TWI716702B - Engineered chitosanase and method of producing chitotriose - Google Patents

Abstract

The present invention discloses a protein engineered chitosanase. The preferred embodiment of the protein engineered chitosanase is the wild-type chitosanase form Bacillus circulans inserted with one or more amino acid between Histidine and Proline or their mutants.

Description

重組幾丁聚醣酶及其幾丁三醣的製造方法 Recombinant chitosanase and method for producing chitotriose

本發明係提供一種幾丁聚醣酶，尤其指一種經基因重組的芽孢桿菌屬幾丁聚醣酶。 The present invention provides a chitosanase, especially a Bacillus chitosanase that has been genetically recombined.

幾丁質(chitin)廣泛存在節肢動物的外骨骼，例如蝦殼、蟹殼、烏賊軟骨及真菌的細胞壁中，是由N-乙醯葡萄糖胺(N-acetylglucosamine)以β-1,4醣苷鍵所聚合而成的天然聚合物，也是產量僅次於纖維素與木質素的生物質。但由於其結構排列緊密而不溶於水，不利於人們利用，所以過去常被認定為是廢棄物之一。直到它的衍生物解決了溶解度的問題，才展開了另一個應用的局面，不但能降低廢棄物的處理，還在經濟上發掘出新的價值。幾丁聚醣(chitosan)就是其中一個例子。在以往的研究發現，幾丁質去乙醯化後的產物幾丁聚醣(chitosan)，在酸環境下能夠溶於水中，大幅提升能被利用的程度，現已被廣泛利用到各種產業上，例如保養品、保健食品、藥物、生醫器材及農業等。 Chitin is widely found in the exoskeletons of arthropods, such as shrimp shells, crab shells, squid cartilage and fungal cell walls. It is composed of N-acetylglucosamine with β-1,4 glycosidic bonds. The natural polymer produced by polymerization is also a biomass whose yield is second only to cellulose and lignin. However, because its structure is tightly arranged and insoluble in water, it is not conducive to people's use, so it was often regarded as one of wastes in the past. It wasn't until its derivatives solved the solubility problem that another application situation was launched, which not only reduced waste disposal, but also discovered new economic value. Chitosan (chitosan) is one example. In previous studies, it was found that chitosan, the product of deacetylation of chitin, can be dissolved in water in an acidic environment, greatly increasing the degree of utilization, and has been widely used in various industries. , Such as skin care products, health foods, drugs, biomedical equipment and agriculture.

幾丁聚醣經水解後產生約由2~20個葡萄醣胺單體所 鍵結而成的分子稱為幾丁寡醣(chito-oligosaccharides)。其分子量遠小於聚醣體，約數佰至數仟之間，比幾丁聚醣的水溶性更好。目前研究已知幾丁寡醣在植物防禦系統中扮演舉足輕重的角色，除了可活化植物體中的抗病基因大量表現之外，還具有各種生物活性，例如降低膽固醇、抗微生物與提升免疫機能，更有研究指出六個單體之幾丁寡醣具有抗腫瘤細胞等。 After being hydrolyzed, chitosan produces about 2-20 glucosamine monomers. The bonded molecules are called chito-oligosaccharides. Its molecular weight is much smaller than that of polysaccharides, between hundreds and thousands, and it has better water solubility than chitosan. Current studies have shown that chitin oligosaccharides play a pivotal role in plant defense systems. In addition to activating a large number of disease resistance genes in plants, they also have various biological activities, such as lowering cholesterol, anti-microbial and improving immune function. More studies have pointed out that the six monomeric chitosan oligosaccharides have anti-tumor cells and so on.

一般而言，製造幾丁寡醣之方式主要分為化學法、物理法及酵素法三大類。物理法因為效率不高，所以目前市面上販售之幾丁寡醣多為化學法水解之產物。但是此方法不僅須使用大量強酸、強鹼，且其水解產物不具專一性，且以一醣為主要產物，不利於特定長度幾丁寡醣的生產。反之，酵素則對生成物具有高專一性，所以以酵素法水解幾丁聚醣較有機會得到特定比例之幾丁寡醣，以適用於特定生物活性的發展。 Generally speaking, the methods of producing chitosan oligosaccharides are mainly divided into three categories: chemical method, physical method and enzyme method. Because the physical method is not efficient, most of the chitosan oligosaccharides currently on the market are the products of chemical hydrolysis. However, this method not only requires the use of a large amount of strong acids and bases, and its hydrolysate is not specific, and uses monosaccharide as the main product, which is not conducive to the production of chitosan oligosaccharides of specific length. On the contrary, enzymes are highly specific to the product, so the enzymatic hydrolysis of chitosan has a better chance of obtaining a specific proportion of chitosan oligosaccharides, which is suitable for the development of specific biological activities.

本發明係提供一種重組幾丁聚醣酶，其係在芽孢桿菌屬幾丁聚醣酶組胺酸(Histidine)至脯胺酸(Proline)或由其突變之胺基酸間***一到數個胺基酸。 The present invention provides a recombinant chitosanase in which one to several amino acids are inserted between histidine (Histidine) and proline (Proline) or the amino acid mutated from the Bacillus chitosanase Amino acid.

本發明之申請專利範圍係係另揭露，該***之胺基酸係選自由甘胺酸(Glycine)、丙胺酸(Alanine)、纈胺酸(Valine)、白胺酸(Leucine)、異白胺酸(Isoleucine)、***酸(Phenylalanine)、酪胺酸(Tyrosine)、色胺酸(Tryptophan)、組胺酸 (Histidine)、天門冬胺酸(Aspartic acid)、天門冬醯胺酸(Asparagine)、麩胺酸(Glutamic acid)、麩醯胺酸(Glutamine)、離胺酸(Lysine)、精胺酸(Arginine)、絲胺酸(Serine)、穌胺酸(Threonine)、羥脯胺酸(Hydroxyproline)、甲基胺酸(Methionine)、半胱胺酸(Cysteine)、胱胺酸(Cystine)、脯胺酸(Proline)所組成的群組。 The scope of patent application of the present invention is disclosed separately. The inserted amino acid is selected from Glycine, Alanine, Valine, Leucine, and Isoleucine. Acid (Isoleucine), Phenylalanine (Phenylalanine), Tyrosine (Tyrosine), Tryptophan (Tryptophan), Histidine (Histidine), Aspartic acid (Aspartic acid), Asparagine (Asparagine), Glutamic acid (Glutamic acid), Glutamine, Lysine (Lysine), Arginine (Arginine) ), Serine, Threonine, Hydroxyproline, Methionine, Cysteine, Cystine, Proline (Proline) group.

本發明之申請專利範圍係另揭露，該***之胺基酸係至少有一帶負電的胺基酸 The patent application scope of the present invention is disclosed separately, the inserted amino acid is at least one negatively charged amino acid

本發明之申請專利範圍係另揭露，該帶負電的胺基酸係選自由天門冬胺酸(Asparatic acid；D)、麩胺酸(Glutamic acid；E)以及組胺酸(Histidine；H)所組成的群組。 The scope of patent application of the present invention is disclosed separately. The negatively charged amino acid is selected from the group consisting of Asparatic acid (D), Glutamic acid (E) and Histidine (H). Groups formed.

本發明之申請專利範圍係另揭露，該酵素包含與SEQ ID NO：1之胺基酸至少95%一致的胺基酸序列。 The patent application scope of the present invention is disclosed separately, and the enzyme contains an amino acid sequence that is at least 95% identical to the amino acid of SEQ ID NO:1.

本發明之申請專利範圍係另揭露，該酵素在酸鹼值pH5到10之間，能保持80%的活性。 The patent application scope of the present invention is another disclosure that the enzyme can maintain 80% activity at a pH between 5 and 10.

本發明之申請專利範圍係另揭露，該酵素與幾丁聚醣反應的主要水解產物為三醣。 The patent application scope of the present invention discloses separately that the main hydrolysis product of the reaction between the enzyme and chitosan is trisaccharide.

本發明之申請專利範圍係另揭露，該酵素與幾丁聚醣反應的水解產物二醣：三醣：四醣的比例為1：8：1。 The scope of patent application of the present invention is another disclosure that the ratio of the hydrolysate of the enzyme and chitosan, disaccharide:trisaccharide:tetrasaccharide, is 1:8:1.

如前文所述幾丁寡醣具有許多生物活性，尤其是特定長度的幾丁寡醣能有更多發展，達成量產便是很重要的研究目標。現階段常見的量產產物多為幾丁二醣以下的產物，就算是生 技公司所能購買的幾丁寡醣也都要價不斐。本發明揭露之重組幾丁聚醣酶酵素，係藉由限制酵素與基質，影響產物的形成，並藉此生成特定長度的幾丁寡醣的酵素。 As mentioned above, chitin oligosaccharides have many biological activities, especially chitin oligosaccharides of a specific length can have more development, and achieving mass production is a very important research goal. The common mass-produced products at this stage are mostly products below chitobiose, even if it is raw Chito-oligosaccharides that can be purchased by technology companies are also expensive. The recombinant chitosanase enzyme disclosed in the present invention is an enzyme that restricts the enzyme and the substrate to affect the formation of the product, and thereby generates chito-oligosaccharides of a specific length.

關於本發明之優點與精神可以藉由以下的發明詳述及所附圖式得到進一步的瞭解。 The advantages and spirit of the present invention can be further understood from the following detailed description of the invention and the accompanying drawings.

第1圖為pET20b-csn-TI之質體圖譜；第2圖(a)為其為突變株誘導2.5小時後以20mM磷酸緩衝溶液(pH 7)破菌回收菌體之突變株粗酵素液SDS-PAGE圖；(b)為突變株誘導2.5小時後以0.3M NaH₂PO₄(pH 4.5)破菌後離心沉澱取蛋白質上清液之突變株粗酵素液SDS-PAGE圖；第3圖為各種突變幾丁聚醣水解酶之水解產物分析圖；第4圖為各種突變幾丁聚醣水解酶之水解產物比例圖(24小時)；第5圖為酵素pH耐受性圖；第6圖為蛋白質調整pH值後上清液蛋白質含量變化圖；第7圖為酵素最適pH圖；第8圖為酵素溫度耐受性圖；第9圖為酵素最適溫度圖；第10圖為野生型BWT與突變株TI-D長時間水解反應圖；第11圖為野生型BWT與突變株TI-D水解型式圖； 第12圖(a)-(d)，其為幾丁聚醣酶TI-D的開口與閉口型結構模擬圖；第13圖為幾丁聚醣酶BWT與基質結合模擬圖；第14圖為幾丁聚醣酶TI-D與基質結合模擬圖；第15圖為幾丁聚醣酶TI-D水解基質產生三醣之模擬示意圖；第16圖為幾丁聚醣酶TI-D水解基質產生四醣之模擬示意圖；第17圖為野生型BWT與突變株TI-D長時間水解產物走勢圖；第18圖為回收寡醣粉末產物分析圖；以及第19圖為為回收產物的二、三、四醣之比例圖。 The first picture shows the plastid map of pET20b-csn-TI; the second picture (a) shows the crude enzyme solution SDS of the mutant strain recovered by breaking the bacteria with 20mM phosphate buffer solution (pH 7) after 2.5 hours of induction of the mutant strain -PAGE chart; (b) is the mutant strain's crude enzyme solution SDS-PAGE chart of the mutant strain's crude enzyme solution after 2.5 hours after induction of the mutant strain with 0.3M NaH ₂ PO ₄ (pH 4.5) and centrifuged to precipitate the protein supernatant; Analysis diagram of the hydrolysate of various mutant chitosan hydrolases; Figure 4 is the ratio of hydrolysates of various mutant chitosan hydrolases (24 hours); Figure 5 is the enzyme pH tolerance diagram; Figure 6 It is the graph of the change of the supernatant protein content after the pH value of the protein is adjusted; Figure 7 is the optimal pH graph of the enzyme; Figure 8 is the temperature tolerance graph of the enzyme; Figure 9 is the optimal temperature graph of the enzyme; Figure 10 is the wild-type BWT Figure 11 shows the hydrolysis pattern of wild-type BWT and mutant TI-D; Figure 12 (a)-(d), which are chitinase TI-D Figure 13 is a simulation diagram of the binding of chitosanase BWT to the substrate; Figure 14 is a simulation diagram of the binding of chitosanase TI-D to the substrate; Figure 15 is a simulation diagram of the binding of chitinase TI-D to the substrate; Carbohydrase TI-D hydrolyzes the substrate to produce trisaccharides; Figure 16 is the chitosanase TI-D hydrolyzes substrate to produce tetrasaccharides; Figure 17 is the wild-type BWT and the mutant strain TI-D for a long time The hydrolysate trend chart; Picture 18 is the analysis chart of the recovered oligosaccharide powder product; and Picture 19 is the ratio chart of the di-, tri-, and tetra-saccharide of the recovered product.

