KR20240042273A - Corynebacterium glutamicum(C. glutamicum)-based glycotransferase expression body and method for producing high-functional oligosaccharides with enhanced digestive delay effectiveness using thereof - Google Patents
Corynebacterium glutamicum(C. glutamicum)-based glycotransferase expression body and method for producing high-functional oligosaccharides with enhanced digestive delay effectiveness using thereof Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
- C12N9/1051—Hexosyltransferases (2.4.1)
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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
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
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- C12Y204/00—Glycosyltransferases (2.4)
- C12Y204/01—Hexosyltransferases (2.4.1)
- C12Y204/01024—1,4-Alpha-glucan 6-alpha-glucosyltransferase (2.4.1.24)
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- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
Abstract
소화지연 효능을 가지는 유효한 올리고당을 포함하는 L-IMO를 제조하는 효소를 생산하는 산업적 균주개발을 위하여 GRAS 인정을 통해 그 안정성이 입증된 C. glutamicum 균주 기반의 발현체(CgTG)를 구축하였고, C. glutamicum은 균주 기반의 발현체를 통해 생산한 소화 지연 효능을 갖춘 지소화성 올리고당(L-IMO) 소재 및 그 제조 방법을 개시하였다.In order to develop an industrial strain that produces an enzyme that produces L-IMO, which contains effective oligosaccharides with digestion delay effect, an expression body (CgTG) based on a C. glutamicum strain whose stability was proven through GRAS recognition was constructed, and C glutamicum disclosed a lipolytic oligosaccharide (L-IMO) material with digestion-retarding effect produced through a strain-based expression material and a method for manufacturing the same.
Description
본 발명은 코리네박테리움(C. glutamicum) 기반 당전이효소 발현체 및 이를 이용한 소화지연 유효성이 강화된 고기능성 올리고당 제조 방법에 관한 것이다.The present invention relates to a Corynebacterium ( C. glutamicum )-based glycosyltransferase expression form and a method for producing highly functional oligosaccharides with enhanced digestion delay effectiveness using the same.
단맛을 내는 물질인 당류는 당분자의 갯수에 따라 단당류, 이당류, 다당류로 나뉜다. 포도당(glucose)과 과당(fructose)은 분자가 하나로 된 단당류이고, 설탕(sucrose sugar)은 분자가 2개인 이당류, 올리고당(oligosaccharide)은 3개 이상의 분자가 뭉쳐진 다당류에 속한다. 다당류는 α-1,4 결합 및/또는 α-1,6 결합 등으로 이어져 있고, 상기 α-1,4 결합은 α-D 포도당 분자가 인접한 α-D 포도당 분자 고리와 탄소 1 및 4를 통해 서로 연결된 공유 결합의 유형을 지칭하고, 상기 α-1,6 결합은 α-D 포도당 분자가 인접한 α-D 포도당 분자 고리와 탄소 1 및 6을 통해 서로 연결된 공유 결합의 유형을 지칭한다.Sugars, which are substances that produce sweet taste, are divided into monosaccharides, disaccharides, and polysaccharides depending on the number of sugar molecules. Glucose and fructose are monosaccharides with one molecule, sucrose is a disaccharide with two molecules, and oligosaccharide is a polysaccharide with three or more molecules bundled together. The polysaccharide is connected by an α-1,4 bond and/or an α-1,6 bond, and the α-1,4 bond connects the α-D glucose molecule through the adjacent α-D glucose molecule ring and carbons 1 and 4. The α-1,6 bond refers to a type of covalent bond in which an α-D glucose molecule is linked to an adjacent α-D glucose molecule ring through carbons 1 and 6.
당류는 대부분의 과일, 유제품, 곡류 등에 함유되어 있기 때문에 식품을 통하여 섭취될 수 있으며 에너지를 공급하고, 정신적인 만족감을 주는 기능을 한다. 그러나 입자가 작은 단당류일수록 소화와 섭취가 빨라 급격하게 혈당수치를 높이고, 혈중 중성 지방을 높여 비만, 당뇨병, 심혈관 질환 등 성인병 발생의 위험성을 증가시킨다. 또한 어린이의 경우에는 충치, 과잉행동장애와 같은 질병 발생 위험과 관련이 있는 것으로 보고되고 있는 등 유해성 논란이 계속되면서, 건강을 위해 단맛을 내기 위한 설탕의 대체제로 올리고당을 사용하는 소비자가 늘어나고 있다.Since sugars are contained in most fruits, dairy products, and grains, they can be consumed through food and function to supply energy and provide mental satisfaction. However, the smaller the particle size of monosaccharides, the faster they are digested and consumed, rapidly raising blood sugar levels and increasing triglycerides in the blood, increasing the risk of developing adult diseases such as obesity, diabetes, and cardiovascular disease. In addition, in the case of children, as controversy over its harmfulness continues, as it is reported to be related to the risk of developing diseases such as tooth decay and hyperactivity disorder, the number of consumers using oligosaccharides as a sweetener substitute for health is increasing.
올리고당이 포함되어 있는 다당류는 소화와 섭취가 어려워 설탕의 3분의 1 수준으로 칼로리가 낮고, 장내에 공생하는 또는 유리한 미생물의 성장을 자극시키고, 체내에서 수용성 식이섬유와 같은 작용을 한다고 알려져 있다.Polysaccharides containing oligosaccharides are difficult to digest and ingest, are low in calories (one-third of sugar), stimulate the growth of symbiotic or beneficial microorganisms in the intestines, and are known to act like soluble dietary fiber in the body.
특히 올리고당은 영양, 면역, 노화, 장내 부패물질 억제, 비타민 합성 등 숙주의 건강과 밀접한 관계를 갖는 장내 비피더스균을 증식시키기 위해 비피더스 균의 생육 촉진물질로서 상업적으로 제조되고 있다. In particular, oligosaccharides are commercially manufactured as growth promoting substances for bifidobacteria to proliferate intestinal bifidobacteria, which are closely related to the health of the host, such as nutrition, immunity, aging, inhibition of intestinal decay substances, and vitamin synthesis.
올리고당 중 이소말토올리고당(Isomaltooligosaccharide, IMO)은 포도당이 α-(1,4)- 및/또는 α-(1,6)-글루코시드 결합 형태의 분지결합인 올리고 당류를 말하는 것으로 단쇄 탄수화물의 혼합물이다. IMO에서 발견되는 올리고당의 대부분은 서로 연결된 세 개 내지 여섯 개의 단당류(글루코스) 단위로 구성된다. IMO의 이당류 분획은 주로 α(1,6)-연결된 이소말토스(isomaltose)로 구성되며, 말토트리오스, 파노스(panose), 이소말토트리오스(isomaltotriose)는 삼당류 분획을 구성한다. 이러한 이소말토스 (isomaltose), 이소말토테트라오스(isomaltotetraose) 및 이소파노스(isopanose)는 고급 분지형 올리고당이다.Among oligosaccharides, Isomaltooligosaccharide (IMO) refers to oligosaccharides in which glucose is branched in the form of α-(1,4)- and/or α-(1,6)-glucosidic bonds and is a mixture of short-chain carbohydrates. . Most of the oligosaccharides found in IMO are composed of three to six monosaccharide (glucose) units linked together. The disaccharide fraction of IMO mainly consists of α(1,6)-linked isomaltose, while maltotriose, panose, and isomaltotriose make up the trisaccharide fraction. These isomaltose, isomaltotetraose and isopanose are highly branched oligosaccharides.
이소말토올리고당(IMO) 제조에 사용되는 원료는 전분이다. 밀, 보리, 맥박 (완두콩, 콩, 렌즈 콩), 귀리, 타피오카, 쌀, 감자 등과 같은 시리얼 작물에서 가공 된 전분이 사용될 수 있다. 이러한 전분은 효소적으로 이소말토올리고당의 혼합물로 전환된다. 전분은 먼저 단순한 효소 가수분해를 통해 인간의 장에서 쉽게 소화될 수 있는 α1-4 글리코시드 결합을 갖는 di-, tri- 및 올리고당을 갖는 말토오스 시럽으로 전환된다. 이들 α(1,4)-글리코시드 결합은 소화 내성 α(1,6)-글리코시드 결합으로 전환되어 글루코스 잔기 사이에 "이소" 결합을 생성하여 이소말토올리고당(IMO)을 형성한다.The raw material used to manufacture isomaltooligosaccharide (IMO) is starch. Processed starch from cereal crops such as wheat, barley, pulses (peas, beans, lentils), oats, tapioca, rice, potatoes, etc. can be used. These starches are enzymatically converted into a mixture of isomaltooligosaccharides. Starch is first converted through simple enzymatic hydrolysis into maltose syrup, which has di-, tri- and oligosaccharides with α1-4 glycosidic linkages, which can be easily digested in the human intestine. These α(1,4)-glycosidic bonds are converted to digestion-resistant α(1,6)-glycosidic bonds, creating “iso” bonds between glucose residues to form isomaltooligosaccharides (IMOs).
전분 원료를 기질로 당 전이 반응이 일으키는 효소로는 크게 glycosyl transferase(GTase), transglucosidases(TGase), glycoside hydrolases(GHase) 및 glycoside phosphorylase(GPase) 등 네 가지로 분류된다. glycosyl transferase(GTase)는 당수용체(탄수화물 등)에 글루코실기를 전달하는 효소이고, transglucosidases(TGase)는 글리코겐 사슬의 α-1,4 결합으로 연결된 포도당을 α-1,6 결합으로 변환시켜 글리코겐에 가지 형성 반응을 매개하는 효소로서, 아밀로오스형(노르말 사슬형) 다당류에서 아밀로펙틴형(분기형) 다당류를 생성하는 효소이고, glycoside hydrolases(GHase)는 sugar와 다른 molecule 사이에 있는 결합을 가수분해하는 효소이며, glycoside phosphorylase(GPase)는 글리코시드 결합의 가역적 형성 및 절단을 촉매하는 효소로서 글리코시드 가수분해효소(GH)와 글라이코트랜스퍼라제(GT)의 이중 기능을 겸한다. GTase는 당 전이 효율이 좋고 다양한 수용체에 당을 전이할 수 있는 능력이 뛰어나지만, UDP-glucose와 같은 값이 상당히 비싼 기질을 사용해야 한다. 이에 반해 TGase, GPase, GHase는 전분이나 설탕 같은 비교적 값이 저렴한 기질을 이용해 당 전이 반응을 수행하기 때문에 화장품 및 식품 산업 등에 활용 가능성이 높은 효소이다. 본 발명은 써모안아에로박터 써모코프리애 (Thermoanaerobacter thermocopriae) 균주 유래 당전이효소(TtTG; Thermoanaerobacter thermocopriae transglucosidases) 유전자를 코리네박테리움 글루타미쿰(Corynebacterium glutamicum) 발현체를 이용하여 이소말토올리고당을 제조한다. Enzymes that cause the sugar transfer reaction using starch as a substrate are broadly classified into four types: glycosyl transferase (GTase), transglucosidases (TGase), glycoside hydrolases (GHase), and glycoside phosphorylase (GPase). Glycosyl transferase (GTase) is an enzyme that transfers glucosyl groups to sugar receptors (carbohydrates, etc.), and transglucosidases (TGase) convert glucose linked by α-1,4 bonds of the glycogen chain into α-1,6 bonds to form glycogen. As an enzyme that mediates the branching reaction, it is an enzyme that produces amylopectin-type (branched) polysaccharide from amylose-type (normal chain) polysaccharide, and glycoside hydrolases (GHase) are enzymes that hydrolyze bonds between sugar and other molecules. Glycoside phosphorylase (GPase) is an enzyme that catalyzes the reversible formation and cleavage of glycosidic bonds and has the dual function of glycoside hydrolase (GH) and glycotransferase (GT). GTase has good sugar transfer efficiency and the ability to transfer sugar to various receptors, but it requires the use of quite expensive substrates such as UDP-glucose. On the other hand, TGase, GPase, and GHase are enzymes with high potential for use in the cosmetics and food industries because they perform a sugar transfer reaction using relatively inexpensive substrates such as starch or sugar. The present invention is an isomaltooligosaccharide using a Corynebacterium glutamicum expression body of the glycosyltransferase ( Tt TG; Thermoanaerobacter thermocopriae transglucosidases) gene derived from the Thermoanaerobacter thermocopriae strain. manufactures.
이소말토올리고당(IMO)이 당전이 효소를 이용하여 상업적으로 많이 제조되는 이유는 IMO의 소화지연 효과 및 프리바이오틱스 역할 때문이다. The reason why isomaltooligosaccharide (IMO) is commercially manufactured using glycosyltransferase is because of IMO's digestion delay effect and prebiotic role.
이소말토올리고당(IMO)은 난소화성과 소화성의 중간 위치에 있으므로 장관에 대한 자극이 약하여 최대 무작용량(no observable effect level)이 설탕과 유사한 수준인 1.5g/kg 정도로 안전성이 뛰어난 것으로 알려져 있다. 이러한 이소말토올리고당의 소화지연 효과는 인간이 가지고 있는 탄수화물 분해 효소의 특성에서 유래한다.Isomaltooligosaccharide (IMO) is located between indigestibility and digestibility, so it is less irritating to the intestinal tract and is known to have excellent safety with a maximum no observable effect level of 1.5 g/kg, which is similar to sugar. This digestion-delaying effect of isomaltooligosaccharide derives from the characteristics of carbohydrate decomposition enzymes in humans.
