所述的重组表达菌株，其特征在于，对重组细胞进行培养的条件为：
    采用LB液体培养基，培养温度37℃，摇床培养，培养5-7h，离心分离得到重组表达菌株。
  15. 一种胶原蛋白酶，其特征在于，该胶原蛋白酶由权利要求13所述的重组表达菌株表达得到。
  16. 权利要求15所述的胶原蛋白酶，在胶原蛋白降解中的应用。
  17. 一种胶原蛋白的降解方法，包括：
    使用如SEQ ID NO.2所示的核苷酸序列，将所述核苷酸序列克隆到质粒中作为重组载体，该重组载体转化到宿主细胞中，构建得到重组细胞；在发酵培养基中培养重组细胞，得到重组表达菌株；重组表达菌株表达得到胶原蛋白酶，胶原蛋白酶对胶原蛋白进行降解。
  18. 根据权利要求17所述的胶原蛋白的降解方法，其特征在于，所述的质粒为质粒pET-22b。
  19. 根据权利要求17所述的胶原蛋白的降解方法，其特征在于，所述的宿主细胞为大肠杆菌，进一步优选为大肠杆菌BL21(DE3)。
  20. 根据权利要求17所述的胶原蛋白的降解方法，其特征在于，在发酵培养基中培养重组细胞的条件为：
    采用LB液体培养基，培养温度37℃，摇床培养，培养5-7h，离心分离得到重组表达菌株。
  21. 根据权利要求17所述的胶原蛋白的降解方法，其特征在于，胶原蛋白酶对胶原蛋白进行降解至寡肽分子量小于5000Da的肽段。
  22. 根据权利要求17所述的胶原蛋白的降解方法，其特征在于，所述的胶原蛋白来源于牛骨或鱼皮。
  23. 根据权利要求17所述的胶原蛋白的降解方法，其特征在于，胶原蛋白酶对胶原蛋白进行降解的步骤如下：
    向胶原蛋白中加入胶原蛋白酶液，在45～55℃的条件下反应2～3h，经离心后去除沉淀，得到的上清液即为胶原蛋白酶解液。
  24. 根据权利要求17所述的胶原蛋白的降解方法，其特征在于，离心的转速为6000～8000r/min，离心时间为8～12min。
  25. 根据权利要求17所述的胶原蛋白的降解方法，其特征在于，胶原蛋白酶液的加入量为1300～1700U/g的酶料比(E/S)。
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