芽孢桿菌屬MH-K1幾丁聚醣酶對幾丁聚醣的水解產物為幾丁二醣到五醣的混合物。為了降低產物中的二醣比例，首先設計破壞酵素上相對應於基質結合位-2醣基位置的第216與217個胺基酸，並以飽和突變法成功取得一可降低對幾丁四醣的分解能力的突變株A216T/L217I(以TI表示此突變株)。為了進一步提高幾丁三醣的比例並降低二醣的產物的產生，設計將TI***一個帶負電檔板，使酵素與基質的結合位置受到限制，並增加對基質吸引力。 The hydrolysis product of chitosan by Bacillus MH-K1 chitinase is a mixture of chitobiose to pentasaccharide. In order to reduce the ratio of disaccharides in the product, first designed to destroy the 216th and 217th amino acids corresponding to the -2 glycosyl position of the substrate binding site on the enzyme, and successfully achieved a reduction of chitotetraose by the saturation mutation method. The mutant strain A216T/L217I (this mutant strain is represented by TI). In order to further increase the ratio of chitotriose and reduce the production of disaccharide products, TI was designed to be inserted into a negatively charged baffle plate to limit the binding position of the enzyme and the substrate and increase the attraction to the substrate.

經蛋白質結構模擬後決定在第117與118胺基酸中間***擋板，以定點突變得到突變株TI-D、TI-HD、TI-EHD。所有實驗用酵素包括野生型(BWT)都是由大腸桿菌生產，並用0.3M Na₂HPO₄選擇性沉澱法純化。以TLC計算總產物中三醣的莫耳 比例，BWT為30%、TI為58%、TI-D為84%、TI-HD為34%、TI-EHD為2%。將酵素TI-D以液相層析法純化後，與選擇性沉澱法比較酵素活性與產物差異。決定以選擇性沉澱法做為主要實驗純化方式。以BWT做為對照進行特性測定。TI-D與BWT兩種酵素在溫度50℃與pH6.5都有最佳活性。在溫度20~40℃與pH4~10環境經過一小時後，BWT酵素活性能保留80%以上。而TI-D在溫度20~40℃與pH 5~10環境一小時後，酵素活性能保留80%以上。 After protein structure simulation, it was decided to insert a baffle between the 117th and 118th amino acids to obtain mutant strains TI-D, TI-HD, and TI-EHD by site-directed mutation. All experimental enzymes including wild type (BWT) are produced by E. coli and purified by selective precipitation with 0.3M Na ₂ HPO ₄ . Calculating the molar ratio of trisaccharides in the total product by TLC, BWT was 30%, TI was 58%, TI-D was 84%, TI-HD was 34%, and TI-EHD was 2%. After the enzyme TI-D was purified by liquid chromatography, the enzyme activity and product difference were compared with the selective precipitation method. It was decided to use selective precipitation as the main experimental purification method. Use BWT as a control for characterization. Both TI-D and BWT enzymes have the best activity at a temperature of 50°C and pH 6.5. After one hour at a temperature of 20-40℃ and a pH of 4-10, BWT enzyme activity can retain more than 80%. And TI-D can retain more than 80% of the enzyme activity after one hour at a temperature of 20-40°C and a pH of 5-10.

取2500U的酵素TI-D對5%幾丁聚醣10L溶液進行72小時反應。能得到80%為三醣，平均寡醣長度為3.8的幾丁聚醣水解產物。10L的幾丁聚醣反應產物利用IPA沉澱，能回收約8成的產物，產物平均寡醣長度約6，其中70%為三醣產物。 Take 2500U of enzyme TI-D to react with 5% chitosan 10L solution for 72 hours. A chitosan hydrolysate with 80% trisaccharides and an average oligosaccharide length of 3.8 can be obtained. The 10L chitosan reaction product is precipitated by IPA, and about 80% of the product can be recovered. The average oligosaccharide length of the product is about 6, of which 70% are trisaccharide products.

以下為詳細實驗方法及實驗結果。 The following are detailed experimental methods and experimental results.

實驗方法 experimental method

1.藥品配製 1. Pharmaceutical preparation

LB培養基：每1L含10g Tryptone、5g Yeast extract及5g NaCl。(LBA培養基為其含有0.1mg/mL Ampicillin) LB medium: each 1L contains 10g Tryptone, 5g Yeast extract and 5g NaCl. (LBA medium contains 0.1mg/mL Ampicillin)

LB培養盤：LB培養基每1L添加20g Agar。(LBA培養盤為其含有0.1mg/mL Ampicillin) LB culture plate: add 20g Agar per 1L of LB medium. (LBA culture dish contains 0.1mg/mL Ampicillin)

5x M9培養基：每1L含有30g Na₂HPO₄、15g KH₂PO₄、2.5g NaCl及5g NH₄Cl。 5x M9 medium: each 1L contains 30g Na ₂ HPO ₄ , 15g KH ₂ PO ₄ , 2.5g NaCl and 5g NH ₄ Cl.

TSS：每1L含73mL LB培養基、20mL 50% PEG8000、5mL DMSO及2mL 1M MgSO₄。 TSS: Each 1L contains 73mL LB medium, 20mL 50% PEG8000, 5mL DMSO and 2mL 1M MgSO ₄ .

5×KCM：每1L含37.25g KCl、22.05g CaCl₂及5.075g MgCl₂。 5×KCM: Each 1L contains 37.25g KCl, 22.05g CaCl ₂ and 5.075g MgCl ₂ .

Phosphate buffer(pH 7)：1M NaH₂PO₄和1M Na₂HPO₄依1：2.24比例混合，此為1M儲存液，使用時稀釋至20mM。 Phosphate buffer (pH 7): 1M NaH ₂ PO ₄ and 1M Na ₂ HPO ₄ are mixed at a ratio of 1:2.24. This is a 1M stock solution, which is diluted to 20mM when used.

退染劑：50% Ethanol、40% Acetic acid、10% ddH₂O。 Destaining agent: 50% Ethanol, 40% Acetic acid, 10% ddH ₂ O.

1%幾丁聚醣：每100mL含有1g幾丁聚醣和0.7mL Acetic acid。 1% Chitosan: Each 100mL contains 1g chitosan and 0.7mL Acetic acid.

DNS試劑：每1L含300g酒石酸鉀鈉、4.8g 3,5-Dinitrosalicylic acid及25.35g NaOH。 DNS reagent: each 1L contains 300g sodium potassium tartrate, 4.8g 3,5-Dinitrosalicylic acid and 25.35g NaOH.

TLC展開液：28% Amino water和1-Propanol以2：1.5的比例混合使用。 TLC developing solution: 28% Amino water and 1-Propanol are mixed in a ratio of 2:1.5.

2.質體的選用 2. Selection of plastids

本實驗選用的質體為pET20b-csn-TI，請參見第1圖，其為pET20b-csn-TI之質體圖譜。質體全長為4373bp，其包含783bp的MH-K1幾丁聚醣酶基因片段包含一個轉譯起始密碼子ATG，經轉譯轉錄作用後可生成260個胺基酸，分子量約29.1kDa。質體上帶有從MH-K1選殖過來的幾丁聚醣酶基因片段，並且經過飽和突變(引子序列係如序列識別號(SEQ ID NO)：6及7所示)，挑選出的A216T/L217I之突變株。 The plastid used in this experiment is pET20b-csn-TI, please refer to Figure 1, which is the plastid map of pET20b-csn-TI. The plastid has a full length of 4373bp, which contains a 783bp MH-K1 chitinase gene fragment containing a translation initiation codon ATG, which can generate 260 amino acids after translation and transcription, with a molecular weight of about 29.1kDa. The plastid contains the chitosanase gene fragment selected from MH-K1, and has undergone saturation mutation (the primer sequence is shown in SEQ ID NO: 6 and 7), and the selected A216T /L217I mutant strain.

3.基因定點突變 3. Gene site-directed mutation

以pET20b-csn-TI質體做為模板，使用設計的引子(如表1)進行PCR。PCR條件設定為：95℃加熱5分鐘，95℃變性30秒、55℃黏合1分鐘、68℃延長10分鐘，重復16個循環，10分鐘內從68℃降溫至15℃。PCR產物取20μL，加入8μL 10倍的緩衝溶液、補充無菌水至80μL及0.5μL DpnI混勻，置於37℃反應2小時。以55℃加熱10分鐘後冰浴。加入20μL之5×KCM，將混合液加入E.coli DH5α勝任細胞中，使質體轉形到勝任細胞中。於37℃震盪培養1小時後，將所有菌液塗於LBA培養盤上，置於37℃隔夜培養。 The pET20b-csn-TI plastid was used as a template and the designed primers (as shown in Table 1) were used for PCR. The PCR conditions were set as follows: heating at 95°C for 5 minutes, denaturing at 95°C for 30 seconds, bonding at 55°C for 1 minute, and extending at 68°C for 10 minutes, repeating 16 cycles, and cooling from 68°C to 15°C within 10 minutes. Take 20μL of PCR product, add 8μL of 10-fold buffer solution, add sterile water to 80μL and 0.5μL Dpn I to mix well, and place at 37°C for 2 hours. Heat at 55°C for 10 minutes and then ice bath. Add 20μL of 5×KCM, add the mixture to E.coli DH5α competent cells to transform the plastids into competent cells. After shaking culture at 37°C for 1 hour, spread all the bacterial liquid on the LBA culture plate and place it at 37°C for overnight culture.

挑選培養盤上單一菌落，接種到5mL LBA培養基中，於37℃培養約12小時後，取1mL的菌液抽取其質體。將質體轉形至E.coli BL21(DE3)中誘導表現並篩選突變之幾丁聚醣酶。突變株進行核酸定序，確認突變後的胺基酸序列。 Pick a single colony on the culture plate, inoculate it into 5mL LBA medium, culture at 37°C for about 12 hours, then take 1mL of bacterial solution to extract its plastids. Transform the plastids into E.coli BL21 (DE3) to induce expression and screen for mutant chitosanase. The mutant strains undergo nucleic acid sequencing to confirm the amino acid sequence after mutation.

4.質體的轉形 4. Transformation of plastids

4.1 勝任細胞的製備 4.1 Preparation of competent cells

取單一勝任細胞的菌落(E.coli DH5α或E.coli BL21(DE3))接種至5mL LB培養基中，於37℃培養約12小時。取1mL培養後菌液至100mL LB培養基進行放大培養，於37℃震盪培養至OD600nm=0.5(約2小時)冰浴5分鐘，以5,000rpm離心10分鐘並去除上清液，加入1mL冰的TSS並懸浮菌體。每100μL分裝成1管，急速冷凍(鹽酒精或液態氮)後保存於-80℃。 A single competent cell colony ( E.coli DH5α or E.coli BL21(DE3)) was inoculated into 5mL LB medium and cultured at 37°C for about 12 hours. Take 1 mL of the cultured bacteria liquid to 100 mL of LB medium for amplification culture, shake culture at 37°C until OD600nm=0.5 (about 2 hours) in an ice bath for 5 minutes, centrifuge at 5,000 rpm for 10 minutes, remove the supernatant, and add 1 mL of ice TSS And suspend the bacteria. Each 100μL aliquoted into 1 tube, quickly frozen (salt alcohol or liquid nitrogen) and stored at -80°C.