탄수화물을 분해하는 소화효소로는 대표적으로 아밀라아제(Amylase)가 있다. 아밀라아제(Amylase)는 우리 몸의 침샘과 췌장에서 분비되는 소화효소로 다당류를 가수분해하여 이당류로 전환하는 효소이다. 아밀라아제에는 α, β, γ형의 세 종류가 있으며, 인체의 침과 췌장에서 분비되는 아밀라아제는 모두 α-아밀라아제이다. α-아밀라아제는 α-1, 4-글리코시드결합만을 무작위로 분해하는 것으로 α-1,6 글리코시드 결합은 분해하지 못한다. A representative digestive enzyme that breaks down carbohydrates is amylase. Amylase is a digestive enzyme secreted by our body's salivary glands and pancreas that hydrolyzes polysaccharides and converts them into disaccharides. There are three types of amylase: α, β, and γ types, and the amylase secreted from the human body's saliva and pancreas is all α-amylase. α-Amylase randomly decomposes only α-1,4-glycosidic bonds and does not decompose α-1,6 glycosidic bonds.
따라서 α-1,6 글리코시드 결합을 많이 가지고 있는 당(L-IMO 등)의 경우 소화 지연 특성을 보인다.또한 이소말토올리고당은 장내 유용균으로 대표되는 비피더스균의 증식인자로 프리바이오틱스의 역할을 하는 것으로 알려져 있다. 특히, 중합도(DP, degree of polymeriztion)가 높은 이소말토올리고당은 건강기능성 효능이 있는 것으로 알려져 있고(Toshiyuki Kaneko et al.Biosci. Biotech. Biochem., 58(12): 2288-2290, 1994), 비피더스 생육 촉진 물질로 음료, 유제품, 장류 등 다양한 종류의 식품에 첨가되고 있다.Therefore, sugars (such as L-IMO) that have a lot of α-1,6 glycosidic bonds show digestion delay properties. In addition, isomaltooligosaccharides are growth factors for bifidobacteria, which are representative of useful intestinal bacteria, and play the role of prebiotics. It is known to do so. In particular, isomaltooligosaccharides with a high degree of polymerization (DP) are known to have health functional effects (Toshiyuki Kaneko et al.Biosci. Biotech. Biochem., 58(12): 2288-2290, 1994), and Bifidus As a growth-promoting substance, it is added to various types of foods such as beverages, dairy products, and sauces.
본 발명에서는 써모안아에로박터 써모코프리애 (Thermoanaerobacter thermocopriae) 균주 유래 당전이효소(TtTG; Thermoanaerobacter thermocopriae transglucosidases) 유전자를 포함하는 벡터로 코리네박테리움 글루타미쿰(Corynebacterium glutamicum)을 형질 전환시킨 신규한 당전이 효소 발현체(형질전환체)를 사용하면, 소화지연 효과가 있는 유효한 올리고당 (L-IMO)을 대량으로 신속하게 제조할 수 있음을 확인하여 본 발명을 완성하였다.In the present invention , Corynebacterium glutamicum is transformed with a vector containing a glycosyltransferase ( Tt TG; Thermoanaerobacter thermocopriae transglucosidases) gene derived from the Thermoanaerobacter thermocopriae strain. The present invention was completed by confirming that an effective oligosaccharide (L-IMO) with a digestion delay effect can be rapidly produced in large quantities by using a novel glycosyltransferase expression product (transformant).
전분 유래 고기능성 이소말토올리고당 제조를 위한 안전한 발현체 구축을 위하여 코돈 최적화화된 신규한 당전이효소 유전자를 제공하고 이 유전자를 포함하는 벡터를 만들고, 이 벡터로 현질 전환된 발현체 (형질전환체)를 배양하여 소화지연 효과가 있고, 혈당 증가 지연 효능을 갖춘 지소화성 올리고당을 제조한다.In order to construct a safe expression product for producing highly functional isomaltooligosaccharide derived from starch, a novel codon-optimized glycosyltransferase gene was provided, a vector containing this gene was created, and an expression product (transformant) transformed with this vector was provided. ) is cultured to produce lipodigestible oligosaccharides that have a digestion-delaying effect and an effect of delaying the increase in blood sugar levels.
본 발명은 신규한 당전이효소를 제공한다. The present invention provides a novel glycosyltransferase.
본 발명은 상기 신규한 당전이효소 유전자의 발현에 최적화된 벡터를 제공한다. The present invention provides a vector optimized for expression of the novel glycosyltransferase gene.
본 발명은 상기 벡터로 형질 전환된 발현체(형질전환체)를 제공한다 The present invention provides an expression body (transformant) transformed with the above vector.
본 발명은 상기 신규한 당전이효소를 이용하는 고기능성 이소말토올리고당 (L-IMO) 제조방법을 제공한다. 본 발명은 상기 신규한 당전이효소 발현체를 이용하는 고기능성 이소말토올리고당 (L-IMO) 제조방법을 제공한다. The present invention provides a method for producing highly functional isomaltooligosaccharide (L-IMO) using the novel glycosyltransferase. The present invention provides a method for producing highly functional isomaltooligosaccharide (L-IMO) using the novel glycosyltransferase expression form.
본 발명은 상기 신규한 당전이 효소를 이용하는 고기능성 이소말토올리고당 제조방법을 통해 제조된 다양한 중합도 및 결합형태의 올리고당 중 강력한 소화지연 효과가 있고, 혈당 증가 지연 효능을 갖춘 고기능성 올리고당을 제공한다.The present invention provides a highly functional oligosaccharide with a strong digestion delay effect and an effect of delaying the increase in blood sugar levels among oligosaccharides of various degrees of polymerization and bonding forms prepared through a high-functional isomaltooligosaccharide production method using the novel glycosyltransferase.
본 발명의 신규한 당전이 효소 발현체(코리네박테리움 글루타미쿰(Corynebacterium glutamicum)을 이용한 이소말토올리고당(L-IMO) 제조방법은 전분 원료의 효소적 구조변형을 통하여 α-1,6 결합의 비율이 높은 이소말토올리고당 (L-IMO)을 제조하며 세포반응 (whole cell)을 통해 이소말토올리고당 (L-IMO)을 생산할 수 있어, 기존의 방법(E.coli 이용) 에서 세포를 파쇄하여 효소를 얻고 이 효소를 농축하여 고기능성 이소말토올리고당(L-IMO)을 제조하는 방법보다 공정을 단축할 수 있고 대량으로 생산할 수 있다. The method for producing isomaltooligosaccharide (L-IMO) using the novel glycosyltransferase expression form of the present invention ( Corynebacterium glutamicum ) is α-1,6 bonded through enzymatic structural modification of starch raw materials. It manufactures isomaltooligosaccharide (L-IMO) with a high ratio of isomaltooligosaccharide (L-IMO) and can produce it through a cell reaction (whole cell) by crushing cells using the existing method (using E.coli ). The process can be shortened and produced in large quantities compared to the method of obtaining enzymes and concentrating these enzymes to produce highly functional isomaltooligosaccharide (L-IMO).
또한 본 발명의 방법으로 제조된 이소말토올리고당 (L-IMO)은 우수한 혈당증가 지연 효과를 나타낸다. 기존 당전이효소 작용결과 생성된 말토덱스트린 유래 L-IMO 생성물을 구성하는 다양한 올리고당을 분획화하여 지소화 특성에 기여하는 유효분획을 확인함으로써 유효물질의 구성 및 구조적 특징을 제시하여 고기능성 올리고당을 특정하였다.In addition, isomaltooligosaccharide (L-IMO) prepared by the method of the present invention exhibits an excellent effect of delaying the increase in blood sugar levels. By fractionating the various oligosaccharides that make up the maltodextrin-derived L-IMO product generated as a result of the action of existing glycosyltransferases and identifying the effective fractions that contribute to lipolytic properties, the composition and structural characteristics of the effective substances are presented to identify highly functional oligosaccharides. did.
도1 L-IMO 분획을 위해 사용한 레진, column 장치
도2 LIMO 분획의 TLC 분석
도3 총 LIMO 분획의 분자량 분포
도4 L-IMO 분획물(No.13,19,29, main 3 region of L-IMO Fraction)의 분자량 구성비교
도5 Human pancreatic amylase 작용 기작과 분해 결과
도6 주요 분획별 Rat 유래 소화효소처리에 따른 포도당 분해결과 확인
도7 (가)시판 IMO 및 LIMO 의 소화율 비교, (나) LIMO 분획별 소화율 비교
도8 Biobrick을 이용한 코리네박테리움 발현 시스템 구축 및 발굴한 당전이효소 최적 발현 시스템
(가) pHCP-H36-T7-RBS-TtTG(opti)-his)을 구축 모식도
(나)서로 다른 프로모터 (H4, H28, H30, H36)와 BCD2와 목적 유전자의 N 말단에 6X histidine tag를 가지는 발현 시스템.
도9 pHCP-H4-BCD2-6xhis-TtTG(opti), pHCP-H28-BCD2-6xhis-TtTG(opti), pHCP-H30-BCD2-6xhis-TtTG(opti), pHCP-H36-BCD2-6xhis-TtTG(opti) 을 가지는 코리네박테리움 글루타미쿰(C.glutamicum)의 SDS-PAGE 결과
도10 pHCP 플라스미드에서 H36 프로모터를 가지고, 목적 유전자인 TtTG의 N 말단에 6X his tag를 가지고, T7 RBS 또는 BCD2를 가지는 시스템의 발현 결과. ((가) 30℃ 배양 결과, (나) 25℃ 배양 결과)
도11 pHCP 플라스미드에서 H36 프로모터와 T7 RBS를 가지면서, N 말단 또는 C 말단에 6X his tag을 가지는 TtTG 발현 시스템의 모식도 ((가) C 말단에 6X his tag을 가지는 pHCP-H36-T7RBS-TtTG(opti)-6Xhis (나) N 말단에 6X his tag을 가지는 pHCP-H36-T7RBS-6Xhis-TtTG(opti)
도12
(가) pHCP-H36-T7RBS-TtTG(opti)-6Xhis 과 pHCP-H36-T7RBS-6Xhis-TtTG(opti)의 30℃와 25℃에서의 발현 결과.
(나) 온도에 따른 코리네박테리움 기반의 TtTG 발현
도13 CgTG 세포반응을 통한 L-IMO 제조시간별 당전이 생성물 구성당 변화
도14 CgTG 세포반응을 통한 L-IMO 제조시간별 분자량 구성변화
도15 L-IMO 생성물의 소화율 평가Figure 1 Resin and column device used for L-IMO fractionation
Figure 2 TLC analysis of LIMO fractions
Figure 3 Molecular weight distribution of total LIMO fraction
Figure 4 Comparison of molecular weight composition of L-IMO fractions (No.13, 19, 29, main 3 region of L-IMO Fraction)
Figure 5 Human pancreatic amylase action mechanism and degradation results
Figure 6 Confirmation of glucose decomposition results according to rat-derived digestive enzyme treatment for each major fraction
Figure 7 (A) Comparison of digestibility of commercially available IMO and LIMO, (B) Comparison of digestibility by LIMO fraction
Figure 8 Construction of Corynebacterium expression system using Biobrick and discovery of optimal glycosyltransferase expression system
(A) Schematic diagram of constructing pHCP-H36-T7-RBS- Tt TG(opti)-his)
(B) Expression system with different promoters (H4, H28, H30, H36), BCD2, and a 6X histidine tag at the N terminus of the target gene.
Figure 9 pHCP-H4-BCD2-6xhis- Tt TG(opti), pHCP-H28-BCD2-6xhis- Tt TG(opti), pHCP-H30-BCD2-6xhis- Tt TG(opti), pHCP-H36-BCD2- SDS-PAGE results of Corynebacterium glutamicum ( C.glutamicum ) with 6xhis- Tt TG(opti)
Figure 10 Expression results of a system with the H36 promoter in the pHCP plasmid, a 6X his tag at the N terminus of the target gene, Tt TG, and T7 RBS or BCD2. ((A) 30℃ culture results, (B) 25℃ culture results)
Figure 11 Schematic diagram of the Tt TG expression system with an H36 promoter and T7 RBS in the pHCP plasmid and a 6X his tag at the N or C terminus ((A) pHCP-H36-T7RBS- Tt with a 6X his tag at the C terminus TG(opti)-6Xhis (B) pHCP-H36-T7RBS-6Xhis- Tt TG(opti) with 6X his tag at the N terminus
Figure 12
(A) Expression results of pHCP-H36-T7RBS- Tt TG(opti)-6Xhis and pHCP-H36-T7RBS-6Xhis- Tt TG(opti) at 30℃ and 25℃.
(B) Corynebacterium-based Tt TG expression according to temperature
Figure 13 Changes in sugar content of glycotransfer products according to L-IMO production time through Cg TG cell reaction
Figure 14 Molecular weight composition change according to L-IMO production time through Cg TG cell reaction
Figure 15 Evaluation of digestibility of L-IMO product
탄수화물을 분해하는 소화효소로는 대표적으로 침샘과 췌장에서 분비되는 아밀라아제(Amylase)가 있다. 아밀라아제에는 α, β, γ형의 세 종류가 있으며, 인체의 침과 췌장에서 분비되는 아밀라아제는 모두 α-아밀라아제이다. α-아밀라아제는 α-1, 4-글리코시드결합만을 무작위로 분해하는 것으로, α-1,6 글리코시드 결합은 미생물 유래 효소는 분해하지 못하며, 고등생물체 (Mammalian) 유래 소화효소는 아주 천천히 분해하는 것으로 알려져 있다. 따라서 α-1,6 글리코시드 결합을 많이 가지고 있는 이소말토올리고당(L-IMO)의 경우 Rat 유래 소화효소 반응시 소화 지연 특성을 보인다.A representative digestive enzyme that breaks down carbohydrates is amylase, which is secreted by the salivary glands and pancreas. There are three types of amylase: α, β, and γ types, and the amylase secreted from the human body's saliva and pancreas is all α-amylase. α-Amylase randomly decomposes only α-1,4-glycosidic bonds, and enzymes derived from microorganisms cannot decompose α-1,6 glycosidic bonds, and digestive enzymes derived from higher organisms (Mammalians) decompose very slowly. It is known that Therefore, in the case of isomaltooligosaccharide (L-IMO), which has many α-1,6 glycosidic bonds, it shows digestion delay characteristics when reacted with rat-derived digestive enzymes.