4.2 質體的轉形 4.2 Transformation of plastids

使用熱休克法(Heat shock)轉形。混合1μL(約60μg)質體、20μL 5×KCM及79μL無菌水，並冰浴5分鐘。將100μL質體混合液加入100μL勝任細胞中，輕輕攪拌混勻後冰浴20分鐘，再以42℃恆溫水浴加熱45秒後，迅速置於冰上2分鐘。加入800μL LB培養基於37℃輕搖培養1小時。以10至1,000倍體積LB培養基序列稀釋，再分別塗100μL稀釋菌液於LBA培養盤，於37℃隔夜培養。 Use heat shock to transform. Mix 1 μL (about 60 μg) plastids, 20 μL 5×KCM and 79 μL sterile water, and ice bath for 5 minutes. Add 100 μL of the plastid mixture to 100 μL of competent cells, gently stir and mix, and then ice-bath for 20 minutes, then heat it in a 42°C constant temperature water bath for 45 seconds, and then quickly place it on ice for 2 minutes. Add 800μL of LB medium and culture at 37°C for 1 hour with gentle shaking. Dilute serially with 10 to 1,000 times the volume of LB medium, and then apply 100 μL of diluted bacterial solution to LBA culture plate, and incubate overnight at 37°C.

5.原位篩選法(In-situ screening) 5. In-situ screening

5.1 幾丁聚醣水解酶檢測盤(Chitosanase-detection agar plate)的製備 5.1 Preparation of Chitosanase-detection agar plate

秤取3.20g之幾丁聚醣於含有338mL無菌水的1L血清瓶中混勻，另外秤取8g洋菜膠(Agar)含有300mL無菌水的1L血清瓶中混勻。再取160mL的5x M9培養基於250mL之血清瓶中，上述溶液皆以121℃滅菌20分鐘處理，滅菌後溶液皆以70℃保溫。 一邊攪拌幾丁聚醣溶液，一邊緩緩加入1.7mL冰醋酸，再使用磁石快速攪拌溶解後之幾丁聚醣溶液，同時緩慢加入滅菌後的160mL 5x M9培養基，此時溶液呈白色混濁狀。再加入1.6mL 1M MgSO₄與80μL 1M CaCl₂並混勻，確保此混合液為70℃，並與保溫於70℃之洋菜膠混合均勻(使用磁石攪拌)，一邊攪拌一邊使用1mL微量吸取器將氣泡取出。去除氣泡之溶液每15mL分裝至1盤培養盤中，待其凝固並使用UV照射20分鐘後，置於4℃備用。此為0.4%幾丁聚醣水解酶檢測盤(Cheng and Li,2000)。 Weigh 3.20 g of chitosan in a 1 L serum bottle containing 338 mL of sterile water and mix well. In addition, weigh 8 g of agar gum (Agar) in a 1 L serum bottle containing 300 mL of sterile water and mix well. Then take 160 mL of 5x M9 medium in a 250 mL serum bottle. The above solutions were sterilized at 121°C for 20 minutes. After sterilization, the solutions were all incubated at 70°C. While stirring the chitosan solution, slowly add 1.7mL of glacial acetic acid, then use a magnet to quickly stir the dissolved chitosan solution, and slowly add 160mL of sterilized 5x M9 medium. At this time, the solution is white and turbid. Then add 1.6mL 1M MgSO ₄ and 80μL 1M CaCl ₂ and mix well to ensure that the mixture is at 70°C, and mix well with the agar gum which is kept at 70°C (use a magnet for stirring), use a 1mL micropipette while stirring Take out the bubbles. Dispense the bubble-removing solution into a culture dish every 15mL, wait for it to solidify and irradiate it with UV for 20 minutes, then place it at 4°C for use. This is a 0.4% chitosan hydrolase test panel (Cheng and Li, 2000).

5.2 原位篩選法的產物組成分析 5.2 Product composition analysis of in-situ screening method

將幾丁聚醣水解酶檢測盤以每格1平方公分畫分為36格，以接種環取單一菌落，並點於畫分完成之幾丁聚醣水解酶檢測盤，靜置於37℃反應。待反應區域產生透明環(Clear zone)後，挖取約5μL體積之透明環邊緣的膠體於1.5mL微量離心管中，加入5μL之無菌水並搗碎膠體。以微量吸取器吸取0.5μL之搗碎液，並點於TLC上，重覆吸取搗碎液共5次。再以體積比2：1之氨水及正丙醇於55℃展開20分鐘，而後使用100℃加熱板烤乾。使用0.1% Ninhydrin浸濕後，於100℃加熱板加熱至呈現紫色。 Divide the chitosan hydrolase test disk into 36 cells with 1 square centimeter per cell, take a single colony with an inoculation loop, and click on the finished chitosan hydrolase test disk, and place it at 37℃ for reaction . After a clear zone is formed in the reaction area, pick up about 5 μL of the gel at the edge of the clear ring in a 1.5 mL microcentrifuge tube, add 5 μL of sterile water and mash the gel. Use a micropipette to suck up 0.5 μL of the mashed liquid and place it on the TLC. Repeat the mashed liquid 5 times. Then expand the ammonia water and n-propanol with a volume ratio of 2:1 at 55°C for 20 minutes, and then use a 100°C hot plate to bake. After soaking in 0.1% Ninhydrin, heat it on a hot plate at 100°C until it appears purple.

5.蛋白質的誘導表達 5. Induced expression of protein

取單一菌落於含5mL LBA培養基的試管中，經過37℃震盪培養12小時後，將菌液加入到50mL LBA培養基中並加入50μL 1M IPTG，於37℃誘導2.5小時，最後以4℃、12,000rpm離 心10分鐘，取得誘導後之菌體。 Take a single colony in a test tube containing 5mL LBA medium. After shaking at 37°C for 12 hours, add the bacterial solution to 50mL LBA medium and add 50μL 1M IPTG. Induce at 37°C for 2.5 hours, and finally at 4°C, 12,000rpm from Heart for 10 minutes, get the induced bacteria.

7.酵素的取得與純化 7. Acquisition and purification of enzymes

7.1 破菌取得酵素 7.1 Obtaining enzymes by breaking bacteria

將誘導表達後的菌塊用5mL 20M磷酸緩衝液(phosphate buffer，pH 7)進行懸浮。以超音波細胞破碎機破菌，設定為功率20%(最高功率400W)，每次作功1秒停止2秒，作功時間10分鐘。將破菌液於4℃、12,000rpm離心20分鐘，取上清液。以相同條件再次離心，取得之上清液即粗萃酵素。 The bacterial block after the induction of expression was suspended in 5 mL 20M phosphate buffer (pH 7). Use an ultrasonic cell crusher to break the bacteria, set the power to 20% (maximum power 400W), and stop for 2 seconds every 1 second, and the working time is 10 minutes. The bacteriolytic solution was centrifuged at 4°C and 12,000 rpm for 20 minutes, and the supernatant was taken. Centrifuge again under the same conditions to obtain the supernatant, the crude enzyme.

7.2 選擇性沉澱法 7.2 Selective precipitation method

將誘導表達後之離心菌塊用5mL 0.3M NaH₂PO₄(pH 4.5)懸浮。以超音波細胞破碎機破菌，設定為功率20%(最高功率400W)，每次作功1秒停止2秒，作功時間10分鐘。細胞裂解液於4℃、12,000rpm離心20分鐘，取上清液在4℃冰箱靜置12小時。以相同條件再次離心，取得之上清液即經過簡單純化的酵素。 The centrifuged bacterial block after the induction of expression was suspended with 5 mL 0.3M NaH ₂ PO ₄ (pH 4.5). Use an ultrasonic cell crusher to break the bacteria, set the power to 20% (maximum power 400W), and stop for 2 seconds every 1 second, and the working time is 10 minutes. The cell lysate was centrifuged at 4°C and 12,000 rpm for 20 minutes, and the supernatant was taken and placed in a refrigerator at 4°C for 12 hours. Centrifuge again under the same conditions to obtain the supernatant, which is simply purified enzyme.

7.3 FPLC液相層析法 7.3 FPLC liquid chromatography

取得粗萃酵素經0.22μm濾膜過濾後，利用MWCO 6KD的半透膜與20mM pH 7.0之磷酸緩衝溶液透析12小時，離心備用。再利用陰離子交換樹脂管柱(HiTrap^TM in 5ml Q-Sepharose,Amersham Biosciences)純化目標蛋白質。先以20mM pH 7.0之磷 酸緩衝溶液25mL平衡管柱，再將透析後的粗萃酵素通入管柱中，並以相同緩衝溶液流洗至A280訊號平穩。之後以含有0至1000mM氯化鈉之20mM pH 7.0磷酸緩衝溶液梯度流洗，並收集具有幾丁聚醣水解酶活性之波峰。 After the obtained crude enzyme was filtered through a 0.22μm filter membrane, it was dialyzed with a MWCO 6KD semipermeable membrane and 20mM pH 7.0 phosphate buffer solution for 12 hours, and centrifuged for use. Then use anion exchange resin column (HiTrap ^TM in 5ml Q-Sepharose, Amersham Biosciences) to purify the target protein. First equilibrate the column with 25mL of 20mM pH 7.0 phosphate buffer solution, then pass the dialyzed crude enzyme into the column, and wash with the same buffer solution until the A280 signal is stable. Then, it was washed with a gradient flow of 20 mM pH 7.0 phosphate buffer solution containing 0 to 1000 mM sodium chloride, and the peaks with chitosan hydrolase activity were collected.

8.蛋白質分析 8. Protein analysis

為了對酵素蛋白質的純度及分子量大小分析。以12% SDS-PAGE分離不同分子量之蛋白質，再以Coomassie brilliant blue G-250染色，之後以退染劑與水交互退染至背景透明。利用影像分析儀分析目標蛋白佔總蛋白量的百分比。蛋白質濃度使用protein assay kit(Bio-Rad)測定。以小牛血清蛋白(Bovine Serum Albumin)繪製標準曲線。 In order to analyze the purity and molecular weight of the enzyme protein. Separate proteins of different molecular weights by 12% SDS-PAGE, stain them with Coomassie brilliant blue G-250, and then de-stain them with a destaining agent and water until the background is transparent. Use an image analyzer to analyze the percentage of target protein to total protein. The protein concentration was measured using protein assay kit (Bio-Rad). The standard curve was drawn with Bovine Serum Albumin.

9.酵素活性分析 9. Enzyme activity analysis

使用DNS(Dinitrosalicylic-acid)測定幾丁聚醣經酵素反應後的還原醣含量作為酵素活性分析方法。適當稀釋之酵素與1%幾丁聚醣分別於37℃預熱3分鐘後，以1：1比例混勻，於37℃反應30分鐘。加入等體積之DNS試劑混勻，以95℃加熱15分鐘後，4℃、12,000rpm離心5分鐘。取200μL上清液以微量分析儀測定OD540nm之吸光值。以2~12mM的葡萄糖胺做標準品，繪製標準曲線。酵素活性1U的定義為：於37℃下每分鐘產生相當於1μmol還原端之酵素量。 DNS (Dinitrosalicylic-acid) was used to measure the reducing sugar content of chitosan after enzymatic reaction as an enzyme activity analysis method. Appropriately diluted enzyme and 1% chitosan were preheated at 37°C for 3 minutes, then mixed at a ratio of 1:1, and reacted at 37°C for 30 minutes. Add an equal volume of DNS reagent and mix well, heat at 95°C for 15 minutes, and centrifuge at 4°C and 12,000 rpm for 5 minutes. Take 200μL of the supernatant and measure the absorbance at OD540nm with a microanalyzer. Use 2~12mM glucosamine as standard to draw a standard curve. Enzyme activity 1U is defined as the amount of enzyme that produces 1μmol of reducing end per minute at 37°C.