이소말토올리고당(IMO)은 전분 원료에 액화-당화효소를 처리하여 생성된 말토오스 (maltose)를 주요 기질로 하여 상업용 당전이효소 (Transglucosidase from Asperguillus niger) 를 통해 제조되는 올리고당으로서, 이소말토스, 이소말토트리오스, 이소말토테트라오스 등 포도당 분자간에 α-1,6결합이 1개 이상 존재하고 있는 올리고당으로 정의된다.Isomaltooligosaccharide (IMO) is an oligosaccharide manufactured through commercial glycosyltransferase (Transglucosidase from Asperguillus niger ) using maltose, which is produced by treating starch raw materials with liquefaction-saccharification enzyme, as the main substrate. It is defined as an oligosaccharide that has one or more α-1,6 bonds between glucose molecules, such as maltotriose and isomaltotetraose.
그러나 시판 IMO는 효소작용의 한계로 인해 중합도(degree of polymerization)가 DP 2~3이고, α-1,6결합이 1-2개 수준인 이소말토스, 파노스, 이소말토트리오스로 구성되며, 반응 생성물의 포도당 함량이 50% 수준으로 높다.However, due to limitations in enzyme action, commercially available IMO has a degree of polymerization of DP 2-3 and is composed of isomaltose, panose, and isomaltotriose with 1-2 α-1,6 bonds. , the glucose content of the reaction product is as high as 50%.
<화학식><Chemical formula>
반면 본 발명을 통해 생성된 고중합 이소말토올리고당(Long chian IMO, L-IMO)는 말토덱스트린을 주요기질로 하여 신규 당전이 효소(TtTG) 를 통해 제조되었다. 신규 당전이 효소(TtTG)를 통해 제조된 L-IMO 생성물을 구성하는 다양한 당전이 생성물은 α-1,6 결합 비율이 높고 중합도 (Degree of Polymerization ,DP)가 다양한 당전이 생성물을 포함한다. 여러 실시예를 통해 제조된 L-IMO 생성물은 α-1,6결합이 1,2개 뿐인 시판 IMO에 비해 고등생물체 유래의 소화효소에도 잘 분해되지 않는 소화지연 특성을 가짐을 확인하였다. 또한 상기 다양한 당전이 생성물 중 우수한 소화지연 효과를 보이는 성분을 특정하였다. On the other hand, the highly polymerized isomaltooligosaccharide (Long chian IMO, L-IMO) produced through the present invention was manufactured through a novel glycosyltransferase ( Tt TG) using maltodextrin as the main substrate. The various glycosyltransferase products constituting the L-IMO product prepared through a novel glycosyltransferase ( Tt TG) include glycosyltransfer products with a high α-1,6 bond ratio and various degrees of polymerization (DP). It was confirmed that the L-IMO product prepared through several examples has a digestion delay property that is not easily decomposed even by digestive enzymes derived from higher organisms compared to commercially available IMO, which has only 1 or 2 α-1,6 bonds. In addition, among the various glycotransfer products, ingredients showing excellent digestion delay effects were identified.
한편, 효소적 합성을 통해 제조되는 올리고당의 경우 E.coli, bacillus 및 corynebacterium 등 효소 발현체 내에 생성된 목적 효소을 회수하기 위해 cell lysis - soluble protein 회수 - 농축 및 정제 과정을 거친다. 본원 발명은 발현체 cell 과 기질을 직접적으로 반응시켜 기질이 cell 내부로 들어가게 하고, cell 내에 생성된 효소와 직접 반응하여 생성물이 만들어지는 반응을 통해 L-IMO를 제조하는 기술이다. 따라서 기존의 방법보다 간편하고 신속하게 L-IMO를 제조할 수 있다. Meanwhile, in the case of oligosaccharides manufactured through enzymatic synthesis, they undergo cell lysis - soluble protein recovery - concentration and purification processes to recover the target enzyme produced in enzyme expression bodies such as E. coli, bacillus , and corynebacterium . The present invention is a technology for producing L-IMO through a reaction in which an expression cell and a substrate are directly reacted to allow the substrate to enter the cell, and the product is produced by direct reaction with the enzyme produced within the cell. Therefore, L-IMO can be manufactured more simply and quickly than existing methods.
이때 사용한 발현체는 corynebacterium 을 host로 사용하였으며, 이 균주는 목적 효소 유전자를 균주가 선호하는 codon으로 optimization 하였고, strong promoter 및 ribosomal binding sequence(RBS) library를 조합하여 선발된 발현시스템을 적용하였다. 상기 발현체의 배양결과 얻어진 cell 을 회수하여 기질반응에 사용하는 경우, α-1,6 결합 생성물의 함량이 증가하였으며, 포도당의 함량이 감소하였다. The expression body used at this time was corynebacterium as a host, and this strain optimized the target enzyme gene to the codon preferred by the strain, and applied an expression system selected by combining a strong promoter and ribosomal binding sequence (RBS) library. When the cells obtained as a result of culturing the expression body were recovered and used for substrate reaction, the content of α-1,6 bond product increased and the content of glucose decreased.
L-IMO 내에 지소화성 특성을 가지는 유효성분을 특정하기 위해 당 전이 활성이 있는 유전자를 포함한 효소 발현체(E.coli) 유래 효소를 제조하여 maltodextrin (DE 13-18) 수용액을 기질로 하여 L-IMO (Long-chain isomatooligosaccharide)를 제조하였다. In order to identify active ingredients with lipolytic properties in L-IMO, an enzyme derived from an enzyme ( E. coli ) containing a gene with transglycosylation activity was prepared and L- IMO (Long-chain isomatooligosaccharide) was prepared.
말토덱스트린(maltodextrin)은 포도당, 말토오스 등 단당, 이당류에서부터 DP 20 수준의 말토올리고당 등 다양한 길이의 포도당 사슬형으로 구성된 혼합물이다. L-IMO는 이러한 다양한 복합물 형태의 기질과 당전이 반응 생성물로 만들어진 결과 다양한 결합 및 DP를 가진 올리고당 혼합물 형태로 존재한다. 따라서 L-IMO 내에 소화 지연 효과 성분을 특정하기 위해 분자량 별로 분획화 하였다. Maltodextrin is a mixture of glucose chains of various lengths, from monosaccharides and disaccharides such as glucose and maltose to maltooligosaccharides with a DP level of 20. L-IMO exists in the form of a mixture of oligosaccharides with various bonds and DPs as a result of being made from these various complex substrates and transglycosylation reaction products. Therefore, in order to identify components with a digestion delay effect in L-IMO, they were fractionated by molecular weight.
또한 당류의 분자 간 결합 위치를 확인하기 위하여 H-NMR 분석을 통해 α-1,4/ α-1,6 결합의 비율을 측정하였다. Additionally, in order to confirm the intermolecular bonding position of the saccharide, the ratio of α-1,4/α-1,6 bonds was measured through H-NMR analysis.
이에. 소화지연 효과는 올리고당 구성의 중합도 뿐만 아니라 결합형태 (α-1,4/ α-1,6 결합의 비율)가 큰 영향을 미침을 확인하였다. 중합도(DP)가 7 이상의 물질로 구성되어 있으며 α-1,6 결합의 비율이 23% 정도 함유한 분획물보다Therefore. It was confirmed that the digestion delay effect is greatly influenced by not only the degree of polymerization of the oligosaccharide composition but also the bond type (ratio of α-1,4/α-1,6 bonds). It is composed of substances with a degree of polymerization (DP) of 7 or higher and contains about 23% of α-1,6 bonds.
중합도 (DP) 7 수준으로 α-1,6결합의 비율이 70% 이상인 분획물이 더 소화지연 효과가 뛰어난 경향을 보인다.Fractions with a degree of polymerization (DP) of 7 and a ratio of α-1,6 bonds of 70% or more tend to have a better digestion delay effect.
소화지연 특성이 있는 L-IMO 제조방식을 개선하기 위하여 코리네박테리움 글루타미쿰(Corynebacterium glutamicum) 균주에 당전이효소 유전자를 넣어 발현체를 구축하였으며, 이때 발현 promoter 및 RBS를 우수한 조건으로 선발하였다.In order to improve the production method of L-IMO, which has the property of delaying digestion, an expression body was constructed by inserting a glycosyltransferase gene into a Corynebacterium glutamicum strain, and the expression promoter and RBS were selected under excellent conditions. .
코리네박테리움 글루타미쿰(Corynebacterium glutamicum)은 사람 몸에 존재하는 정상세균총(normal flora) 균주 중 하나로 우리 몸의 면역계에도 적응되어 있어 안정성과 안전성이 인정되고, GRAS (Generally recognized as safe) 스트레인(strain) 중 하나로 아미노산, 핵산 생산에 주로 이용되어 산업적으로 활용도가 높은 균주이다(Nakayama K et al., Adv.Exp. Med. Biol., 105:649-661(1978)) Corynebacterium glutamicum is one of the normal flora strains present in the human body and has been adapted to our body's immune system, so its stability and safety are recognized, and it is a GRAS (Generally recognized as safe) strain ( strain) and is a strain with high industrial utility as it is mainly used for producing amino acids and nucleic acids (Nakayama K et al., Adv.Exp. Med. Biol., 105:649-661 (1978))
또한 본 발명자들이 발명한 기존 E.coli 균주 기반 발현체에서는 대장균을 배양한 후, 유효서열이 삽입된 플라스미드를 induction시키기 위해 저온에서 다시 배양하고, cell lysis 를 거쳐 Amicon tube를 사용하여 농축시키는 단계를 거친다.In addition, in the existing E. coli strain-based expression product invented by the present inventors, E. coli is cultured, then cultured again at low temperature to induce the plasmid into which the effective sequence is inserted, and then concentrated using an Amicon tube after cell lysis. It's rough.
반면, 본원 발명의 C.glutamicum 효소 발현체를 이용하는 경우 Whole cell 반응을 이용해 cell lysis 필요 없이 고중합 이소말토올리고당을 제조 할 수 있어 E.coli 균주 기반 발현체의 반응 대비 L-IMO 제조 공정이 단축된다.On the other hand, when using the C. glutamicum enzyme expression product of the present invention, highly polymerized isomaltooligosaccharide can be produced without the need for cell lysis using whole cell reaction, shortening the L-IMO manufacturing process compared to the reaction of the E. coli strain-based expression product. do.
그러나 이러한 장점에도 불구하고 C. glutamicum에서의 단백질 발현 조절 시스템은 대장균만큼 다양하게 연구되지 않은 실정이다.However, despite these advantages, the protein expression control system in C. glutamicum has not been studied as extensively as in E. coli.
C. glutamicum에서 써모안아에로박터 써모코프리애 (Thermoanaerobacter thermocopriae) 균주 유래 당전이효소 (TtTG)를 발현하기 위해서는 C. glutamicum 균주의 코돈 선호도에 따라 아미노산 서열은 유지하면서, 단백질의 염기 서열을 최적화하는 과정이 필요하다. 이는 단백질 발현에 있어서 미생물 균주간에 선호하는 코돈이 편향되어 있기 때문이다.In order to express the glycotransferase ( Tt TG) derived from the Thermoanaerobacter thermocopriae strain in C. glutamicum , the nucleotide sequence of the protein must be changed while maintaining the amino acid sequence according to the codon preference of the C. glutamicum strain. An optimization process is necessary. This is because preferred codons are biased among microbial strains in protein expression.
또한 목적 단백질에 따라 최적의 발현을 보이는 promoter, RBS 조합은 상이하며, 단순히 그 세기에 비례하여 단백질 발현이 증가하지 않는다는 특성이 있어 고중합 이소말토올리고당 제조효소(TtTG)가 C. glutamicum 균주에서 소화지연 효과가 있는 당 전이 생성물(L-IMO)을 발현하기 위해서는 최적의 promoter, RBS 조합을 선택하는 것이 중요하다. In addition, the promoter and RBS combination showing optimal expression varies depending on the target protein, and protein expression does not simply increase in proportion to the intensity, so highly polymerized isomaltooligosaccharide synthase ( Tt TG) is used in C. glutamicum strains. In order to express the glycotranslation product (L-IMO), which has a digestion delay effect, it is important to select the optimal promoter and RBS combination.
1.본 발명은 신규한 당전이효소를 제공한다. 1. The present invention provides a novel glycosyltransferase.
전분 원료를 기질로 당 전이 반응이 일으키는 당전이 효소는 크게 glycosyl transferase(GTase), transglucosidases(TGase), glycoside phosphorylase(GPase) 및 glycoside hydrolases(GHase) 등 네 가지로 분류된다. Glycosyltransferases, which cause a glycosyltransferase reaction using starch as a substrate, are broadly classified into four types: glycosyl transferase (GTase), transglucosidases (TGase), glycoside phosphorylase (GPase), and glycoside hydrolases (GHase).
전분 원료를 기질로 당 전이 반응이 일으키는 효소로는 크게 glycosyl transferase(GTase), transglucosidases(TGase), glycoside hydrolases(GHase) 및 glycoside phosphorylase(GPase) 등 네 가지로 분류된다. glycosyl transferase(GTase)는 당수용체(탄수화물 등)에 글루코실기를 전달하는 효소이고, transglucosidases(TGase)는 글리코겐 사슬의 α-1,4 결합으로 연결된 포도당을 α-1,6 결합으로 변환시켜 글리코겐에 가지 형성 반응을 매개하는 효소로서, 아밀로오스형(노르말 사슬형) 다당류에서 아밀로펙틴형(분기형) 다당류를 생성하는 효소이고, glycoside hydrolases(GHase)는 sugar와 다른 molecule 사이에 있는 결합을 가수분해하는 효소이며, glycoside phosphorylase(GPase)는 글리코시드 결합의 가역적 형성 및 절단을 촉매하는 효소로서 글리코시드 가수분해효소(GH)와 글라이코트랜스퍼라제(GT)의 이중 기능을 겸한다. Enzymes that cause the sugar transfer reaction using starch as a substrate are broadly classified into four types: glycosyl transferase (GTase), transglucosidases (TGase), glycoside hydrolases (GHase), and glycoside phosphorylase (GPase). Glycosyl transferase (GTase) is an enzyme that transfers glucosyl groups to sugar receptors (carbohydrates, etc.), and transglucosidases (TGase) convert glucose linked by α-1,4 bonds of the glycogen chain into α-1,6 bonds to form glycogen. As an enzyme that mediates the branching reaction, it is an enzyme that produces amylopectin-type (branched) polysaccharide from amylose-type (normal chain) polysaccharide, and glycoside hydrolases (GHase) are enzymes that hydrolyze bonds between sugar and other molecules. Glycoside phosphorylase (GPase) is an enzyme that catalyzes the reversible formation and cleavage of glycosidic bonds and has the dual function of glycoside hydrolase (GH) and glycotransferase (GT).