10.薄層層析法(TLC)分析產物組成 10. Analysis of product composition by thin layer chromatography (TLC)

TLC片(Merck，TLC Silica gel 60)裁切成8cm×8cm高，於展開下方1cm處畫樣品起始線。將0.2U/mL酵素與1%幾丁聚醣1：1混合於37℃反應，取不同反應時間的產物，取適當體積(0.5~1μL)點於TLC片並以80℃烘乾。TLC片置於展開槽內使用展開液進行展開，當展開液到達TLC片頂端時停止反應。最後以120℃烤乾並噴0.3%Ninhydrin再次烤乾呈色。分別以1μL 2、4、6mM葡萄糖胺作為定量標準。 The TLC sheet (Merck, TLC Silica gel 60) was cut into 8cm×8cm high, and the starting line of the sample was drawn 1cm below the unfolding. Mix 0.2U/mL enzyme and 1% chitosan 1:1 to react at 37°C, take the products with different reaction times, take appropriate volume (0.5~1μL) and place on TLC tablets and dry at 80°C. The TLC film is placed in the developing tank and the developing solution is used for developing, and the reaction is stopped when the developing solution reaches the top of the TLC film. Finally, it was baked at 120°C and sprayed with 0.3% Ninhydrin and baked again to give it a color. 1μL 2, 4, and 6mM glucosamine were used as quantitative standards.

呈色後的TLC片使用影像分析軟體量化。以葡萄糖胺定量2至6聚合度之幾丁寡醣的莫爾數後，計算TLC片上的產物的濃度，並換算成百分比。 The developed TLC film is quantified using image analysis software. After quantifying the molar number of chitosan oligosaccharides with a degree of polymerization of 2 to 6 with glucosamine, the concentration of the product on the TLC chip was calculated and converted into a percentage.

11.蛋白質結構模擬與預測 11. Protein structure simulation and prediction

11.1 蛋白質三級結構模擬 11.1 Simulation of protein tertiary structure

以PyMOL觀看B.circulans MH-K1幾丁聚醣酶在GH46家族中已有的蛋白質結晶結構1QGI與4OLT。尋找適合***擋板結構的位置，並利用SWISS-MODEL(http：//swissmodel.expasy.org/)模擬MH-K1幾丁聚醣酶***設計檔板後，酵素的結構變化以及對基質結合的影響。 View the protein crystal structures 1QGI and 4OLT of B.circulans MH-K1 chitinase in the GH46 family with PyMOL. Find a suitable position for inserting the baffle structure, and use SWISS-MODEL (http://swissmodel.expasy.org/) to simulate the structural changes of the enzyme and the binding to the substrate after the MH-K1 chitosanase is inserted into the design baffle. influences.

11.2 酵素與基質對接模擬(docking) 11.2 Docking simulation of enzyme and substrate

利用分子對接軟體iGEMDOCK Version 2.1模擬基質於酵素水解時的位置。在Protein Ligand Docking下，於Prepare Binding Site下選擇酵素結構(.pdb)，於Prepare Compounds下選擇基質結構(.mol)。於Docking Accuracy Setting分別設定Population size、Generations及Number of solutions為400、80及10，並選擇進行隨機對接模擬。 Use the molecular docking software iGEMDOCK Version 2.1 to simulate the position of the substrate during enzyme hydrolysis. Under Protein Ligand Docking, in Prepare Select the enzyme structure (.pdb) under Binding Site, and select the matrix structure (.mol) under Prepare Compounds. Set Population size, Generations and Number of solutions to 400, 80, and 10 respectively in Docking Accuracy Setting, and choose random docking simulation.

12.酵素的最適pH與溫度之耐受性測試 12. The optimum pH and temperature tolerance test of the enzyme

使用britton-robinson buffer做為pH緩衝溶液。配製100ml三酸混合液(磷酸、乙酸、硼酸，濃度均為0.04mol/L)中，加入指定體積的0.2M NaOH，配置出相應pH值的緩衝溶液。 Use britton-robinson buffer as the pH buffer solution. Prepare 100ml of the triacid mixture (phosphoric acid, acetic acid, boric acid, all with a concentration of 0.04mol/L), add a specified volume of 0.2M NaOH to prepare a buffer solution with corresponding pH value.

12.1 pH值穩定度 12.1 pH stability

將測定樣品在不同pH值緩衝溶液依比例1：2混合(例如：酵素100μL+緩衝溶液200μL)，在37℃放置60分鐘。反應後之酵素以pH值緩衝溶液調回pH 6.0，再分析其酵素活性(詳見9.酵素活性分析)。 Mix the measurement samples in buffer solutions with different pH values in a ratio of 1:2 (for example: enzyme 100μL + buffer solution 200μL), and place at 37°C for 60 minutes. After the reaction, the enzyme is adjusted back to pH 6.0 with a pH buffer solution, and then its enzyme activity is analyzed (see 9. Enzyme Activity Analysis for details).

12.2 最佳活性pH值測定 12.2 Determination of optimal active pH

將測定樣品在不同pH值緩衝溶液依比例1：2混合(例如：酵素100μL+緩衝溶液200μL)，在37℃放置5分鐘，與1%幾丁聚醣以1：1(例如：調整過pH的酵素300μL+1%幾丁聚醣300μL)比例加入混勻，於37℃反應30分鐘，加入等體積之DNS試劑混勻，以95℃加熱15分鐘後，4℃、12,000rpm離心5分鐘。取200μL上清液以微量分析儀測定OD540nm之吸光值。 Mix the test sample in a buffer solution with different pH values in a ratio of 1:2 (for example: enzyme 100μL+buffer solution 200μL), place it at 37°C for 5 minutes, and 1% chitosan at a ratio of 1:1 (for example: pH adjusted Enzyme 300μL + 1% chitosan 300μL) was added and mixed, reacted at 37°C for 30 minutes, added an equal volume of DNS reagent and mixed well, heated at 95°C for 15 minutes, and centrifuged at 4°C and 12,000 rpm for 5 minutes. Take 200μL of the supernatant and measure the absorbance at OD540nm with a microanalyzer.

12.3 酵素熱穩定度 12.3 Enzyme thermal stability

將測定酵素與1%幾丁聚醣個別放置於不同的溫度下60分鐘，之後分析其酵素活性(詳見9.酵素活性分析)。 Place the assay enzyme and 1% chitosan separately at different temperatures for 60 minutes, and then analyze the enzyme activity (see 9. Enzyme Activity Analysis for details).

12.4 最佳溫度測定 12.4 Determination of optimal temperature

將測定酵素與1%幾丁聚醣以1：1比例加入混勻，於不同溫度下反應30分鐘，加入等體積之DNS試劑混勻，以95℃加熱15分鐘後，4℃、12,000rpm離心5分鐘。取200μL上清液以微量分析儀測定OD540nm之吸光值。 Add assay enzyme and 1% chitosan in a ratio of 1:1 and mix, react at different temperatures for 30 minutes, add an equal volume of DNS reagent and mix well, heat at 95°C for 15 minutes, and centrifuge at 4°C, 12,000rpm 5 minutes. Take 200μL of the supernatant and measure the absorbance at OD540nm with a microanalyzer.

13.量產幾丁三醣 13. Mass production of chitosan

13.1 量產幾丁聚醣酵素 13.1 Mass production of chitosan enzyme

取單一菌落於含5mL LBA培養基的試管中，經過37℃震盪培養12小時後，將菌液加入50mL LBA培養基中於37℃繼續培養8小時，之後將全部的菌液在常溫下以5,000rpm離心10分鐘去除上液，將離心下來的菌塊加入到500mL LBA培養基並加入500μL 1M IPTG，於37℃誘導2.5小時，最後以4℃、12,000rpm離心10分鐘，取得誘導後之菌體。 Take a single colony in a test tube containing 5mL LBA medium. After shaking and culture at 37°C for 12 hours, add the bacterial solution to 50mL LBA medium and continue culturing at 37°C for 8 hours, and then centrifuge the entire bacterial solution at 5,000rpm at room temperature. Remove the supernatant in 10 minutes, add the centrifuged bacterial mass to 500mL LBA medium and add 500μL 1M IPTG, induce at 37°C for 2.5 hours, and finally centrifuge at 4°C and 12,000rpm for 10 minutes to obtain the induced bacteria.

13.2 量產幾丁寡醣 13.2 Mass production of oligochitosan

取500g幾丁聚醣加入9.65L的水攪拌懸浮，攪拌過程中加入350mL的醋酸。加強攪拌強度直到液體變成黏稠狀。取2500U的幾丁聚醣酵素加入溶液中，於常溫下反應48-72小 時。定時取樣保存於-20℃冰箱，以TLC分析酵素的產物。 Take 500g of chitosan and add 9.65L of water to stir and suspend, add 350mL of acetic acid during stirring. Increase the stirring intensity until the liquid becomes viscous. Add 2500U of chitosan enzyme to the solution and react for 48-72 hours at room temperature Time. Regular samples are stored in the refrigerator at -20℃, and the enzyme products are analyzed by TLC.

13.3 IPA沉澱法 13.3 IPA precipitation method

將反應後的幾丁聚醣產物，以NaOH調整pH值成4、7、12三種不同酸鹼。取適當的反應產物與異丙醇用1：7的比例分別於室溫與低溫的環境進行沉澱。將沉澱物以4℃、12,000rpm離心後去除上清液體，利用冷凍乾燥機進行凍乾。 The reacted chitosan product was adjusted to pH 4, 7, 12 with NaOH. Take the appropriate reaction product and isopropanol in a ratio of 1:7 to precipitate at room temperature and low temperature respectively. The precipitate was centrifuged at 4°C and 12,000 rpm, and the supernatant liquid was removed, and then lyophilized using a freeze dryer.

實驗結果 Experimental results

根據前述實驗設計及方法，相關之實驗結果如下： According to the aforementioned experimental design and method, the relevant experimental results are as follows:

1 幾丁聚醣酶之結構模擬與突變 1 Structure simulation and mutation of chitosanase

1.1 幾丁聚醣酶的選擇 1.1 The choice of chitosanase

B.circulans MH-K1幾丁聚醣酶(GenBank：BAA01474.2)原本的開讀框(Open reading frame)為301個胺基酸。實驗用的野生型幾丁聚醣酶(BWT)重組酵素因其去除前端由42個胺基酸組成之訊息胜肽，並加入一個轉譯起始密碼子ATG，因此表現於E.coli BL21(DE3)時會形成由260個胺基酸組成之胞內蛋白，其全長胺基酶序列如SEQ ID NO：2，分子量約29.1kDa，胺基酸變異請參照表2。 The original open reading frame of B. circulans MH-K1 chitinase (GenBank: BAA01474.2) is 301 amino acids. The wild-type chitosanase (BWT) recombinant enzyme used in the experiment is expressed in E.coli BL21(DE3) because it removes the message peptide composed of 42 amino acids at the front end and adds a translation start codon ATG. ) Will form an intracellular protein composed of 260 amino acids. The full-length aminase sequence is as SEQ ID NO: 2, and the molecular weight is about 29.1kDa. Please refer to Table 2 for amino acid variation.

以野生型幾丁聚醣酶(BWT)為基礎，利用飽和突變及原位篩選法挑選出A216T、L217I的TI突變株，其表現於E.coli BL21(DE3)時形成由260個胺基酸組成之胞內蛋白，胺 基酸變異請參照表2。突變株TI可將水解產物中三醣莫耳數的比例由野生型的30%提高至58%，因此，被作為爾後定點突變設計的基本模板以繼續進行基因突變的酵素改造。本實施方式所提及的突變株與原生Bacillus circulans MH-K1 chitosanase序列(GenBank：BAA01474.2)之胺基酸變異整理請參照表2。 On the basis of wild-type chitosanase (BWT), the TI mutants of A216T and L217I were selected by saturation mutation and in situ screening methods, which formed 260 amino acids when expressed in E.coli BL21(DE3). Please refer to Table 2 for the composition of intracellular proteins and amino acid variation. Mutant TI can increase the proportion of trisaccharide moles in the hydrolysate from 30% of the wild type to 58%. Therefore, it is used as the basic template for site-directed mutagenesis design to continue genetic mutation enzyme modification. Please refer to Table 2 for the amino acid variation of the mutant strain and the native Bacillus circulans MH-K1 chitosanase sequence (GenBank: BAA01474.2) mentioned in this embodiment.

1.2 擋板結構設計 1.2 Baffle structure design

由以前的實驗得知，設計添加的胺基酸外切檔板可以用來限制酵素與基質結合後的水解產物，以及帶有電荷的胺基酸殘基會影響酵素與基質的結合方式。為了進一步改變此酵素的水解產物，決定在酵素上***帶有負電的胺基酸擋版限制酵素水解基質，造成外切的效果。以SWISS-MODEL網站模擬在GenBank：BAA01474.2之胺基酸序列的第73/74個與第184/185個胺基酸(相當於在SEQ ID NO：2之胺基酸序列的第32/33個及第143/144個胺基酸)附近***不同位置的擋板結構。最後決定在 GenBank：BAA01474.2之胺基酸序列的第117和118個胺基酸(相當於在SEQ ID NO：2之胺基酸序列的第76和77個胺基酸)中間位置***擋板。 From previous experiments, it is known that the designed and added amino acid exoside baffle can be used to limit the hydrolysis products after the enzyme binds to the substrate, and the charged amino acid residues will affect the way the enzyme binds to the substrate. In order to further change the hydrolysate of this enzyme, it was decided to insert a negatively charged amino acid block on the enzyme to restrict the enzyme from hydrolyzing the substrate, resulting in an exocytosis effect. Use the SWISS-MODEL website to simulate the 73/74th and 184/185th amino acids of the amino acid sequence in GenBank: BAA01474.2 (equivalent to the 32/th amino acid sequence in SEQ ID NO: 2 33 and 143/144th amino acids) are inserted into baffle structures at different positions. Finally decided on GenBank: The 117th and 118th amino acids of the amino acid sequence of BAA01474.2 (corresponding to the 76th and 77th amino acids of the amino acid sequence of SEQ ID NO: 2) insert a baffle at the middle position.

為了做出具有負電的外切擋板，選用帶有負電的胺基酸，天門冬胺酸與麩胺酸這兩者，並以一個帶正電的組胺酸作為中間聯接。以TI做為模板，將E、H、D三種胺基酸***GenBank：BAA01474.2之胺基酸序列的第117和118個胺基酸(相當於在SEQ ID NO：2之胺基酸序列的第76和77個胺基酸)中間，用SWISS-MODEL網站模擬其***後的結構。 In order to make a negatively charged circumscribed baffle, a negatively charged amino acid, aspartic acid and glutamic acid are selected, and a positively charged histidine is used as an intermediate connection. Using TI as a template, insert the three amino acids E, H, D into GenBank: the 117th and 118th amino acids of the amino acid sequence of BAA01474.2 (equivalent to the amino acid sequence of SEQ ID NO: 2 Between the 76th and 77th amino acid), use the SWISS-MODEL website to simulate its inserted structure.

1.3突變與結果定序 1.3 Sequencing of mutations and results

依照要***胺基酸序列來構築突變株引子(表1)，使用符合胺基酸序列的遺傳密碼子進行基因定點突變。在本實驗中是要額外***胺基酸片段，且實驗中需要改變9個核酸長度。為了要確保突變的成功率，在引子設計上採用循序漸進的突變方式。一開始先以引子TI-D進行突變，再以此突變株作為模板使用引子TI-HD突變。最後以引子TI-EHD突變成第三種突變株。每次突變完後均會送定序，以確保突變序列的正確性，將定序結果轉譯成胺基酸序列，並與做為突變用的模板水解酶進行多序列比對。結果請參照表3，突變成功的菌株分別命名為TI-D、TI-HD、TI-EHD。 Construct the mutant primers according to the amino acid sequence to be inserted (Table 1), and use the genetic code corresponding to the amino acid sequence for gene-directed mutation. In this experiment, additional amino acid fragments are inserted, and the length of 9 nucleic acids needs to be changed in the experiment. In order to ensure the success rate of mutation, a gradual mutation method is adopted in primer design. In the beginning, the primer TI-D was used for mutation, and then the mutant strain was used as a template to use the primer TI-HD mutation. Finally, the primer TI-EHD was mutated into the third mutant strain. After each mutation, the sequence will be sent to ensure the correctness of the mutation sequence, the sequence result will be translated into amino acid sequence, and multiple sequence comparison will be performed with the template hydrolase used as the mutation. Please refer to Table 3 for the results. The successfully mutated strains were named TI-D, TI-HD, and TI-EHD.

2.幾丁聚醣酶的生產與純化 2. Production and purification of chitosanase

將實驗菌株的質體轉形到E.coli BL21(DE3)之中，接菌放大培養離心後，分別使用20mM磷酸緩衝溶液(pH 7)與選擇性沉澱法0.3M NaH₂PO₄(pH 4.5)兩種不同緩衝液作為破菌的溶液進行超音波破菌，取得兩種不同純度的酵素液。以SDS-PAGE蛋白質電泳分析，請參閱第2(a)圖及第2(b)圖，其分別為突變株誘導2.5小時後以20mM磷酸緩衝溶液(pH 7)破菌回收菌體之突變株粗酵素液SDS-PAGE圖，及突變株誘導2.5小時後以0.3M NaH₂PO₄(pH 4.5)破菌後離心沉澱取蛋白質上清液之突變株粗酵素液SDS-PAGE圖。並以BWT、TI、NDTI(p.[H2_A42del；A216T；L217I；T222N；G223D]，序列如SEQ ID NO：14所示)來跟TI-D、TI-HD、TI-EHD突變株比較。計算酵素的蛋白質分子量，BWT的分子量為29082Da，突變株TI-D、TI-HD、TI-EHD的分子量分別為29227、29364、29493Da。對照第2(a)(b)圖之電泳圖上標準品的大小，確認兩種方式取得的酵素都有目標蛋白，電泳圖上突變株酵素隨著突變胺基酸的增加，分子量有逐漸上升的趨勢。從第2(a)圖來看，不管是原始的BWT、當作模板的TI，還是突變後的三株突變株都是胞內可溶的酵素。 Transform the plastids of the experimental strains into E.coli BL21(DE3), and after inoculation to enlarge culture and centrifugation, use 20mM phosphate buffer solution (pH 7) and selective precipitation method 0.3M NaH ₂ PO ₄ (pH 4.5 ) Two different buffer solutions are used as a solution for bacteriostasis to perform ultrasonic bacteriostasis, and two enzyme solutions of different purity are obtained. For protein electrophoresis analysis by SDS-PAGE, please refer to Figure 2(a) and Figure 2(b). They are the mutant strains that were recovered with 20mM phosphate buffer solution (pH 7) after 2.5 hours of induction of the mutant strain. SDS-PAGE chart of crude enzyme solution and SDS-PAGE chart of mutant strain crude enzyme solution of mutant strain after being induced 2.5 hours after induction with 0.3M NaH ₂ PO ₄ (pH 4.5) and centrifugal precipitation. BWT, TI, NDTI (p. [H2_A42del; A216T; L217I; T222N; G223D], the sequence is shown in SEQ ID NO: 14) were compared with the TI-D, TI-HD, and TI-EHD mutant strains. Calculating the protein molecular weight of the enzyme, the molecular weight of BWT is 29082Da, and the molecular weights of mutant strains TI-D, TI-HD, and TI-EHD are 29227, 29364, 29493Da, respectively. Compare the size of the standard on the electropherogram in Figure 2(a)(b) to confirm that the enzymes obtained by the two methods have the target protein. As the mutant amino acid increases, the molecular weight of the mutant enzyme on the electropherogram increases gradually. the trend of. From Figure 2(a), whether it is the original BWT, TI used as a template, or the three mutant strains after mutation, they are all intracellular soluble enzymes.

請參見第2(b)圖，選擇性沉澱法得到的酵素比20mM磷酸緩衝溶液乾淨許多。使用選擇性沉澱法破菌時，會發現沉澱物的總量會增加，而我們的目標蛋白幾丁聚醣酶不被沉澱。而宿主細胞原有的其他蛋白質則會被沉澱，達到保留酵素活性並去除非目標蛋白的目的，在用來大量製備酵素以生產幾丁寡醣時，可以做為快速純化的方式。 Please refer to Figure 2(b), the enzyme obtained by selective precipitation method is much cleaner than 20mM phosphate buffer solution. When using the selective precipitation method to break bacteria, you will find that the total amount of precipitate will increase, and our target protein chitosanase will not be precipitated. Other proteins in the host cell will be precipitated to achieve the purpose of retaining enzyme activity and removing non-target proteins. When used to prepare large quantities of enzymes to produce chitin oligosaccharides, it can be used as a rapid purification method.

3.幾丁聚醣酶的水解產物分析 3. Analysis of the hydrolysate of chitosanase

為了對TI-D、TI-HD、TI-EHD水解產物進行比較，我們挑選了BWT、TI、NDTI三種酵素來跟突變株進行對照。請參見第3圖，其為各種突變株之水解產物圖，各種突變株酵素以0.02U/mL在反應溫度為37℃條件下與1%幾丁聚醣溶液反應24小時後，取1μl以TLC分析水解產物組成。由第3圖得知TI-D的水解產物能夠將主要產物為三醣，TI-HD的主要產物為二、三醣，TI-EHD的主要產物為二醣，與模板TI的產物相比都有發生改變。在以TLC分析產物的同時，我們以2、4、6mM的葡萄糖胺作標準品，用來對產物的濃度進行定量並繪製成長條圖。請參見第4圖，其為各種突變幾丁聚醣水解酶之水解產物比例圖(24小時)，將TLC分析水解產物組成後的數據使用軟體重新計算濃度後，換算成百分比以長條圖的方式來顯示。從產物的長條圖來看，突變後的TI-D酵素生產的三醣比例高達90%，跟野生型的BWT或者是突變株的TI與NDTI相比都明顯上升。TI-HD的反應產物比例與BWT相似並沒有 太突出的變化。TI-EHD的產物是很專一的二醣產物，甚至可達到90%以上的比例。 In order to compare the hydrolysates of TI-D, TI-HD, and TI-EHD, we selected three enzymes, BWT, TI, and NDTI, to compare with the mutant strain. Please refer to Figure 3, which shows the hydrolyzed products of various mutant strains. After reacting with 1% chitosan solution at 0.02U/mL at a reaction temperature of 37℃ for 24 hours, take 1μl of the mutant strains and use TLC. Analyze the composition of the hydrolysate. Figure 3 shows that the main product of TI-D hydrolysate can be trisaccharide, the main product of TI-HD is disaccharide and trisaccharide, the main product of TI-EHD is disaccharide, compared with the product of template TI Has changed. While analyzing the product by TLC, we used 2, 4, and 6 mM glucosamine as standards to quantify the concentration of the product and draw a bar graph. Please refer to Figure 4, which is the ratio of hydrolysates of various mutant chitosan hydrolases (24 hours). After TLC analysis of the hydrolysate composition data, use software to recalculate the concentration and convert it to a percentage as a bar graph Way to display. From the bar graph of the product, the proportion of trisaccharides produced by the mutant TI-D enzyme is as high as 90%, which is significantly higher than the wild-type BWT or the TI and NDTI of the mutant strain. The reaction product ratio of TI-HD is similar to that of BWT. Changes that are too prominent. The product of TI-EHD is a very specific disaccharide product, even reaching a ratio of more than 90%.

4.突變株幾丁聚醣酶TI-D特性分析 4. Characteristic analysis of mutant chitosanase TI-D

4.1 幾丁聚醣酶TI-D液相層析法純化 4.1 Chitosanase TI-D liquid chromatography purification

為了得到更乾淨的TI-D酵素，將酵素以液相層析法進行純化(FPLC)，本發明使用純化管柱為陰離子交換樹脂管柱。先用20mM pH 7.0磷酸緩衝溶液流洗去除未吸附的蛋白質，接著以0M至1M NaCl的20mM pH 7.0磷酸緩衝溶液流洗出具有幾丁聚醣酶活性者的波峰。含有酵素活性的波鋒約於導電度12.5mS/cm時流出，此時磷酸緩衝液的鹽濃度約為200mM NaCl。 In order to obtain a cleaner TI-D enzyme, the enzyme is purified by liquid chromatography (FPLC). The present invention uses a purification column as an anion exchange resin column. First, wash with a flow of 20mM pH 7.0 phosphate buffer solution to remove unadsorbed proteins, and then wash out the peaks with chitosanase activity with a flow of 20mM pH 7.0 phosphate buffer solution of 0M to 1M NaCl. The wave front containing enzyme activity flows out when the conductivity is about 12.5mS/cm. At this time, the salt concentration of the phosphate buffer is about 200mM NaCl.