본 발명에서는 소화지연성이 있는 이소말토올리고당(L-IMO)을 제조하기 위하여 써모안아에로박터 써모코프리애 (Thermoanaerobacter thermocopriae) 균주 유래 당전이효소(TtTG; Thermoanaerobacter thermocopriae transglucosidases) 유전자를 포함하는 벡터를 사용하여 코리네박테리움 글루타미쿰(Corynebacterium glutamicum)에서 발현시킨 신규한 당전이 효소를 사용한다.In the present invention, in order to produce isomaltooligosaccharide (L-IMO) with digestion delay , a glycosyltransferase ( Tt TG; Thermoanaerobacter thermocopriae transglucosidases) gene derived from the Thermoanaerobacter thermocopriae strain is used. A novel glycosyltransferase expressed in Corynebacterium glutamicum using a vector is used.
단백질 발현에서 미생물 균주간에 선호하는 코돈이 편향되어 있어 상기 목적 단백질(TtTG)의 최적의 발현 수준을 이끌어내기 위해서 TtTG 염기 서열의 최적화 과정을 진행하였다.Since the preferred codons are biased among microbial strains in protein expression, an optimization process of the TtTG base sequence was performed to derive the optimal expression level of the target protein (TtTG).
써모안아에로박터 써모코프리애 (Thermoanaerobacter thermocopriae) 균주 유래 당전이효소(TtTG; Thermoanaerobacter thermocopriae transglucosidases)의 아미노산 서열은 서열번호 5 이다. The amino acid sequence of Thermoanaerobacter thermocopriae transglucosidases (TtTG) derived from the Thermoanaerobacter thermocopriae strain is SEQ ID NO: 5.
상기 당전이효소(TtTG)는 '변이체'를 포함하며, '변이체'는 상기 서열번호 5의 아미노산 서열을 갖는 당전이효소(TtTG)와 90% 이상의 상동성을 가지는 단백질을 말한다. The glycosyltransferase ( Tt TG) includes 'variants', and the 'variant' refers to a protein that has more than 90% homology to the glycosyltransferase ( Tt TG) having the amino acid sequence of SEQ ID NO: 5.
"상동성"이란 야생형(wild type) 단백질의 아미노산 서열과의 유사한 정도를 나타내기 위한 것으로서, 본 발명 의 당전이효소(TtTG)를 코딩하는 아미노산 서열(서열번호 5)과 바람직하게는 90%이상, 보다 바람직하게는 95% 이상, 더욱 바람직하게는 98%이상 동일할 수 있는 아미노산 서열을 포함한다. 이러한 상동성의 비교는 육안으로나 구입이 용이한 비교 프로그램을 이용하여 수행할 수 있다. 시판되는 컴퓨터 프로그램은 2개 이상의 서열 간의 상동성을 백분율(%)로 계산할 수 있으며, 상동성(%)은 인접한 서열에 대해 계산될 수 있다. “Homology” refers to the degree of similarity with the amino acid sequence of a wild type protein, preferably 90% with the amino acid sequence (SEQ ID NO: 5) encoding the glycosyltransferase ( Tt TG) of the present invention. It includes amino acid sequences that may be at least 95% identical, more preferably at least 95% identical, and even more preferably at least 98% identical. This comparison of homologies can be performed with the naked eye or using a comparison program that is easily purchased. Commercially available computer programs can calculate the percent homology between two or more sequences, and the percent homology can be calculated for adjacent sequences.
또한, 본 발명의 당전이효소(TtTG) 또는 이의 상동체는 효소 활성을 가지는 한 아미노산 서열 변이체를 포함한다. 서열 변이체란 천연의 아미노산 서열과 하나 이상의 아미노산 잔기가 상이한 서열을 가지는 단백질을 의미한다. 변이체 단백질은 야생형 단백질과 동일한 생물학적 활성을 나타내나, 단백질의 특성이 변형된 변이체일 수 있다. 바람직하게는 아미노산 서열상의 변이와 수식으로 단백질의 열, pH 등에 대한 구조적 안정성이 증대될 수 있다. 예를 들어, 천연의 단백질이 효소 활성을 보이지 않는 강산성에서도 효소 활성을 나타낼 수 있고, 저온이나 고온에서도 효소 활성을 나타낼 수 있다. 또한 아미노산 서열상의 변이와 수식으로 당전이효소의 기질 특이성을 강화되거나 효소와 반응하는 기질의 종류가 확대된 변이체일 수 있다.In addition, the glycosyltransferase ( Tt TG) or its homolog of the present invention includes amino acid sequence variants as long as they have enzymatic activity. A sequence variant refers to a protein having a sequence that differs from the natural amino acid sequence by one or more amino acid residues. A mutant protein exhibits the same biological activity as the wild-type protein, but may be a mutant with modified protein characteristics. Preferably, the structural stability of the protein against heat, pH, etc. can be increased by mutations and modifications in the amino acid sequence. For example, it can show enzyme activity even in strong acids where natural proteins do not show enzyme activity, and it can also show enzyme activity at low or high temperatures. In addition, it may be a variant in which the substrate specificity of the glycosyltransferase is strengthened or the type of substrate that reacts with the enzyme is expanded through mutations and modifications in the amino acid sequence.
2.본 발명은 상기 신규한 당전이효소 유전자의 발현에 최적화된 벡터를 제공한다. 2. The present invention provides a vector optimized for expression of the novel glycosyltransferase gene.
본 발명의 당전이효소(TtTG)는 서열번호 5 로 나타내는 아미노산 서열 또는 이를 암호화하는 뉴클레오타이드 서열(서열번호 4)을 이용하여 유전공학적 방법을 통해 생산될 수 있다. 본 발명에서 상기 재조합 벡터는 상기 당전이효소 (TtTG) 유전자의 효율적인 발현을 위해 발현벡터를 사용하는 것이 바람직하다. The glycosyltransferase ( Tt TG) of the present invention can be produced through genetic engineering methods using the amino acid sequence shown in SEQ ID NO: 5 or the nucleotide sequence encoding it (SEQ ID NO: 4). In the present invention, it is preferable to use an expression vector as the recombinant vector for efficient expression of the glycosyltransferase ( Tt TG) gene.
본 발명에서 "발현벡터"란 적당한 숙주세포에서 목적 펩타이드를 발현할 수 있는 재조합 벡터로서, 유전자 삽입물이 발현되도록 작동하게 연결된 필수적인 조절 요소를 포함하는 유전자 제작물을 말한다. 본 발명의 발현벡터는 적합한 발현 벡터가 일반적으로 가지고 있는 요소로서 프로모터, RBS, 개시코돈 등과 같은 발현조절 요소들을 포함한다. In the present invention, “expression vector” refers to a recombinant vector capable of expressing a target peptide in a suitable host cell, and refers to a gene construct containing essential regulatory elements operatively linked to express the gene insert. The expression vector of the present invention includes expression control elements such as a promoter, RBS, start codon, etc., which are elements that suitable expression vectors generally have.
“프로모터”는 목적 유전자의 전사에 관여하는 부분으로 적절한 발현 세기와 조절 기작을 가지므로 유전자 발현에 중요한 인자이고, “RBS”는 목적 유전자의 번역 과정을 시작할 수 있도록 해주어 목적 유전자의 발현에 중요한 영향을 미치는 인자이며, 특히 “bicistronic 파트”는 목적 유전자 서열의 앞에 짧은 펩타이드 서열을 포함시켜서 목적 유전자가 안정적으로 발현되도록 도와주는 요소이다. “Promoter” is a part involved in the transcription of the target gene and is an important factor in gene expression as it has appropriate expression intensity and control mechanism, and “RBS” allows the translation process of the target gene to start and has an important influence on the expression of the target gene. In particular, the “bicistronic part” is an element that helps the target gene to be expressed stably by including a short peptide sequence in front of the target gene sequence.
본 발명에서 사용한 "발현벡터"는 pHCP plasmid이다. The “expression vector” used in the present invention is pHCP plasmid.
pHCP plasmid (Choi, Yim, & Jeong, 2018, 한국 공개 공보 10-2018-0092110)는 pCES208을 모체로 하고, 복제수를 가지도록 orfA2의 전체가 결실, 미발현, 또는 저발현 되도록 하나 이상의 뉴클레오티드가 결실 또는 치환된 벡터이다. pHCP 플라스미드는 rep 복제 단백질과 ORFa, ORFa2를 포함하는 pCG1 복제원점을 가진다. 본 플라스미드는 목적 단백질 발현에 필요한 프로모터, 리보좀 결합부위와 같은 다양한 발현 조절 인자들을 포함할 수 있고, 플라스미드를 포함하는 숙주 세균을 선별하기 위한 항생제 내성, 영양 요구성, 세포 독성 내성과 같은 선택성 마커가 포함될 수 있으며, 그 종류는 유동적이다.The pHCP plasmid (Choi, Yim, & Jeong, 2018, Korean Publication No. 10-2018-0092110) is based on pCES208, and one or more nucleotides are added to ensure that the entire orfA2 is deleted, not expressed, or underexpressed to have a copy number. It is a deleted or substituted vector. The pHCP plasmid has a pCG1 origin of replication containing the rep replication proteins and ORFa and ORFa2. This plasmid may contain various expression control factors such as a promoter and ribosome binding site required for expression of the target protein, and may include selectable markers such as antibiotic resistance, auxotrophy, and cytotoxic resistance to select host bacteria containing the plasmid. It can be included, and its type is flexible.
본 발명에서는 목적 유전자인 TtTG의 발현을 최적화하기 위해 High-copy number plasmid인 pHCP (Choi, Yim, & Jeong, 2018) 내에서 목적 유전자의 발현에 영향을 미칠 수 있는 요소 중 유전자 발현을 미세 조절하는 프로모터와 RBS 등의 BioBrick 파트들을 조합하고, 다양한 세기를 가지는 프로모터와 RBS와 bicistronic 파트 및 fusion tag의 위치를 조합하였다.In the present invention, in order to optimize the expression of the target gene, Tt TG, gene expression is finely controlled among the factors that can affect the expression of the target gene within pHCP, a high-copy number plasmid (Choi, Yim, & Jeong, 2018). BioBrick parts such as the promoter and RBS were combined, and the positions of the promoter, RBS, bicistronic part, and fusion tag with various strengths were combined.
합성 프로모터 라이브러리 중, 서로 다른 발현 세기를 가지는 H4, H28, H30, H36 (Yim, An, Kang, Lee, & Jeong, 2013)가 있으며, 바람직하기로는 H36이 이용될 수 있다. Among the synthetic promoter libraries, there are H4, H28, H30, and H36 (Yim, An, Kang, Lee, & Jeong, 2013) with different expression intensities, and H36 is preferably used.
bicistronic 파트인 BCD2 또는 RBS인 T7 Ribosome binding site (T7 RBS)가 사용될 수 있으며, 바람직하게는 T7 RBS가 이용될 수 있다.BCD2, which is a bicistronic part, or T7 Ribosome binding site (T7 RBS), which is RBS, can be used, preferably T7 RBS.
3. 본 발명은 상기 벡터로 형질 전환된 발현체 (KACC 81221BP)를 제공한다.3. The present invention provides an expression body (KACC 81221BP) transformed with the above vector.
써모안아에로박터 써모코프리애 (Thermoanaerobacter thermocopriae) 균주 유래 당전이효소(TtTG; Thermoanaerobacter thermocopriae transglucosidases) 유전자를 포함하는 벡터를 사용하여 코레네박테리움 글루타미쿰(Corynebacterium glutamicum)을 형질 전환시켜 얻은 발현체를 제공한다.Obtained by transforming Corynebacterium glutamicum using a vector containing a glycosyltransferase ( Tt TG; Thermoanaerobacter thermocopriae transglucosidases) gene derived from the Thermoanaerobacter thermocopriae strain. An expression body is provided.
TtTG 유전자 발현을 SDS-PAGE로 분석하여 가장 발현양이 많은 프로모터, RBS, fusion tag 등의 위치조합 구성을 선택할 수 있다.By analyzing Tt TG gene expression using SDS-PAGE, you can select the positional combination of promoter, RBS, and fusion tag with the highest expression amount.
pHCP-H36-T7 RBS-6Xhis-TtTG(opti) 시스템으로 C. glutamicum ATCC 13032 균주를 형질 전환하여 발현체(Corynebacterium glutamicum C-TtTG)를 얻을 수 있다.(KACC 81221BP)The expression body ( Corynebacterium glutamicum C - Tt TG) can be obtained by transforming the C. glutamicum ATCC 13032 strain with the pHCP-H36-T7 RBS-6Xhis- Tt TG (opti) system. (KACC 81221BP)
본 발명의 형질전환체를 배양하는 방법은, 숙주의 배양에 사용되는 통상의 방법을 사용하면 된다. 배양방법은, 배치(batch)식, 유동배치식, 연속배양, 리액터형식 등 통상의 미생물의 배양에 사용하는 어떠한 방법도 사용할 수 있다.The method for cultivating the transformant of the present invention may be a conventional method used for host culture. The culture method can be any method used for cultivating ordinary microorganisms, such as batch culture, flow-batch culture, continuous culture, or reactor culture.