將FPLC純化後的酵素、選擇性沉澱法還有20mM磷酸緩衝溶液的酵素，以SDS-PAGE確認蛋白質純度，並以影像分析軟體計算出磷酸緩衝溶液、選擇性沉澱法、液相層析法純化之蛋白質純度，分別約為35%、55%及95%。 The enzymes purified by FPLC, selective precipitation method, and enzymes in 20mM phosphate buffer solution were used to confirm the protein purity by SDS-PAGE, and the phosphate buffer solution, selective precipitation method, and liquid chromatography purification were calculated by image analysis software. The protein purity is approximately 35%, 55% and 95% respectively.

4.2 不同方式得到幾丁聚醣酶TI-D比較 4.2 Comparison of Chitosanase TI-D obtained by different methods

表4是經過三種方式得到的酵素液活性表現，BWT與TI-D的蛋白質總量與酵素總活性隨著純化都會逐漸下降，而比活性會隨著純度增加而上升，最乾淨的FPLC純化倍率能達到近2倍。 Table 4 shows the activity performance of the enzyme solution obtained through three methods. The total protein and total enzyme activity of BWT and TI-D will gradually decrease with purification, while the specific activity will increase with the increase in purity. The cleanest FPLC purification rate Can reach nearly 2 times.

表4、菌株在0.5L培養基培養後生產和純化的酵素

Table 4. Enzymes produced and purified by the strain after cultured in 0.5L medium

調整三種方式所得到的酵素量，以相同酵素活性測定其水解產物後發現，三者的產物組成與濃度並沒有太大差異。為了減少純化時使用的步驟，加上選擇性沉澱法取得酵素的步驟基本上與20mM磷酸緩衝液相同，並且選擇性沉澱法得到的酵素比較乾淨，比活性也較高，因此後續的實驗將以選擇性沉澱法進行酵素純化。 After adjusting the amount of enzymes obtained in the three ways, and measuring the hydrolysate with the same enzyme activity, it was found that the composition and concentration of the three products did not differ much. In order to reduce the steps used in purification, plus the selective precipitation method to obtain the enzyme steps are basically the same as 20mM phosphate buffer, and the enzyme obtained by the selective precipitation method is relatively clean, and the specific activity is also higher, so subsequent experiments will be Selective precipitation method for enzyme purification.

4.3 pH耐受性與最適pH值 4.3 pH tolerance and optimal pH

作為量產前的條件測試，我們對酵素在不同溫度或酸鹼環境的耐受性進行測試，並找到最適合反應的條件。請參照第5圖，其為酵素pH耐受性圖，酵素處於各種pH環境下一小時後再調回原始的pH值與1%幾丁聚醣溶液進行反應並測定酵素活性。在第5圖發現，野生型BWT酵素活性在pH值4到10之間有著良好的酵素活性並能維持在80%以上，突變的TI-D酵素酸鹼耐受性在pH 5到10之間能保持80%以上。突變株酵素要進行保存時環境酸鹼 值必須在pH值5到10之間，若環境pH值低於4以下不利於酵素保存。 As a condition test before mass production, we test the tolerance of the enzyme at different temperatures or acid-base environments and find the most suitable conditions for the reaction. Please refer to Figure 5, which is a graph of enzyme pH tolerance. After the enzyme is exposed to various pH environments for one hour, it is adjusted back to the original pH value and reacted with 1% chitosan solution to determine the enzyme activity. In Figure 5, it is found that the wild-type BWT enzyme activity has good enzyme activity between pH 4 and 10 and can be maintained above 80%, and the acid-base tolerance of the mutant TI-D enzyme is between pH 5 and 10. Can maintain more than 80%. Environmental acid-base when the mutant enzyme needs to be preserved The value must be between pH 5 and 10. If the pH of the environment is lower than 4, it is not conducive to the preservation of enzymes.

其中特別的是在較低的pH值附近，不論是野生型或是突變型的酵素活性都會有下降再上升的趨勢。實驗中觀察到當酵素在pH 3附近時，還沒有加入做為基質的幾丁聚醣，酵素就已經有沉澱物的產生。即使將酵素的pH值調整回原始的酸鹼，酵素的沉澱物依然存在不會回溶到液體中。 Especially in the vicinity of a lower pH value, the activity of both wild-type and mutant enzymes will decrease and then increase. In the experiment, it was observed that when the enzyme was around pH 3, the enzyme had precipitated without adding chitosan as the substrate. Even if the pH value of the enzyme is adjusted back to the original acid-base value, the precipitate of the enzyme still exists and will not re-dissolve into the liquid.

為了確定這種現象，當沉澱物產生時將酵素進行離心，取上清液進行蛋白質測試。請參見第6圖，其為蛋白質調整pH值後上清液蛋白質含量變化圖。酵素處於各種pH環境下一小時後離心取上清液後，調回原始的pH值並測定蛋白質含量。從第6圖可以發現接近pH 3的時候蛋白含量有明顯的下降的現象，可以推斷在pH值3時酵素會發生蛋白質沉澱的現象，導致酵素在pH 3的活性比pH 2來的更差。 To determine this phenomenon, the enzyme was centrifuged when the precipitate was produced, and the supernatant was taken for protein testing. Please refer to Figure 6, which is a graph showing changes in the protein content of the supernatant after adjusting the pH of the protein. After the enzyme was placed in various pH environments for one hour, the supernatant was collected by centrifugation, then adjusted back to the original pH value and determined the protein content. From Figure 6, it can be found that the protein content is significantly decreased when the pH is close to 3, and it can be inferred that the enzyme will precipitate protein at pH 3, which causes the enzyme's activity at pH 3 to be worse than pH 2.

接著請參見第7圖，其為酵素最適pH圖。酵素處於各種pH環境下與1%幾丁聚醣溶液進行反應30分鐘並測定酵素活性。從第7圖可以發現，野生型與突變型酵素的最佳活性都是在接近中性的pH 6附近有著最高活性。當酵素偏向酸性時活性會漸降低，而偏向鹼性時做為基質的幾丁聚醣不溶於鹼，酵素無法對基質進行良好的水解反應。 Then please refer to Figure 7, which is the optimal pH diagram for enzymes. The enzyme is reacted with 1% chitosan solution under various pH environments for 30 minutes and the enzyme activity is measured. It can be found from Figure 7 that the best activity of wild-type and mutant enzymes is near neutral pH 6 with the highest activity. When the enzyme tends to be acidic, its activity will gradually decrease, and when it tends to be alkaline, the chitosan used as the substrate is insoluble in alkali, and the enzyme cannot perform a good hydrolysis reaction on the substrate.

在先前的耐受性實驗中我們注意到酵素在pH 3的時候活性下降，而在最適pH值實驗裡這個現象沒有出現。這是 因為最適pH值實驗的酵素在調整完pH值後就立刻與基質進行反應，這麼一來即使在pH 3酵素開始沉澱失活，在完全失活前酵素已經先水解完一部份的幾丁聚醣。這使得反應完成後在pH 3依然能測到有酵素反應。 In the previous tolerance experiment, we noticed that the enzyme activity decreased at pH 3, but this phenomenon did not appear in the optimal pH experiment. this is Because the enzyme for the optimum pH experiment immediately reacts with the substrate after adjusting the pH value, even if the enzyme begins to precipitate and inactivate at pH 3, the enzyme has already hydrolyzed a part of the chitin before it is completely inactivated. sugar. This makes it possible to detect an enzyme reaction at pH 3 after the reaction is complete.

4.4溫度耐受性與最適溫度 4.4 Temperature tolerance and optimum temperature

為了測試酵素對溫度的耐受性，將酵素處於各種溫度環境下一小時後，等回到室溫再進行反應後測定活性。請參見第8圖，其為酵素溫度耐受性圖。酵素處於各種溫度下一小時後，等回到室溫後與1%幾丁聚醣溶液進行反應並測定酵素活性。結果可以發現野生型與突變型酵素在20~40℃之間酵素都能維持80%的活性。而當溫度超過40℃之後，酵素的活性便會大幅度下降，即使回到原溫度也無法還原，TI-D甚至在50℃就已經沒有了活性。因此酵素保存要在溫度較低的環境下。 In order to test the temperature tolerance of the enzyme, the enzyme was exposed to various temperature environments for one hour, and then the activity was measured after returning to room temperature and reacting. Please refer to Figure 8, which is a graph of enzyme temperature tolerance. After the enzyme is kept at various temperatures for one hour, it will react with 1% chitosan solution after returning to room temperature and measure the enzyme activity. As a result, it can be found that both wild-type and mutant enzymes can maintain 80% of the activity between 20-40°C. When the temperature exceeds 40°C, the activity of the enzyme will drop drastically, and it cannot be reduced even if it returns to the original temperature. TI-D has no activity even at 50°C. Therefore, the enzyme should be stored in a lower temperature environment.

將野生型與突變型酵素直接與基質在不同的溫度下測試活性，結果發現在50℃最有利於這兩種酵素進行反應。請參見第9圖，其為酵素最適溫度圖。酵素處於各種溫度下與1%幾丁聚醣溶液進行反應30分鐘並測定酵素活性。但是考慮到酵素溫度耐受性超過40℃的結果，如果長時間處於較高溫的環境下會使酵素的活性下降，因此要長時間進行反應必須要降低一些溫度。若在37℃進行長時間反應，能夠有著良好的酵素活性並延長酵素的活性存留時間。在本篇中各種實驗時的溫度也依據此特性 做為參考。 The wild-type and mutant enzymes were directly tested for activity with the substrate at different temperatures, and it was found that 50°C was the most favorable for the reaction of these two enzymes. Please refer to Figure 9, which is the optimum temperature diagram for enzymes. The enzyme is reacted with 1% chitosan solution at various temperatures for 30 minutes and the enzyme activity is measured. However, considering the result that the temperature tolerance of the enzyme exceeds 40°C, the activity of the enzyme will decrease if it is exposed to a higher temperature for a long time, so the temperature must be lowered for a long time. If the reaction is carried out for a long time at 37°C, it can have good enzyme activity and prolong the retention time of enzyme activity. The temperature during various experiments in this article is also based on this characteristic For reference.

4.5 幾丁聚醣酶BWT與TI-D的長時間水解反應 4.5 Long-term hydrolysis reaction of chitosanase BWT and TI-D

為了將酵素用於大量生產上，有必要對量產時長時間的產物組成進行分析。以野生型BWT與主產物為三醣的TI-D突變株來進行測試，以10公升的5%幾丁聚醣溶液作為量產的基質。並持續取樣達到72小時後進行TLC分析大量產物的比例(圖10)。請參見第10圖，其為野生型(BWT)與突變株TI-D長時間水解反應圖。取酵素活性2500U的野生型BWT與突變株TI-D，對10公升的5%幾丁聚醣溶液進行反應，並持續取樣達到72小時來進行TLC分析大量產物的比例。長時間產物來看BWT一開始的產物並不明顯，在反應初期會有少量五醣的存在，到了反應後期產物只剩下二、三、四醣。而TI-D一開始就有著很高三醣的出現，並且三醣產物的比使終是最高的，我們懷疑TI-D的水解方式突變成外切的形式。 In order to use enzymes for mass production, it is necessary to analyze the product composition for a long time during mass production. The wild-type BWT and the TI-D mutant strain with trisaccharide as the main product were tested, and 10 liters of 5% chitosan solution was used as the matrix for mass production. After continuous sampling for 72 hours, TLC was performed to analyze the proportion of a large number of products (Figure 10). Please refer to Figure 10, which shows the long-term hydrolysis reaction between wild-type (BWT) and mutant strain TI-D. Take the wild-type BWT with enzyme activity of 2500U and the mutant strain TI-D to react with 10 liters of 5% chitosan solution, and continue sampling for 72 hours to analyze the proportion of a large number of products by TLC. From a long-term product perspective, the initial product of BWT is not obvious. A small amount of pentasaccharides will be present in the early stage of the reaction, and only di-, tri-, and tetra-saccharides will remain in the late stage of the reaction. However, TI-D had very high trisaccharides from the beginning, and the ratio of trisaccharide products was the highest in the end. We suspect that the hydrolysis of TI-D has mutated into an exoform form.

4.6 幾丁聚醣酶BWT與TI-D水解型式 4.6 Chitosanase BWT and TI-D hydrolysis type

為了證實酵素究竟為內切還是外切的型式，利用測定酵素截切還原端的能力以及濁度曲線關係進行判斷。DNS的成色反應是與醣類基質的還原端進行反應，可以用來做為做為截切能力的測量。酵素在水解過程時若用鹼終止反應，會使還沒被水解的幾丁聚醣釋出，透過濁度(OD600nm)的測量，可以得知剩下多少的幾丁聚醣未被水解。利用酵素的DNS與濁度作為 座標軸的話就可得到一張酵素的水解型式圖。請參見第11圖，其為野生型BWT與突變株TI-D水解型式圖。取相同酵素活性的野生型BWT與突變株TI-D，以兩者活性與濁度型做圖。由此種方式判斷酵素的水解型式究竟是哪種。 In order to confirm whether the enzyme is an internal or external type, the ability of the enzyme to cut the reducing end and the relationship between the turbidity curve are used to determine. The color reaction of DNS is to react with the reducing end of the carbohydrate matrix, which can be used as a measure of the cutting ability. If the enzyme terminates the reaction with alkali during the hydrolysis process, the chitosan that has not been hydrolyzed will be released. Through the measurement of turbidity (OD600nm), it can be known how much chitosan is left unhydrolyzed. Use enzyme DNS and turbidity as If you use the coordinate axis, you can get a picture of the enzyme hydrolysis pattern. Please refer to Figure 11, which shows the hydrolysis patterns of wild-type BWT and mutant TI-D. Take wild-type BWT and mutant strain TI-D with the same enzyme activity, and plot the activity and turbidity type of both. In this way, it is judged which type of enzyme is hydrolyzed.

取同樣活性的內切與外切酵素進行反應，可以得到DNS與濁度的曲線，因為取用相同的酵素活性，所以DNS的曲線幅度是相同的。內切型的酵素因為水解是從基質內部去水解，使得長鏈狀的幾丁聚醣變成許多短鏈的型式。以濁度來看，混濁的長鏈狀幾丁聚醣會立刻變成澄清透明的短鏈。此外變成透明後酵素依然持續進行水解，所以DNS依然在上升，但是濁度卻不會再變化，如第11圖中的BWT曲線的末端。外切型的酵素水解型式是由還原端或非還端開始有限制的進行水解。即使用DNS已經測到很多被水解出的還原醣，但是做為基質的幾丁聚醣依然有著長鏈狀的結構在。因此與同活性的內切酵素相比會下降的較為緩慢。 Taking the same activity of endo and exo enzymes to react, the curve of DNS and turbidity can be obtained. Because the same enzyme activity is used, the amplitude of the curve of DNS is the same. Endo-type enzymes are hydrolyzed from the inside of the substrate, so that long-chain chitosan becomes many short-chain forms. In terms of turbidity, the turbid long-chain chitosan will immediately become a clear and transparent short-chain. In addition, the enzyme continues to hydrolyze after it becomes transparent, so the DNS is still rising, but the turbidity does not change anymore, as shown at the end of the BWT curve in Figure 11. The exo-type enzymatic hydrolysis type starts from the reducing end or the non-reduced end with limited hydrolysis. Even though DNS has been used to detect many reducing sugars that have been hydrolyzed, the chitosan used as the matrix still has a long-chain structure. Therefore, compared with the same activity of endoenzyme, it will decrease more slowly.

從第11圖來看，TI-D與BWT走勢十分的相似，因此TI-D與BWT一樣是內切型式。 From Figure 11, TI-D and BWT trend are very similar, so TI-D and BWT are inscribed.

4.7 幾丁聚醣酶TI-D水解基質模擬 4.7 Chitosanase TI-D hydrolysis matrix simulation

當酵素在水解基質時會進行一個類似開閉口的動作產生出開口或閉口的結構模擬。在模擬基質對接時若使用開口結構，酵素的基質結合區域較大，許多酵素胺基酸對基質有影 響的表現並非水解時的情況。並且酵素於閉口型態時原先結構上的胺基酸位置會有所改變，要瞭解這種情形下與基質的對接，必須要重新模擬酵素閉口狀態的結構，才能對基質進行對接模擬。 When the enzyme hydrolyzes the substrate, it will perform a similar opening and closing action to produce an open or closed structure simulation. If an open structure is used when simulating substrate docking, the substrate binding area of the enzyme is larger, and many enzyme amino acids have an impact on the substrate. The performance of the sound is not the case during hydrolysis. In addition, the position of the amino acid in the original structure of the enzyme will change when the enzyme is in the closed state. To understand the docking with the substrate in this situation, it is necessary to re-simulate the structure of the enzyme in the closed state in order to simulate the docking of the substrate.

所以重新選用了4OLT作為模板進行結構模擬，請參見第12圖(a)-(d)，其為幾丁聚醣酶TI-D的開口與閉口型結構模擬圖。其中第12圖(a)為開口狀態的卡通圖，第12圖(b)為閉口狀態卡通圖，第12圖(c)為開口狀態表面電子雲圖，第12圖(d)為閉口狀態表面電子雲圖。請參見第12圖(b)，從卡通圖來看酵素的上下兩部分會互相靠近，將基質結合在中間的空洞中。請參見第12圖(d)，從表面電子雲圖來看閉口狀態的模擬，酵素中間與基質接合的位置被包覆起來。 Therefore, 4OLT was re-selected as a template for structural simulation. Please refer to Figure 12(a)-(d), which are the opening and closing structure simulation diagrams of chitosanase TI-D. Figure 12(a) is a cartoon drawing of an open state, Figure 12(b) is a cartoon drawing of a closed state, Figure 12(c) is an open state surface electron cloud, and Figure 12(d) is a closed state surface electron Cloud Atlas. Please refer to Figure 12(b). From the cartoon, the upper and lower parts of the enzyme will be close to each other, combining the substrate in the cavity in the middle. Please refer to Figure 12(d). From the surface electron cloud image to see the simulation of the closed state, the position between the enzyme and the substrate is covered.

以閉口狀態的結構對基質進行對接模擬，我們模擬了十次酵素與基質的結合情形。可以發現基質結合位置是集中在野生型BWT的中間的位置。請參見第13圖，其為幾丁聚醣酶BWT與基質結合結構模擬圖。幾丁聚醣酶BWT與基質以十個不同結合方式的結構模擬與酵素的基質結合位(+2)~(-4)基本吻合。依照酵素切位對基質水解產物預測，以六醣做基質時會被水解成1.5與4.5個醣的產物。突變株TI-D與基質結合的模擬圖，請參見第14圖，則呈現出基質往左邊偏移了大約一個醣位，模擬水解成2.75與3.25個醣的產物。這表示本發明之較佳實施例成功改變酵素與基質結合的位置。 We simulated the docking of the substrate with the closed structure, and we simulated the binding of the enzyme and the substrate ten times. It can be found that the matrix binding position is concentrated in the middle position of the wild-type BWT. Please refer to Figure 13, which is a simulation diagram of the binding structure of chitosanase BWT and matrix. The structure simulation of chitosanase BWT and the substrate in ten different binding modes is basically consistent with the substrate binding sites (+2)~(-4) of the enzyme. According to the prediction of the substrate hydrolysate based on the enzyme cleavage position, when the hexasaccharide is used as the substrate, it will be hydrolyzed into 1.5 and 4.5 sugar products. For the simulation diagram of the binding of the mutant strain TI-D to the substrate, please refer to Figure 14, which shows that the substrate is shifted by about one sugar position to the left, which simulates hydrolysis into products of 2.75 and 3.25 sugars. This indicates that the preferred embodiment of the present invention successfully changes the position where the enzyme binds to the substrate.

參考實際水解產物情況，計算對接模擬後基質 被酵素水解後的醣類種類。野生型BWT的二醣：三醣：四醣的比例為3：3：1，突變株TI-D的二醣：三醣：四醣的比例為1：3：1。這與由TLC分析出來的產物結果，野生型BWT的二醣：三醣：四醣的比例為4：3：1，突變株TI-D的二醣：三醣：四醣的比例為1：8：1。最高產物的趨勢類似。 Refer to the actual hydrolysate situation, calculate the matrix after docking simulation The type of carbohydrate that is hydrolyzed by enzymes. The ratio of disaccharide:triose:tetrasaccharide of wild-type BWT is 3:3:1, and the ratio of disaccharide:triose:tetrasaccharide of mutant strain TI-D is 1:3:1. This is the result of the product analyzed by TLC. The ratio of disaccharide:trisaccharide:tetrasaccharide of wild-type BWT is 4:3:1, and the ratio of disaccharide:trisaccharide:tetrasaccharide of the mutant strain TI-D is 1: 8:1. The trend for the highest product is similar.

挑選產物為三醣的模擬情形進行分析。請參見第15圖，其為幾丁聚醣酶TI-D水解基質產生三醣的模擬示意圖，模擬幾丁聚醣酶TI-D與基質結合時受到突變胺基酸影響造成基質結合位置改變的結構。當基質以非還原端進入酵素時，觀察到基質原先本來應該是以較水平的方式與酵素結合的。但是產物為三醣的情形卻是基質以傾斜的方式進入酵素之中。並且基質的末端會靠近突變的天門冬胺酸位置上。這是由於本發明較佳實施例中帶負電擋板結構吸引帶正電的幾丁聚醣基質，使基質往擋板靠近。再加上本發明較佳實施例中的突變模板是以TI這個-2醣基結合位被弱化的突變株，使得基質在(-1)~(-3)之間因缺乏向下的吸引力而往上傾斜進入。擋板結構位置大約在(-3)與(-4)之間，由於傾斜與擋板的阻擋剛好使的酵素能夠切出一個三醣的產物出來，故本實施例中酵素幾丁聚醣酶TI-D是內切的酵素，卻有大量三醣產物的產生。幾丁聚醣酶TI-D除了有著三醣的產生外還有二醣與四醣的產生。從結構上發現在突變的天門冬胺酸的後方有著一個帶正電的組胺酸。請參見第16圖，其為幾丁聚醣酶TI-D水解基質產生四醣的模擬示意圖。當基質是由中間被水解或 是如果末端沒有剛好被擋板阻擋而是由下方穿過去，基質會被組胺酸向下推，形成了拱型的樣子讓酵素能與基質結合，回到像BWT的水解型式產生二醣與四醣的產物。 Choose the simulated situation where the product is trisaccharide for analysis. Please refer to Figure 15, which is a schematic diagram of the chitosanase TI-D hydrolyzing the matrix to produce trisaccharides, which simulates the change of the matrix binding position caused by the mutant amino acid when the chitosanase TI-D binds to the matrix. structure. When the substrate enters the enzyme at the non-reducing end, it is observed that the substrate should originally be bound to the enzyme in a relatively horizontal manner. However, when the product is trisaccharide, the substrate enters the enzyme in an oblique manner. And the end of the substrate will be close to the position of the mutant aspartic acid. This is because the negatively charged baffle structure in the preferred embodiment of the present invention attracts the positively charged chitosan matrix and causes the matrix to approach the baffle. In addition, the mutant template in the preferred embodiment of the present invention is a mutant strain in which the -2 glycosyl binding site of TI is weakened, so that the matrix is between (-1) and (-3) due to lack of downward attraction And lean upward to enter. The position of the baffle structure is approximately between (-3) and (-4). Because the inclination and the blocking of the baffle just enable the enzyme to cut out a trisaccharide product, the enzyme chitosanase in this example TI-D is an endogenous enzyme, but it produces a large amount of trisaccharide products. In addition to the production of trisaccharides, chitosanase TI-D also produces disaccharides and tetrasaccharides. From the structure, it is found that there is a positively charged histidine behind the mutant aspartic acid. Please refer to Figure 16, which is a schematic diagram of the chitosanase TI-D hydrolyzing matrix to produce tetrasaccharides. When the substrate is hydrolyzed from the middle or If the end is not just blocked by the baffle but passes through from below, the substrate will be pushed down by histidine, forming an arch shape that allows the enzyme to bind to the substrate, returning to the hydrolysis type like BWT to produce disaccharides and The product of tetrasaccharides.