4. 본 발명은 상기 신규한 당전이효소 또는 발현체를 이용하는 고기능성 이소말토올리고당 제조방법을 제공한다. 4. The present invention provides a method for producing highly functional isomaltooligosaccharide using the novel glycosyltransferase or expression product.
1) C. glutamicum 당전이효소 발현체 (CgTG)를 배양하여 세포를 회수하고, 1) C. glutamicum glycosyltransferase expression ( Cg TG) was cultured to recover cells,
2) 당기질 용액과 단계1)에서 얻은 C. glutamicum 당전이효소 발현체 (CgTG) 세포 회수액과 혼합하여 0-72시간 동안 반응시켜 당전이 생성물 (L-IMO)을 제조하는 것을 특징으로 하는 L-IMO의 제조 방법을 제공한다. 2) The glycolipid solution is mixed with the C. glutamicum glycosyltransferase expression body ( Cg TG) cell recovery solution obtained in step 1) and reacted for 0-72 hours to produce a glycosyltransfer product (L-IMO). A method for producing L-IMO is provided.
목적의 효소인 것의 확인은 통상의 방법, 예를 들면 SDS-폴리아크릴아미드겔 전기영동(SDS-PAGE), 웨스턴블랏팅(western blotting) 등에 의해 행할 수 있다. Confirmation of the enzyme of interest can be performed by conventional methods, such as SDS-polyacrylamide gel electrophoresis (SDS-PAGE), western blotting, etc.
본 발명의 구체적인 일 실시예에서, 본 발명자들은 C. glutamicum에서 써모안아에로박터 써모코프리애 (Thermoanaerobacter thermocopriae) 균주 유래 당전이효소 (TtTG)를 발현하기 위해 pHCP-H36-T7 RBS-6Xhis-TtTG(opti) 재조합 벡터를 사용하였다. In a specific embodiment of the present invention, the present inventors used pHCP-H36-T7 RBS-6Xhis to express glycosyltransferase ( Tt TG) derived from Thermoanaerobacter thermocopriae strain in C. glutamicum. -TtTG(opti) recombinant vector was used.
합성 프로모터 라이브러리 중, 서로 다른 발현 세기를 가지는 H4, H28, H30, H36 (Yim, An, Kang, Lee, & Jeong, 2013) 과 bicistronic 파트인 BCD2를 가지는 TtTG 유전자 발현을 SDS-PAGE로 분석하여 H36 프로모터(서열번호 1)를 선택하였다. Among the synthetic promoter libraries, H4, H28, H30, H36 with different expression intensities (Yim, An, Kang, Lee, & Jeong, 2013) and Tt TG gene expression with the bicistronic part BCD2 were analyzed by SDS-PAGE. The H36 promoter (SEQ ID NO: 1) was selected.
또한 프로모터는 H36으로 고정하고, bicistronic part인 BCD2와 RBS인 T7 Ribosome binding site (T7 RBS)의 TtTG의 발현에 미치는 영향을 비교하여 T7 Ribosome binding site (T7 RBS) (서열번호 2)를 사용하였다. In addition, the promoter was fixed with H36, and the T7 Ribosome binding site (T7 RBS) (SEQ ID NO: 2) was used to compare the effects of the bicistronic part, BCD2, and the RBS, Tt TG, on the expression of Tt TG. .
정제를 위한 6X his histidine tag (서열번호 3)의 결합 위치에 따른 발현양을 비교하여 H36 프로모터와 T7 RBS를 가지는 TtTG 발현 시스템에서 6X his tag이 N-terminus 혹은 C-terminus 부분에 부착된 시스템을 구축하였다. By comparing the expression level according to the binding site of the 6X his histidine tag (SEQ ID NO. 3) for purification, a system in which the 6X his tag is attached to the N-terminus or C-terminus in the Tt TG expression system with an H36 promoter and T7 RBS was built.
상기 과정을 거쳐 pHCP-H36-T7 RBS-6Xhis-TtTG(opti) 플라스미드 시스템을 구축하였다.Through the above process, the pHCP-H36-T7 RBS-6Xhis- Tt TG(opti) plasmid system was constructed.
상기 재조합 벡터를 C. glutamicum에 도입하여 제작한 형질전환체(GgTG)와 maltodextrin 30%(w/v) 기질용액을 0-72시간 동안 반응시킨 결과, α-1,6결합 비율이 높아 소화지연 효과가 있는 이소말토올리고당(L-IMO)을 생성할 수 있는 것을 확인하였다.As a result of reacting the transformant ( Gg TG) prepared by introducing the above recombinant vector into C. glutamicum with maltodextrin 30% (w/v) substrate solution for 0-72 hours, the α-1,6 binding ratio was high, leading to digestion. It was confirmed that isomaltooligosaccharide (L-IMO), which has a delay effect, can be produced.
C. glutamicum에 의해 이소말토올리고당을 제조 방법은 E.coli에 의해 제조하는 방법에 비해 cell lysis 과정이 필요 없는 등 제조 공정을 단축할 수 있고, GRAS (Generally recognized as safe) 스트레인(strain) 중 하나인 C. glutamicum에서 발현 시킴에 따라 인체에 더욱 안전한 이소말토올리고당을 얻을 수 있다.The method of producing isomaltooligosaccharide by C. glutamicum can shorten the manufacturing process by not requiring a cell lysis process compared to the method of producing isomaltooligosaccharide by E. coli , and is one of the GRAS (Generally recognized as safe) strains. By expressing it in C. glutamicum , it is possible to obtain isomaltooligosaccharide that is safer for the human body.
이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail by examples. However, the following examples only illustrate the present invention, and the content of the present invention is not limited to the following examples.
<실험예 1> 당전이효소 반응 생성물 (L-IMO) 내 소화지연 효과를 가지는 올리고당 탐색<Experimental Example 1> Search for oligosaccharides with digestion delay effect in glycosyltransferase reaction product (L-IMO)
<실험예 1-1> L-IMO 제조 및 소화지연 특성이 있는 올리고당 확인을 위한 분획화<Experimental Example 1-1> Production of L-IMO and fractionation to identify oligosaccharides with digestion delay properties
당전이 활성이 있는 유전자를 포함한 효소 발현체(E.coli) 유래 효소를 제조하여 maltodextrin (DE 13-18) 30%(w/v) 수용액을 기질로 하여 L-IMO를 제조하였다. 생성된 L-IMO 에 함유된 다양한 올리고당 구성물질 중 소화효소에 의해 잘 분해되지 않는 유효한 특성을 지닌 올리고당을 확인하기 위하여 고농도로 시료를 물에 용해하여 1g/mL을 Bio P-2 Gel 레진을 충진한 컬럼에 로딩 후 올리고당 혼합물 형태로 존재하는 LIMO를 분자량 별로 분획화 하였다. 사용한 레진은 MW 100-1,800 범위의 당류를 분리할 수 있는 polyacrylamid 레진으로 DP 7 내외의 올리고당이 다량 함유된 LIMO 분리에 적합한 레진을 선택하였다. An enzyme derived from E. coli containing a gene with transglycosylation activity was prepared, and L-IMO was prepared using a 30% (w/v) aqueous solution of maltodextrin (DE 13-18) as a substrate. In order to identify oligosaccharides with effective properties that are not easily decomposed by digestive enzymes among the various oligosaccharide components contained in the produced L-IMO, samples were dissolved in water at high concentration and 1g/mL was filled with Bio P-2 Gel resin. After loading on one column , LIMO present in the form of an oligosaccharide mixture was fractionated by molecular weight . The resin used was a polyacrylamid resin that can separate saccharides in the MW range of 100-1,800, and a resin suitable for LIMO separation containing a large amount of oligosaccharides with a DP of around 7 was selected.
도 1과 같이 1m 길이의 컬럼의 말단에 peristatic pump 및 auto fraction collector를 연결하여 시간별로 자동적으로 40개의 분획물을 회수하였으며 농축하여 분자량 분석, 결합분석, TLC 분석에 활용하였다. As shown in Figure 1, a peristatic pump and an auto fraction collector were connected to the end of a 1 m long column, and 40 fractions were automatically recovered per hour, concentrated, and used for molecular weight analysis, binding analysis, and TLC analysis.
<실험예 1-2> LIMO 분획의 분자량 분포 확인; TLC, HPSEC<Experimental Example 1-2> Confirmation of molecular weight distribution of LIMO fraction; TLC, HPSEC
fraction collector를 통해 회수된 총 40개의 분획에 함유된 당을 확인하기 위하여 TLC 에 spotting 후 전개용매를 이용하여 구성당을 분리하여 구성 올리고당을 확인하였다. 그 결과 fraction 중 No. 11 ~ No. 32 분획에 발색용매인 황산용액과 반응하는 당 성분이 함유되어 있는 것으로 확인되었으며, No. 11~ 19 에는 DP 7 이상의 상대적으로 고분자 물질을 포함하고, No. 20 ~ 32 분획에는 중합도 DP 7 이하의 당류로 구성되어있음을 확인하였다. 특히 No. 29 분획에는 DP 1, 2 수준의 물질로만 구성되어있을 것으로 확인되었다. In order to confirm the sugars contained in a total of 40 fractions recovered through a fraction collector, the constituent sugars were separated using a developing solvent after spotting on TLC to confirm the constituent oligosaccharides. As a result, among the fractions, no. 11~No. It was confirmed that fraction 32 contains sugar components that react with sulfuric acid solution, a color development solvent, and No. Nos. 11 to 19 include relatively high molecular materials with a DP of 7 or higher, and No. It was confirmed that fractions 20 to 32 were composed of saccharides with a degree of polymerization of DP 7 or lower. Especially no. It was confirmed that fraction 29 consisted only of DP 1 and 2 level substances.
더욱 자세히는 high-performance size-exclusion chromatography (HPSEC, ThermoFisher Scientific, San Jose, CA) 및 RI detector (RI, Showa Denko, Tokyo, Japan) 시스템 분석을 통해 분획들의 분자량 분포를 확인하였으며, 이때 Shodex OHpak SB-804 HQ and 802.5 HQ (Showa Denko) 컬럼, deionized water (18 mΩ)과 이동상으로 사용하였고, flow rate 0.4 mL/min, 50°C 에서 분석하였다. In more detail, the molecular weight distribution of the fractions was confirmed through high-performance size-exclusion chromatography (HPSEC, ThermoFisher Scientific, San Jose, CA) and RI detector (RI, Showa Denko, Tokyo, Japan) system analysis, and Shodex OHpak SB -804 HQ and 802.5 HQ (Showa Denko) columns were used with deionized water (18 mΩ) as a mobile phase, and the analysis was performed at a flow rate of 0.4 mL/min and 50°C.
각 분획 중 분자량(중합도) 및 결합비율이 상이한 3개의 분획(No. 13, NO. 19, No. 29) 을 선택하여 결합패턴 및 소화율 비교를 위해 사용하였다. (도 2,3,4)Among each fraction, three fractions (No. 13, No. 19, No. 29) with different molecular weights (degree of polymerization) and binding ratios were selected and used for comparison of binding patterns and digestibility. (Figures 2,3,4)
<실험예 1-3> 1H-NMR을 통한 LIMO 분획의 결합비(α-1,4/α-1,6) 확인<Experimental Example 1-3> Confirmation of binding ratio (α-1,4/α-1,6) of LIMO fraction through 1H-NMR
당이 함유된 No.11~32 분획에 존재하는 당류의 분자 간 결합 위치를 확인하기 위하여 H-NMR 분석을 통해 α-1,4/α-1,6 결합의 비율을 나타내었다. (표 1) 이때 대조군으로 maltose에는 0, waxy corn starch(WCS) 및 maltodextrin 에는 3 % 수준의 α-1,6 결합 물질을 포함하는 것을 확인하였다. In order to confirm the intermolecular bonding positions of sugars present in sugar-containing fractions No. 11 to 32 , the ratio of α-1,4/α-1,6 bonds was shown through H-NMR analysis. (Table 1) At this time, as a control, it was confirmed that maltose contained 0% and waxy corn starch (WCS) and maltodextrin contained 3% α-1,6 binding substances.
결론적으로 L-IMO 분획물의 분자량 구성과 α-1,6 결합의 비율을 비교한 결과 위 총 20개의 분획(No.12~32 분획) 중 DP 7 이상의 물질로 구성되어 있으며 α-1,6 결합의 비율이 23% 정도 함유하는 No. 13, DP 7 수준으로 α-1,6결합의 비율이 70% 이상인 No. 19, DP 1,2 수준이며 α-1,6 결합 비율이 17% 수준인 No. 29 분획 3종을 선별하였다. 상기 3종의 차별적인 구성을 나타내는 분획 중에서 어떤 물질 구조 및 구성이 우수한 소화지연 특성을 나타내는 유효성분인지 추후 하기 실험에 1-4를 통해 확인하였다. In conclusion, as a result of comparing the molecular weight composition of the L-IMO fraction and the ratio of α-1,6 bonds , it is composed of substances with a DP of 7 or higher among the total 20 fractions (No. 12 to 32 fractions) above and contains α-1,6 bonds. No. containing about 23% of. 13 , No. 7, with a ratio of α-1,6 bonds of more than 70% at the DP 7 level. 19 , No. DP 1,2 level and α-1,6 binding ratio of 17%. 29 Three types of fractions were selected. Among the fractions showing the three types of differential composition, which material structure and composition was the active ingredient showing excellent digestion delay properties was later confirmed through the following experiments 1-4.
<실험예 1-4> L-IMO 분획물의 소화지연 효과 확인<Experimental Example 1-4> Confirmation of digestion delay effect of L-IMO fraction
1) Human pancreatic amylase 작용 및 처리1) Human pancreatic amylase action and processing
소화효소 처리를 통한 L-IMO 분획물 중 포도당으로 분해되지 않는 유효 올리고당 분획을 탐색하고자 Human pancreatic amylase를 처리하였다. Human pancreatic amylase was used to search for effective oligosaccharide fractions that are not decomposed into glucose among the L-IMO fractions through digestive enzyme treatment.
소화효소는 유래 생물체별 분해특성이 상이하다. 주로 bacteria 유래의 α-amylase는 α-1,6 결합을 분해하지 못하는 특성이 있고, mammalian 수준의 Rat 유래 소화효소, 인체유래 소화효소(HPA)는 일반 전분 및 말토덱스트린의 α-1,4결합 뿐만 아니라 α-1,6 결합 또한 천천히 분해 시키는 특징이 있다. Rat 유래 소화효소, 인체유래 소화효소(HPA)에 의해서도 분지결합 부분 중 효소 접근이 어려워 최종적으로 분해되지 않고 잔존하는 alpha-limit dextrin 또는 α-amylase에 의해 잘 분해되지 않는 α-1,6결합이 증가된 올리고당이 남게 되는 데, 이러한 양상을 보이는 물질을 분자량 변화를 통해 확인하였다. Digestive enzymes have different decomposition characteristics depending on the organism from which they are derived. α-amylase, mainly derived from bacteria, has the characteristic of not being able to break down the α-1,6 bond, while mammalian-level rat-derived digestive enzymes and human-derived digestive enzymes (HPA) have the property of not breaking down the α-1,4 bond of general starch and maltodextrin. In addition, it has the characteristic of slowly decomposing the α-1,6 bond. Even with rat-derived digestive enzymes and human-derived digestive enzymes (HPA), it is difficult for enzymes to access the branch bond, so the α-1,6 bond, which is not decomposed by the remaining alpha-limit dextrin or α-amylase, is ultimately not decomposed. Increased oligosaccharides remain, and substances showing this behavior were confirmed through changes in molecular weight.
3종 분획물(No. 13, No. 19, No. 29)의 유효 올리고당 분획을 조사하기 위하여 Human pancreatic amylse 유전자 발현을 통한 소화효소를 제조하여 소화지연 특성에 대한 실험을 진행하였다. (도5, 도6)In order to investigate the effective oligosaccharide fraction of the three fractions (No. 13, No. 19, and No. 29), digestive enzymes were prepared through human pancreatic amylse gene expression, and experiments were conducted on digestion delay properties. (Figure 5, Figure 6)
이때 도 3, 도 4와 같이 HPSEC 분석 조건을 통해 말토덱스트린(MD)의 분자량 구성(도5의 오른쪽 상단 크로마토그램)과 말토덱스트린(MD) 및 인체유래 소화효소(HPA) 처리 후 분자량 변화 (도5의 오른쪽 하단 크로마토그램)를 측정하였다. 인체유래 소화효소(HPA) 처리에 따라 말토덱스트린(MD)이 대부분 포도당으로 분해되고 일부의 limit dextrin으로 추정되는 구성물 물질만 남는 결과를 확인하였다 (도5). At this time, the molecular weight composition of maltodextrin (MD) (top right chromatogram in Figure 5) and the molecular weight change after treatment with maltodextrin (MD) and human digestive enzyme (HPA) through HPSEC analysis conditions as shown in Figures 3 and 4 (Figure chromatogram at the bottom right of Fig. 5) was measured. Upon treatment with human-derived digestive enzymes (HPA), it was confirmed that most of the maltodextrin (MD) was decomposed into glucose and only some of the components estimated to be limit dextrin remained (Figure 5).
이를 토대로 L-IMO를 분회화한 각 시료 각 No. 13, No, 19, No. 29 에 HPA (500U)를 6.7mM NaCl이 포함된 50mM sodium phosphate buffer (최종 농도, 1%, w/v, pH 6.9) 에서 50℃, 24시간 반응시켰으며, 반응종료 후 5분간 끓여서 효소를 불활성화 시켜 HPSEC 분석에 사용하였다.Based on this, each sample No. was divided into L-IMO. 13, No. 19, No. 29, HPA (500U) was reacted in 50mM sodium phosphate buffer (final concentration, 1%, w/v, pH 6.9) containing 6.7mM NaCl at 50°C for 24 hours. After the reaction was completed, the enzyme was boiled for 5 minutes. It was activated and used for HPSEC analysis.
2) Human pancreatic amylase의 L-IMO 분획별 작용 결과2) Action results of human pancreatic amylase by L-IMO fraction
도6에서 보는 바와 같이 L-IMO분획별 소화효소에 의한 반응 양상이 다르게 나타났다. 특히 도6 B의 No. 19 분획의 경우, HPA 소화효소 처리 후(실선)에도 No.13, No. 29 분획처럼 glucose peak 가 높아지며 포도당이 쉽게 분해되지 않는 것으로 나타났다. No. 19 는 아주 소량만 포도당으로 분해되고 대부분 DP7 수준의 물질이 잔존하여 소화가 잘 되지 않는 구조적 특징을 띠는 물질로 구성된 성분임을 확인하였다. As shown in Figure 6, the reaction pattern by digestive enzymes for each L-IMO fraction was different. In particular, No. 6B in Figure 6B. In the case of fraction 19, No. 13 and No. 1 even after HPA digestive enzyme treatment (solid line). Like the 29 fraction, the glucose peak increased and glucose was not easily decomposed. No. 19 was confirmed to be a component composed of a substance with structural characteristics that makes it difficult to digest, as only a very small amount of it is decomposed into glucose and most of the DP7-level substances remain.
도6 A의 DP 20 수준의 물질을 함유하는 No. 13의 경우, HPA처리 후 No. 19 분획과 같은 위치에 잔존되는 일부 물질과 대부분 포도당으로 분해되는 양상을 보였다. 이는 α-1,4결합 비율이 높으므로 α-1,4결합된 올리고당이 많이 함유되어 지소화 특성이 No. 19 분획대비 낮은 것으로 보인다.No. 6 A, containing material at the DP 20 level. In case 13, after HPA treatment, No. Some substances remained in the same location as fraction 19, and most of them were decomposed into glucose. Since it has a high α-1,4 bond ratio, it contains a lot of α-1,4 bonded oligosaccharides, and has no lipolytic properties. It appears to be low compared to the 19 fraction.
도6 C의 No. 29 분획은 α-1,6결합물질이 17% 정도이나, DP 1-2 물질의 혼합물로 확인된 바와 같이 DP2 물질이 포도당으로 분해되어 glucose 위치에 peak 가 더 높아진 것을 확인하였다.No. 6C of Figure 6C. The 29 fraction contained approximately 17% of the α-1,6 bonded material, but as confirmed to be a mixture of DP 1-2 materials, the DP2 material was decomposed into glucose and the peak at the glucose position was confirmed to be higher.
이와 같이 Human pancreatic amylase 소화효소를 처리하여 포도당 분해 정도를 확인함으로써 다양한 형태로 존재하는 L-IMO 생성물 중 소화지연 효능을 지닌 유효한 올리고당의 구성에 대해 특정하여 제시하였다 (도6)In this way, the degree of glucose decomposition was confirmed by treatment with human pancreatic amylase digestive enzyme, and the composition of effective oligosaccharides with digestion delay effect among L-IMO products existing in various forms was specified and presented (Figure 6)
결론적으로 L-IMO 분획별 소화효소 처리물의 분자량 분포 중 No. 19 분획이 소화 효소에 잘 분해되지 않는 유효 올리고당으로 확인되었으며, 이 물질을 동일한 농도로 녹여 glucose generate rate 분석을 통해 소화율 측정 실험을 진행하였다. In conclusion, among the molecular weight distributions of digestive enzyme treated products for each L-IMO fraction, No. Fraction 19 was confirmed to be an effective oligosaccharide that is not easily decomposed by digestive enzymes, and this substance was dissolved at the same concentration and a digestibility measurement experiment was conducted through glucose generate rate analysis.
3)3) Mammalian 유래 소화효소 처리를 통한 L-IMO 분획별 포도당 분해 결과Glucose decomposition results by L-IMO fraction through treatment with Mammalian-derived digestive enzymes
기존 LIMO 생성물(crude LIMO)과 L-IMO 분획별 소화율 차이를 비교함과 동시에 대조군으로 기질인 MD, 시판 IMO 또한 비교를 위해 실험하였다. (도 7)In addition to comparing the differences in digestibility between existing LIMO products (crude LIMO) and L-IMO fractions, the substrate MD and commercially available IMO were also tested for comparison as controls. (Figure 7)
포유류 유래의 소화효소 중 α-glucosidase 는 α-1,6결합을 분해하는 활성이 있으므로 실제 인체 소장에서 소화 흡수를 지연하는 소재의 평가를 위해서는 통상적으로 사용되는 bacterial 소화 효소 (AMG, 효소 제품명) 등이 아닌 Rat 유래의 소장 내 효소를 이용하여 작용생성물을 분석하는 것이 정확하다. 따라서 Rat intestinal acetone powder (RIAP)를 인체 소장 내 소화효소의 유사체로 활용하여 소화율 측정에 활용하였으며, 이때 효소성분만을 정제하기 위하여 30kDa 이상의 물질(대부분 효소)만 선택적으로 분리하는 centrifugal membrane filter를 사용하였다(Amicon, merck millipore). 정제된 RIAP 10mg/mL (w/v)와 각 분획물 (0.1%, w/v)을 6.7 mM NaCl이 함유된 100mM phosphate buffer (pH 6.0)에 처리하여 37℃ 에서 반응시켰다. 반응시 sodium azide (0.2%, w/v), ampicillin (0.0005%, w/v)를 처리하여 반응 중 미생물 생장을 억제하였으며, 분획물 및 Rat 유래 소화효소를 잘 혼합되도록 하여 0, 0.5, 1, 2, 3, 6시간 반응 후 10분간 끓여 효소작용을 정지시켰다. 반응결과 생성된 포도당의 양을 D-glucose assay kit (Megazyme)를 이용하여 정량하였다. 이때 시판 IMO, MD, maltose를 대조군으로 사용하였다.Among mammalian digestive enzymes, α-glucosidase has the activity of breaking down α-1,6 bonds, so in order to evaluate materials that delay digestion and absorption in the actual human small intestine, commonly used bacterial digestive enzymes (AMG, enzyme product name), etc. Instead, it is more accurate to analyze the action product using rat-derived small intestine enzymes. Therefore, rat intestinal acetone powder (RIAP) was used as an analogue of digestive enzymes in the human small intestine to measure digestibility. At this time, a centrifugal membrane filter was used to selectively separate only substances over 30 kDa (most enzymes) to purify only the enzyme components. (Amicon, merck millipore). Purified RIAP 10mg/mL (w/v) and each fraction (0.1%, w/v) were treated with 100mM phosphate buffer (pH 6.0) containing 6.7mM NaCl and reacted at 37°C. During the reaction, sodium azide (0.2%, w/v) and ampicillin (0.0005%, w/v) were treated to inhibit microbial growth during the reaction, and the fractions and rat-derived digestive enzymes were mixed well to form 0, 0.5, 1, After reaction for 2, 3, and 6 hours, the enzyme action was stopped by boiling for 10 minutes. The amount of glucose produced as a result of the reaction was quantified using a D-glucose assay kit (Megazyme). At this time, commercially available IMO, MD, and maltose were used as controls.
실험결과 대조군으로 사용한 Maltose, MD는 Rat유래 효소반응 결과 0.5시간만에 90% 정도가 포도당으로 급속히 분해되었다. (도 7 왼쪽 그래프) As a result of the experiment, about 90% of maltose and MD used as a control group were rapidly decomposed into glucose in 0.5 hours as a result of a rat-derived enzyme reaction. (Figure 7 left graph)
시판 IMO의 경우는 이보다 천천히 포도당을 방출하였으나, L-IMO 가 가장 천천히 반응 6시간 동안 지속적으로 분해되는 지소화성 소재임을 확인하였다. In the case of commercially available IMO, glucose was released more slowly, but it was confirmed that L-IMO was the least digestible material that decomposed continuously for 6 hours.
더욱 자세히는 분회화된 No. 13, No.19, No.29 시료도 동일한 농도의 조건으로 소화효소 반응시킨 결과, 예상과 같이 중합도 DP 7~12 정도 로 구성된 α-1,6결합이 70% 수준의 올리고당 구성일 경우 가장 천천히 분해되는 지소화 특성이 강력한 성분임을 확인하였다. (도 7 오른쪽 그래프)In more detail, the branched no. 13, No.19, and No.29 samples were also reacted with digestive enzymes under the same concentration conditions. As expected, the α-1,6 bond composed of 70% of the oligosaccharide with a polymerization degree of DP of 7 to 12 was the slowest. It was confirmed that this ingredient has strong decomposition properties. (Figure 7 right graph)
No. 19 분획은 포도당 생성이 서서히 진행되어 가장 소화가 천천히 되며, No.13은 IMO보다 더 빨리 소화됨을 확인하였다. 이는 올리고당 구성의 중합도 뿐만 아니라 결합형태가 소화율에 큰 영향을 미친다고 판단할 수 있다. No. It was confirmed that fraction 19 was digested the slowest as glucose production progressed slowly, and No. 13 was digested faster than IMO. It can be concluded that not only the degree of polymerization of the oligosaccharide composition but also the bond type has a significant impact on digestibility .
No.13은 NMR을 통한 분자간 결합 형태가 α-1,4 결합(76.6%)과 α-1,6결합(23.5%)이 같이 존재하기 때문에 일단 소화효소에 의해 α-1,6결합이 분해되고 나면 일반 maltooligosaccharide와 같이 α-1,4결합이 빠르게 분해가 진행되므로 IMO의 포도당 방출량 보다 그 속도가 빠르게 나타난 것으로 판단된다. In No.13, the intermolecular bond form according to NMR is α-1,4 bond (76.6%) and α-1,6 bond (23.5%), so the α-1,6 bond is first broken down by digestive enzymes. After this, the α-1,4 bond is rapidly decomposed like general maltooligosaccharide, so it is believed that the rate of glucose release is faster than that of IMO.