5.大量產物分析 5. Mass product analysis

5.1 幾丁寡醣量產 5.1 Mass production of chitosan oligosaccharides

為了將我們的酵素能運用到量產上，將BWT酵素與TI-D酵素在大量表現後以選擇性沉澱破菌取得酵素，與基質反應持續到72小時，並分析不同時間取樣的產物。請參見第10圖，其為野生型BWT與突變株TI-D長時間水解反應圖。取酵素活性2500U的野生型BWT與突變株TI-D，對10公升的5%幾丁聚醣溶液進行反應，並持續取樣達到72小時來進行TLC分析大量產物的比例。 In order to apply our enzymes to mass production, BWT enzymes and TI-D enzymes are used to obtain enzymes by selective precipitation after a large number of expressions. The reaction with the substrate lasts for 72 hours, and the products sampled at different times are analyzed. Please refer to Figure 10, which is a long-term hydrolysis reaction diagram of wild-type BWT and mutant TI-D. Take the wild-type BWT with enzyme activity of 2500U and the mutant strain TI-D to react with 10 liters of 5% chitosan solution, and continue sampling for 72 hours to analyze the proportion of a large number of products by TLC.

接著請參見第17圖，其為野生型BWT與突變株TI-D長時間水解產物走勢圖。將TLC產物由軟體定量產物後，對二、三、四醣的走勢進行統計。以產物的走勢圖來看，可以看到野生型BWT的主產物為二、三、四醣，其中二醣的產率一直維持最高，三、四醣的產量較少。並且到了後期四醣的產率會開始變緩，而二醣開始大量增加，這是因為四醣被水解成二醣。突變株TI-D的產物在一開始便有顯著的三醣產生，隨著時間的反應三醣的產量始終維持最高，當72小時反應後三醣的比例可達到總產物的80%。 Next, please refer to Figure 17, which is a trend chart of the long-term hydrolysate of wild-type BWT and mutant TI-D. After the TLC product is quantified by the software, the trend of the di-, tri-, and tetra-sugar is counted. Judging from the product trend chart, it can be seen that the main products of wild-type BWT are disaccharides, trisaccharides, and tetrasaccharides, among which the yield of disaccharides has been maintained at the highest, and the yield of trisaccharides and tetrasaccharides is small. And in the later stage, the yield of tetrasaccharides will start to slow down, and disaccharides will begin to increase a lot, because the tetrasaccharides are hydrolyzed into disaccharides. The product of the mutant strain TI-D has significant trisaccharide production at the beginning, and the yield of trisaccharide always maintains the highest with the time of reaction. After 72 hours of reaction, the proportion of trisaccharide can reach 80% of the total product.

5.2 幾丁三醣量產物回收 5.2 Recovery of Chitotriose Products

為了回收水解後的產物，將反應後的產物調整達到酸性、中性、鹼性三種pH值，並以IPA使產物沉澱。沉澱物離心後去除上清液後，利用冷凍乾燥機進行凍乾。取20mL的產物在不同酸鹼時的回收率，其中酸性與鹼性的產物回收率都接近80%。此外以IPA使產物沉澱時若在-20℃靜置24小時，能夠回收到更多的產物達到90%的回收率。不過鹼性回收的產物回溶後會快速產生褐化的現象，產物中的寡醣比例也容易產生變化，因此在回收時不需要特別去調整酸鹼值，而是要控制溫度在低溫下進行回收。 In order to recover the hydrolyzed product, the reacted product is adjusted to three pH values of acidic, neutral, and alkaline, and the product is precipitated with IPA. After centrifuging the precipitate to remove the supernatant, it was freeze-dried using a freeze dryer. The recovery rate of 20mL of product under different acid and alkali, among which the recovery rate of acidic and basic products are close to 80%. In addition, if the product is allowed to stand at -20°C for 24 hours when the product is precipitated with IPA, more products can be recovered to achieve a recovery rate of 90%. However, the product of alkaline recovery will quickly cause browning after re-dissolution, and the proportion of oligosaccharides in the product is also prone to change. Therefore, there is no need to adjust the pH value during recovery, but to control the temperature at low temperature. Recycling.

為了確認回收後的寡醣是否產物有沒有發生改變，將不同回收方式取得的寡醣粉末，各取0.1g回溶到1mL的無菌水中，並以TLC進行分析。請參照第18圖，其為回收寡醣粉末產物分析圖。將不同條件回收後的寡醣粉末，各取0.1g回溶到1mL的無菌水中，以TLC測定回售的產物比例。 In order to confirm whether the product of the recovered oligosaccharide has changed, 0.1g of the oligosaccharide powder obtained by different recovery methods was re-dissolved into 1mL of sterile water, and analyzed by TLC. Please refer to Figure 18, which is an analysis diagram of recovered oligosaccharide powder products. The oligosaccharide powder recovered under different conditions was re-dissolved in 1 mL of sterile water with 0.1 g of each oligosaccharide powder, and the proportion of products sold back was determined by TLC.

接著請參照第19圖，其為回收產物的二、三、四醣之比例圖。將TLC測定到的回收寡醣產物定量，產物二、三、四醣所占產物總量長條分析圖。從長條圖來看，酵素的將產物調整到鹼性並以-20℃沉澱回收到的三醣總量最高。回收後的幾丁聚醣酶TI-D的寡醣產物中，三醣的比例雖然略有下降，但是依然維持在最高的比例，比例占總量的70%。 Next, please refer to Figure 19, which is the ratio of the di-, tri-, and tetra-saccharides of the recovered product. The recovered oligosaccharide products measured by TLC were quantified, and the long-bar analysis chart of the total product amount of the product di-, tri-, and tetra-saccharide. Judging from the bar graph, the total amount of trisaccharide recovered from the enzyme adjusted to alkaline and precipitated at -20°C is the highest. Although the proportion of trisaccharides in the recovered chitosanase TI-D oligosaccharide product slightly decreased, it still maintained the highest proportion, accounting for 70% of the total.

表5顯示本發明之胜肽序列識別號。 Table 5 shows the peptide sequence identification numbers of the present invention.

以上所述僅為本發明之較佳實施例，凡依本發明申請專利範圍所做之均等變化與修飾，皆應屬本發明之涵蓋範圍。 The foregoing descriptions are only preferred embodiments of the present invention, and all equivalent changes and modifications made in accordance with the scope of the patent application of the present invention shall fall within the scope of the present invention.

<110> 國立高雄科技大學 <110> National Kaohsiung University of Science and Technology

<120> 重組幾丁聚醣酶及其幾丁三醣的製造方法 <120> Recombinant chitosanase and method for producing chitotriose

<130> None <130> None

<140> TW 107123035 <140> TW 107123035

<141> 2018-07-03 <141> 2018-07-03

<160> 14 <160> 14

<210> 1 <210> 1

<211> 261 <211> 261

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<221> 重組芽孢桿菌幾丁聚醣水解酵素TI-D <221> Recombinant Bacillus Chitosan Hydrolytic Enzyme TI-D

<400> 1

<400> 1

<210> 2 <210> 2

<211> 260 <211> 260

<212> PRT <212> PRT

<213> 芽孢桿菌(Bacillus circulans) <213> Bacillus circulans

<400> 2

<400> 2

<210> 3 <210> 3

<211> 262 <211> 262

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<221> 重組芽孢桿菌幾丁聚醣水解酵素TI-HD <221> Recombinant Bacillus Chitosan Hydrolytic Enzyme TI-HD

<400> 3

<400> 3

<210> 4 <210> 4

<211> 263 <211> 263

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<221> 重組芽孢桿菌幾丁聚醣水解酵素TI-EHD <221> Recombinant Bacillus Chitosan Hydrolytic Enzyme TI-EHD

<400> 4

<400> 4

<210> 5 <210> 5

<211> 260 <211> 260

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<221> 重組芽孢桿菌幾丁聚醣水解酵素TI <221> Recombinant Bacillus Chitosan Hydrolytic Enzyme TI

<400> 5

<400> 5

<210> 6 <210> 6

<211> 30 <211> 30

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 突變型TI飽和突變上游引子 <223> Mutant TI saturation mutation upstream primer

<220> <220>

<221> misc_feature <221> misc_feature

<222> (14,15,17,18) <222> (14,15,17,18)

<223> n表示a或g或c或t <223> n means a or g or c or t

<400> 6

<400> 6

<210> 7 <210> 7

<211> 30 <211> 30

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 突變型TI飽和突變下游引子 <223> Downstream primer of mutant TI saturation mutation

<220> <220>

<221> misc_feature <221> misc_feature

<222> (13,14,16,17) <222> (13,14,16,17)

<223> n表示a或g或c或t <223> n means a or g or c or t

<400> 7

<400> 7

<210> 8 <210> 8

<211> 27 <211> 27

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 突變型TI-D定點突變上游引子(TI-D-F) <223> Mutant TI-D site-directed mutagenesis upstream primer (TI-D-F)

<400> 8

<400> 8

<210> 9 <210> 9

<211> 27 <211> 27

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 突變型TI-D定點突變下游引子(TI-D-R) <223> Downstream primer for mutant TI-D site-directed mutagenesis (TI-D-R)

<400> 9

<400> 9

<210> 10 <210> 10

<211> 30 <211> 30

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 突變型TI-ED定點突變上游引子(TI-HD-F) <223> Mutant TI-ED site-directed mutagenesis upstream primer (TI-HD-F)

<400> 10

<400> 10

<210> 11 <210> 11

<211> 30 <211> 30

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 突變型TI-ED定點突變下游引子(TI-HD-R) <223> Downstream primer for mutant TI-ED site-directed mutagenesis (TI-HD-R)

<400> 11

<400> 11

<210> 12 <210> 12

<211> 33 <211> 33

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 突變型TI-EHD定點突變上游引子(TI-EHD-F) <223> Mutant TI-EHD site-directed mutagenesis upstream primer (TI-EHD-F)

<400> 12

<400> 12

<210> 13 <210> 13

<211> 33 <211> 33

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 突變型TI-EHD定點突變下游引子(TI-EHD-R) <223> Downstream primer for mutant TI-EHD site-directed mutagenesis (TI-EHD-R)

<400> 13

<400> 13

<210> 14 <210> 14

<211> 260 <211> 260

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<221> 重組芽孢桿菌幾丁聚醣水解酵素NDTI <221> Recombinant Bacillus Chitosan Hydrolytic Enzyme NDTI

<400> 14

<400> 14

Claims (4)

一種重組幾丁聚醣酶，其胺基酸序列係如序列識別號(SEQ ID NO)：1所示。 A recombinant chitosanase whose amino acid sequence is as shown in SEQ ID NO:1. 如申請專利範圍第1項所述之重組幾丁聚醣酶，其中該重組幾丁聚醣酶之一水解產物包含幾丁寡醣，該幾丁寡醣包含幾丁三醣，且該幾丁三醣之莫耳含量是該幾丁寡醣之莫耳含量的至少90%。 The recombinant chitosanase described in item 1 of the patent application, wherein one of the hydrolysates of the recombinant chitosanase contains chitosan oligosaccharide, the chitosan oligosaccharide contains chitotriose, and the chitin The molar content of the trisaccharide is at least 90% of the molar content of the chitin-oligosaccharide. 如申請專利範圍第1項所述之重組幾丁聚醣酶，其中該重組幾丁聚醣酶在酸鹼值pH 4到7之間具有至少66%之一酵素活性。 The recombinant chitosanase as described in item 1 of the scope of the patent application, wherein the recombinant chitosanase has at least 66% of an enzyme activity between pH 4 and 7. 一種幾丁三醣的製造方法，包含：提供幾丁聚醣水溶液；利用如SEQ ID NO：1所示之重組幾丁聚醣酶水解該幾丁聚醣水溶液，以獲得一水解產物；以及進行一沉澱步驟，以由該水解產物獲得該幾丁寡醣，其中該幾丁寡醣包含該幾丁三醣。 A method for producing chitotriose, comprising: providing an aqueous solution of chitosan; hydrolyzing the aqueous solution of chitosan with a recombinant chitosanase as shown in SEQ ID NO: 1 to obtain a hydrolyzed product; and A precipitation step to obtain the chitosan oligosaccharide from the hydrolysate, wherein the chitosan oligosaccharide comprises the chitotriose.

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