따라서 L-IMO는 소화효소에 의해 포도당으로 잘 분해되지 않는 구조를 지닌 α-1,6결합 올리고당을 유효물질로 포함하고 있는 혼합물이 지소화성 특성을 나타나게한 주요 물질임을 확인하였다. Therefore, it was confirmed that L-IMO , a mixture containing α-1,6-linked oligosaccharides with a structure that is not easily decomposed into glucose by digestive enzymes as an active substance, is the main substance that exhibits lipodigestive properties .
<실험예 2> 코리네박테리움(<Experimental Example 2> Corynebacterium ( C. glutamicumC. glutamicum ) 기반 당전이효소 고발현시스템 구축) Based glycosyltransferase high expression system construction
본 발명은 상기 지소화성 특성을 갖는 유효한 올리고당을 포함하는 L-IMO를 제조하는 효소를 생산하는 산업적 균주개발을 위하여 GRAS 인정을 통해 그 안정성이 입증된 C. glutamicum 균주 기반의 발현체를 구축하고자 하였다. The present invention sought to construct an expression body based on a C. glutamicum strain whose stability has been proven through GRAS recognition in order to develop an industrial strain producing an enzyme that produces L-IMO containing effective oligosaccharides with the above-mentioned lipolytic properties. .
본 발명에서는 다양한 세기를 가지는 프로모터와 RBS와 bicistronic 파트의 조합, 그리고 fusion tag의 조합을 통해서 C. glutamicum에서의 목적 유전자인 TtTG의 발현을 최적화하였다. 구체적으로는 19개의 서로 다른 세기의 합성 promoter와 3개의 biocistronic 파트를 조합한 라이브러리에서 목적 단백질 미세 발현 조절을 위한 최적의 Biobrick 시스템을 개발하였다.In the present invention, the expression of the target gene, TtTG, in C. glutamicum was optimized through a combination of promoters with various strengths, RBS and bicistronic parts, and a fusion tag. Specifically, we developed an optimal Biobrick system for fine control of target protein expression from a library combining 19 synthetic promoters of different strengths and 3 biocistronic parts.
본 발명자들은 Biobrick 시스템을 이용하여 안정성이 확보된 GRAS균주 (C. glutamicum) 기반 신규 당전이 효소 발현체를 구축하였다. C. glutamicum 균주에서 고중합 이소말토올리고당 제조효소 (TtTG)가 포도당이 α-1,4 결합으로 연결된 말토올리고당 형태의 기질인 말토덱스트린(maltodextrin) 수용액과 반응하여 고중합 이소말토올리고당 (L-IMO) 제조 가능함을 확인하여 소화지연 유효성이 강화된 고기능성 올리고당(L-IMO)을 제조하는 방법에 대한 본 발명을 완성하였다.The present inventors used the Biobrick system to construct a novel glycosyltransferase expression body based on GRAS strain ( C. glutamicum ) with guaranteed stability. In the C. glutamicum strain, highly polymerized isomaltooligosaccharide producing enzyme ( Tt TG) reacts with an aqueous solution of maltodextrin, a substrate in the form of maltooligosaccharide in which glucose is linked by an α-1,4 bond, to produce highly polymerized isomaltooligosaccharide (L- By confirming that IMO) can be produced, the present invention regarding a method for producing highly functional oligosaccharide (L-IMO) with enhanced digestion delay effectiveness was completed.
<실험예 2-1> Biobrick을 이용한 코리네박테리움 기반의 최적화된 TtTG 발현 시스템<Experimental Example 2-1> Corynebacterium-based optimized TtTG expression system using Biobrick
1) One) TtTt TG 염기서열의 최적화Optimization of TG sequence
단백질 발현에서 미생물 균주간에 선호하는 코돈이 편향되어있다. 따라서, 목적 단백질의 최적의 발현 수준을 이끌어내기 위해서는 사용할 균주의 코돈 선호도에 따라 아미노산 서열은 유지하면서, 단백질의 염기 서열을 최적화하는 과정이 필수적이다. There is a bias in preferred codons between microbial strains in protein expression. Therefore, in order to achieve the optimal expression level of the target protein, it is essential to optimize the base sequence of the protein while maintaining the amino acid sequence according to the codon preference of the strain to be used.
이에 C. glutamicum에서 당전이효소(TtTG) 발현 시스템을 구축하기에 앞서, TtTG 염기 서열의 최적화 과정을 진행하였다. Accordingly, prior to constructing a glycosyltransferase ( Tt TG) expression system in C. glutamicum , an optimization process of the Tt TG base sequence was performed.
상기 C. glutamicum에서 당전이효소(TtTG) 발현 시스템은 C. glutamicum에서 선호하는 코돈이 편중되어 있는 점을 고려하여 코돈 최적화된 서열(TtTG(opti))을 사용하였다. The glycotransferase ( Tt TG) expression system in C. glutamicum used a codon-optimized sequence ( Tt TG(opti)), considering that the preferred codons in C. glutamicum are biased.
2) 2) TtTt TG의 발현 최적화 시스템 선별Selection of TG expression optimization system
도 8 (가)에서 보는 바와 같이, High-copy number plasmid인 pHCP (Choi, Yim, & Jeong, 2018) 내에서 목적 유전자의 발현에 영향을 미칠 수 있는 요소 중, 각기 다른 세기로 유전자를 발현하는 프로모터와 bicistronic 파트, histidine tag의 위치를 조합하여 발현 시스템을 구축한 후 SDS-PAGE를 통해 발형양을 비교하여 최적의 시스템을 선별하였다. As shown in Figure 8 (a), among the factors that can affect the expression of the target gene within pHCP, a high-copy number plasmid (Choi, Yim, & Jeong, 2018), those that express the gene at different intensities After constructing an expression system by combining the positions of the promoter, bicistronic part, and histidine tag, the optimal system was selected by comparing the expression amount through SDS-PAGE.
프로모터는 목적 유전자의 전사에 관여하는 부분으로 적절한 발현 세기와 조절 기작을 가지므로 유전자 발현에 중요한 인자고, RBS는 목적 유전자의 번역 과정을 시작할 수 있도록 해주어 목적 유전자의 발현에 중요한 영향을 미치는 인자이며, 특히 bicistronic 파트는 목적 유전자 서열의 앞에 짧은 펩타이드 서열을 포함시켜서 목적 유전자가 안정적으로 발현되도록 도와주는 요소이다. 본 발명에서는 다양한 세기를 가지는 프로모터와 RBS와 bicistronic 파트의 조합, 그리고 fusion tag의 조합을 통해서 목적 유전자인 TtTG의 발현을 최적화하였다.The promoter is a part involved in the transcription of the target gene and is an important factor in gene expression because it has an appropriate expression intensity and control mechanism. The RBS is a factor that has an important influence on the expression of the target gene by allowing the translation process of the target gene to start. , In particular, the bicistronic part is an element that helps the target gene to be expressed stably by including a short peptide sequence in front of the target gene sequence. In the present invention, the expression of the target gene, Tt TG, was optimized through a combination of promoters with various strengths, RBS and bicistronic parts, and a fusion tag.
3) 프로모터 선택3) Promoter selection
도 8 (나)에서와 같이 합성 프로모터 라이브러리 중, 서로 다른 발현 세기를 가지는 H4, H28, H30, H36 (Yim, An, Kang, Lee, & Jeong, 2013) 과 bicistronic 파트인 BCD2를 가지는 TtTG 유전자 발현을 SDS-PAGE로 분석하였다As shown in Figure 8 (b), among the synthetic promoter libraries, H4, H28, H30, H36 with different expression intensities (Yim, An, Kang, Lee, & Jeong, 2013) and the Tt TG gene with BCD2, a bicistronic part. Expression was analyzed by SDS-PAGE.
pHCP-promoter-BCD2-6xhis-TtTG(opti) 의 발현 결과 H36 프로모터 하에서 발현되는 TtTG의 양이 가장 많았다. (도 9)As a result of the expression of pHCP- promoter -BCD2-6xhis- Tt TG(opti), the amount of Tt TG expressed under the H36 promoter was the highest. (Figure 9)
프로모터는 H36으로 고정하고, bicistronic part인 BCD2와 RBS인 T7 Ribosome binding site (T7 RBS)의 TtTG의 발현에 미치는 영향을 비교하였다. (도 10)The promoter was fixed with H36, and the effects of the bicistronic part, BCD2, and the RBS, T7 Ribosome binding site (T7 RBS), on the expression of Tt TG were compared. (Figure 10)
도 10에서 보는 바와 같이, pHCP 플라스미드에서 H36 프로모터를 가지고, 목적 유전자인 TtTG의 N 말단에 6X his tag를 가지며, T7 RBS 또는 BCD2를 가지는 시스템의 발현 결과 T7 Ribosome binding site (T7 RBS)를 사용한 경우가 BCD2가 있는 경우보다 배양 온도에 관계없이 발현양이 많았다.As shown in Figure 10, the pHCP plasmid has an H36 promoter, a 6 In cases with BCD2, the expression amount was higher regardless of culture temperature than in cases with BCD2.
도면 11에서 보는 바와 같이, 정제를 위한 6X his histidine tag의 결합 위치에 따른 발현양을 비교하고자, H36 프로모터와 T7 RBS를 가지는 TtTG 발현 시스템에서 6X his tag이 N-terminus 혹은 C-terminus 부분에 부착된 시스템을 구축하였다.As shown in Figure 11, in order to compare the expression level according to the binding site of the 6X his histidine tag for purification, the 6 An attached system was built.
발현 결과를 보기 위해 SDS-PAGE를 수행하였고(도 12), GelAnalyzer를 이용하여 목적 단백질인 TtTG의 함량을 비교하였다. 30℃ 배양하였을 때 N-term-his-tag을 가지는 TtTG는 38.03%, C-term-his-tag을 가지는 TtTG는 28.09% 이었다. 또한 25℃ 배양하였을 때는 N-term-his-tag을 가지는 TtTG는 27.57%, C-term-his-tag을 가지는 TtTG는 22.52% 으로 N-terminus에 his-tag을 가지는 경우에서 더 높은 발형양을 보였다.SDS-PAGE was performed to view the expression results (Figure 12), and the content of the target protein, Tt TG, was compared using GelAnalyzer. When cultured at 30°C, Tt TG with N-term-his-tag was 38.03%, and Tt TG with C-term-his-tag was 28.09%. In addition, when cultured at 25℃, Tt TG with N-term-his-tag was 27.57%, and Tt TG with C-term-his-tag was 22.52%, which was higher in the case with his-tag at N-terminus. It showed shape.
<실험예 3> 형질잔환된 <Experimental Example 3> Transformed C.glutamicumC. glutamicum 제조 및 배양 Manufacturing and cultivation
1)One) Corynebacterium glutamicum C-TtCorynebacterium glutamicum C-Tt TGTG 균주 배양 (KACC 81221BP)Strain culture (KACC 81221BP)
pHCP 기반의 플라스미드 발현 시스템은 모두 C. glutamicum ATCC 13032 균주에서 발현되었다. 모든 코리네박테리움 균주는 전배양 후 본배양을 하였다. 먼저 전배양은 25 ug/ml의 kanamycin을 첨가한 Brain heart infusion (BHI) 액체 배지 5 ml에 균을 접종한 후 30℃에서 18 시간 동안 200 rpm으로 배양하였다. 이후, 본배양은 25 ug/ml의 kanamycin을 첨가한 BHI 액체 배지 50 ml를 250 ml flask에 분주 후, 본배양한 시료를 500 ul 접종하고, 24 시간 동안 200 rpm에서 25℃ 또는 30℃에서 배양하였다. All pHCP-based plasmid expression systems were expressed in C. glutamicum ATCC 13032 strain. All Corynebacterium strains were pre-cultured and then main-cultured. First, for pre-culture, the bacteria were inoculated into 5 ml of Brain heart infusion (BHI) liquid medium containing 25 ug/ml kanamycin and then cultured at 30°C for 18 hours at 200 rpm. Afterwards, for the main culture, 50 ml of BHI liquid medium containing 25 ug/ml of kanamycin was dispensed into a 250 ml flask, then 500 ul of the main culture sample was inoculated and cultured at 25°C or 30°C at 200 rpm for 24 hours. did.
2) 균주 배양 온도 선택2) Selection of strain culture temperature
30℃ 배양하였을 때와 25℃ 배양하였을 때를 비교해 보면, T7 Ribosome binding site (T7 RBS)를 사용한 경우에는 온도의 영향을 받지 않았으나(도 10), 정제를 위한 6X his histidine tag의 결합 위치에 따른 발현양은 차이가 있었다.Comparing when cultured at 30℃ and when cultured at 25℃, when T7 Ribosome binding site (T7 RBS) was used, it was not affected by temperature (Figure 10), but depending on the binding site of the 6X his histidine tag for purification, There was a difference in the amount of expression.
GelAnalyzer를 이용하여 목적 단백질인 TtTG의 함량을 비교한 결과, 30℃ 배양하였을 때 N-term-his-tag을 가지는 TtTG는 38.03%, C-term-his-tag을 가지는 TtTG는 28.09% 이었다. 또한 25℃ 배양하였을 때는 N-term-his-tag을 가지는 TtTG는 27.57%, C-term-his-tag을 가지는 TtTG는 22.52% 으로 30℃ 배양하였을 경우에서 더 높은 발형양을 보였다. (도 12 (가))As a result of comparing the content of Tt TG, the target protein, using GelAnalyzer, when cultured at 30°C, Tt TG with N-term-his-tag was 38.03% and TtTG with C-term-his-tag was 28.09%. . In addition, when cultured at 25℃, TtTG with N-term-his-tag was 27.57% and TtTG with C-term-his-tag was 22.52%, showing a higher growth amount when cultured at 30℃. (Figure 12 (a))
또한 C-말단에 부착된 6X-his tag를 이용해 TALON superflow resin를 활용하여 효소 정제하여 100ml 배양을 통해 총 0.48 mg의 효소를 회수할 수 있었고, 이를 대장균에서 발현된 효소의 전환율과 비교하였다. 대장균에서 발현(37℃에서 OD 600 nm 0.5~0.6 까지 배양 후, IPTG induction 과 함께 25
도 12 (가)에서 30
코리네박테리움 배양은 25
<실험예 4> <Experimental Example 4> Corynebacterium glutamicum C-TtTG Corynebacterium glutamicum C-TtTG 균주 발현 분석 (KACC 81221BP)Strain expression analysis (KACC 81221BP)
1) 폴리아크릴아마이드 겔 전기영동 (SDS-PAGE)1) Polyacrylamide gel electrophoresis (SDS-PAGE)
실험예 3 같은 조건으로 배양이 완료된 시료는 목적 단백질의 발현 확인을 위해 폴리아크릴아마이드 겔 전기영동 (SDS-PAGE)을 진행하였고, SDS-PAGE 분석을 진행하기에 앞서 아래의 전처리 과정을 거쳤다. 각 시료의 OD600 nm을 측정 후, OD600 nm가 4가 되도록 균을 회수하고, T-bfr (20 mM tris, pH 8.0) 300 ul으로 회수된 균을 현탁한 후 초음파파쇄기 (sonicator)로 균을 용해하였다. Experimental Example 3 Samples cultured under the same conditions were subjected to polyacrylamide gel electrophoresis (SDS-PAGE) to confirm the expression of the target protein, and the following pretreatment process was performed prior to SDS-PAGE analysis. After measuring the OD 600 nm of each sample, the bacteria were recovered so that the OD 600 nm was 4, the recovered bacteria were suspended in 300 ul of T-bfr (20 mM tris, pH 8.0), and the bacteria were disrupted using a sonicator. was dissolved.
초음파 파쇄 조건은 시료 한 개 당 5 sec on, 3 sec off, amplitude 25%의 설정값으로 약 10 분간 지속하였으며, 회수된 균이 충분히 용해되어 시료의 색이 투명해질 때까지 진행하였다. The ultrasonic disruption conditions were set at 5 sec on, 3 sec off, and amplitude 25% for each sample and lasted for about 10 minutes until the recovered bacteria were sufficiently dissolved and the color of the sample became transparent.
파쇄가 완료된 시료는 원심 분리하여 전체 단백질과 수용성 단백질, 불용성 단백질을 분획화하였다. 분획된 각 시료는 5X SDS-PAGE loading buffer (250 mM Tris-HCl, pH 6.8, 5% 2-Mercaptoethanol, 10% SDS, 0.2% bromophenol blue, 50% glycerol) 와 1 : 4의 비율로 혼합한 후, 100℃에서 5분간 끓여주었다. 전처리가 완료된 시료는 10 ul를 취하여 10% 폴리아크릴아마이드젤에서 분석하였다. The sample after completion of crushing was centrifuged to fractionate total protein, soluble protein, and insoluble protein. Each fractionated sample was mixed with 5X SDS-PAGE loading buffer (250 mM Tris-HCl, pH 6.8, 5% 2-Mercaptoethanol, 10% SDS, 0.2% bromophenol blue, 50% glycerol) at a ratio of 1:4. , boiled at 100°C for 5 minutes. 10 ul of the pretreated sample was taken and analyzed on a 10% polyacrylamide gel.
Coommasie Briliant Blue로 염색한 후, 목적 단백질의 비율을 확인하기 위해서 GelAnalyzer (GelAnalyzer 19.1 by Istvan Lazar Jr., PhD and Istvan Lazar Sr., PhD, CSc) 가 사용되었다.After staining with Coommasie Briliant Blue, GelAnalyzer (GelAnalyzer 19.1 by Istvan Lazar Jr., PhD and Istvan Lazar Sr., PhD, CSc) was used to confirm the ratio of the target protein.
상기 과정을 거쳐 최종적으로 pHCP-H36-T7 RBS-6Xhis-TtTG(opti) 플라스미드 시스템이 최종 발현 균주로 선별되었다. 최종 시스템의 H36은 서열 번호 1과 같고, T7 RBS는 서열 번호 2와 같고, 6X his tag은 서열 번호 3과 같으며, 코돈 최적화된 TtTG는 서열 번호 4와 같다. Through the above process, pHCP-H36-T7 RBS-6Xhis-TtTG(opti) plasmid system was finally selected as the final expression strain. In the final system, H36 is as in SEQ ID NO: 1, T7 RBS is as in SEQ ID NO: 2, 6X his tag is as in SEQ ID NO: 3, and codon-optimized TtTG is as in SEQ ID NO: 4.
<실험예 5> <Experimental Example 5> CgCg TG 세포 반응을 통한 고중합 이소말토올리고당 제조 방법Method for producing highly polymerized isomaltooligosaccharide through TG cell reaction
1) 형질전환체를 기질과 세포반응1) Cell reaction between the transformant and the substrate
C. glutamicum 당전이효소 발현체 (CgTG)를 배양하여 세포를 회수하고, maltodextrin 30%(w/v) 기질용액과 C. glutamicum 당전이효소 발현체 (CgTG) 세포 회수액을 pH 4 buffer와 혼합하여 0-72시간동안 반응시켰다. HPAEC-PAD를 통해 분석하여 당 전이 생성물 (L-IMO)가 제조됨을 확인하였다.Cells were recovered by culturing C. glutamicum glycosyltransferase expression body ( Cg TG), maltodextrin 30% (w/v) substrate solution and C. glutamicum glycosyltransferase expression body ( Cg TG) cell recovery solution were mixed with pH 4 buffer. Mixed and reacted for 0-72 hours. Analysis through HPAEC-PAD confirmed that a glycotransfer product (L-IMO) was produced.
포도당이 a 1-4 결합으로 연결된 말토올리고당 형태의 기질인 maltodextrin 수용액과 CgTG 세포 배양액을 혼합 반응한 L-IMO 생성물을 반응 시간에 따라 구성당의 peak 변화를 확인한 결과, 반응시간이 증가함에 따라 isomaltooligosaccharide의 구성물인 IG2(maltose), IG3(maltotriose), IG4(maltotetraose), IG5(maltopentaose), IG6(maltoheptaose)의 peak가 증가하여 α-1,6 결합만으로 구성된 물질의 함량이 높아졌다. As a result of checking the peak change in the constituent sugars of the L-IMO product obtained by mixing an aqueous solution of maltodextrin, a substrate in the form of maltooligosaccharide in which glucose is linked by a 1-4 bond, and Cg TG cell culture medium, the change in the peak of the constituent sugars was confirmed according to the reaction time. As the reaction time increased, isomaltooligosaccharide The peaks of the components IG2 (maltose), IG3 (maltotriose), IG4 (maltotetraose), IG5 (maltopentaose), and IG6 (maltoheptaose) increased, increasing the content of substances consisting only of α-1,6 bonds.
<실험예 6> <Experimental Example 6> CgCg TG 세포 반응을 통한 고중합 이소말토올리고당 생성물 분석Analysis of highly polymerized isomaltooligosaccharide products via TG cell reaction
<실험예 6-1> <Experimental Example 6-1> CgCg TG 세포 반응을 통한 고중합 이소말토올리고당 제조Production of highly polymerized isomaltooligosaccharide through TG cell reaction
C.glutamicum 당전이효소 발현체(CgTG)를 배양하여 세포를 회수하여 L-IMO 제조 가능성을 확인하기 위하여 균주를 배양액 기준으로 OD 값 측정 및 반응조건을 표2 와 같이 수립하였다.In order to confirm the possibility of producing L-IMO by cultivating C. glutamicum glycosyltransferase expression ( Cg TG) cells and confirming the possibility of producing L-IMO, OD values were measured and reaction conditions were established as shown in Table 2 based on the culture medium of the strain.
maltodextrin 30%(w/v) 기질용액과 C.glutamicum 발현체 세포 회수액의 세포농도(OD600) 값이 최종 1이 되도록 pH 4 buffer와 혼합하여 pH 4에서 0~ 72시간 동안 반응하여 당전이 생성물 L-IMO가 제조됨을 HPAEC-PAD를 통해 분석하였으며 분자량 구성변화를(GPC) 확인하였다. (도 13)Mix the maltodextrin 30% (w/v) substrate solution with pH 4 buffer so that the final cell concentration (OD 600 ) of the C. glutamicum expression cell recovery solution is 1 and react at pH 4 for 0 to 72 hours to produce a glycotransfer product. The production of L-IMO was analyzed through HPAEC-PAD, and the change in molecular weight composition (GPC) was confirmed. (Figure 13)
포도당이 α-1,4결합으로 연결된 말토올리고당 형태의 기질인 maltodextrin 수용액과 CgTG세포 배양액을 혼합 반응한 L-IMO 생성물을 반응시간에 따라 추적하였다.An aqueous solution of maltodextrin, a substrate in the form of maltooligosaccharide where glucose is linked through an α-1,4 bond. The L-IMO product reacted with mixed Cg TG cell culture medium was tracked according to the reaction time.
구성당의 peak 중 기질과 달리 새로운 peak가 출현하였으며 당전이 반응에 의하여 생성된 물질로 유추할 수 있다. 반응시간이 증가함에 따라 isomaltooligosaccharide 구성물(IG2, IG3, IG4, IG5, IG6)의 peak가 증가하여 α-1,6결합으로만 구성된 물질의 함량이 높아지는 변화를 관찰하였다.(도 13)Among the peaks of the constituent sugars, a new peak, unlike the substrate, appeared and can be inferred to be a substance produced by a sugar transfer reaction. As the reaction time increased, the peaks of isomaltooligosaccharide components (IG2, IG3, IG4, IG5, IG6) increased, and a change in the content of substances consisting only of α-1,6 bonds was observed (Figure 13).
도 14에서 보는 바와 같이, CgTG세포반응 결과 말토덱스트린에 당전이 반응이 일어나며 최고분자량(Mp)이 왼쪽으로 shift되어 분자량이 DP 7내외에서 DP2~4수준 증가하는 것으로 나타나, CgTG세포와의 whole cell 반응으로도 세포질 내 생성된 당전이 효소에 의한 기질과 반응에 의해 당전이 작용이 활발하게 일어나 당전이 반응 생성물로 L-IMO를 생산하는 것으로 확인되었다. 반응 48시간이 경과하면 분자량 구성에는 큰 변화가 없는 것으로 보였다.As shown in Figure 14, as a result of the Cg TG cell reaction, a glycosyltransferase reaction occurs in maltodextrin, and the highest molecular weight (Mp) shifts to the left, showing that the molecular weight increases from around DP 7 to DP 2 to 4 levels, showing the difference between Cg TG cells and Cg TG cells. It was confirmed that even in whole cell reactions, glycosyltransferase activity occurs actively by reacting with the substrate by glycosyltransferase produced in the cytoplasm, producing L-IMO as a glycosyltransferase product. After 48 hours of reaction, there appeared to be no significant change in molecular weight composition.
<실험예 6-2> <Experimental Example 6-2> CgCg TG 세포반응을 통한 L-IMO 제조시간별 분자량 구성변화(H-NMR)Molecular weight composition change by L-IMO manufacturing time through TG cell reaction (H-NMR)
L-IMO 반응물에 함유된 α-1,6결합을 지닌 물질이 주로 소화지연 효과 유효성에 기여하므로 생성물 내 물질의 결합비를 확인하였다.Since the substance with α-1,6 bond contained in the L-IMO reactant mainly contributes to the effectiveness of the digestion delay effect, the binding ratio of the substance in the product was confirmed.
반응시간이 경과함에 따라 α-1,4결합비 대비 α-1,6결합물질의 비율이 점차 증가하였으며, 72시간째 가장 높은 1:2.84 값을 보였다. 기존 E.coli 발현체 및 효소반응법으로 제조한 L-IMO 의 α-1,4 및 1,6결합물질 비율은 1:2.11로 새로운 발현체로 제조한 L-IMO 대비 낮았다.As the reaction time progressed, the ratio of α-1,6-binding material to α-1,4-binding ratio gradually increased, showing the highest value of 1:2.84 at 72 hours. The ratio of α-1,4 and 1,6 binding substances in L-IMO prepared using the existing E. coli expression form and enzyme reaction method was 1:2.11, which was lower than that of L-IMO prepared with the new expression form.
<실험예 6-3> L-IMO 생성물의 소화율 평가<Experimental Example 6-3> Evaluation of digestibility of L-IMO product
위 도 7에서 나타낸 유효한 분획의 소화율 평가방법과 같은 방법으로 CgTG를 이용하여 whole cell 반응으로 제조한 L-IMO 의 소화율을 확인하였다.The digestibility of L-IMO prepared by whole cell reaction was confirmed using Cg TG in the same manner as the digestibility evaluation method of the effective fraction shown in FIG. 7 above.
도 15에서 보는 바와 같이, 기존 L-IMO(□,■) 대비하여 CgTG를 이용하여 제조한 L-IMO가 소화효소 처리시간에 따른 포도당 방출량이 적은 것으로 나타나, 소화지연효과가 높은 것으로 나타났다. 반응 72시간째에 CgTG를 이용하여 제조한 L-IMO의 소화율이 가장 낮았다. As shown in Figure 15, compared to the existing L-IMO (□,■), L-IMO prepared using Cg TG showed less glucose release according to the digestive enzyme treatment time, showing a high digestion delay effect. At 72 hours of reaction, the digestibility of L-IMO prepared using Cg TG was the lowest.
이로써 CgTG를 이용하여 제조한 L-IMO의 지소화특성이 강화된 유효성을 갖는 생성물 구성임을 확인하였으며 α-1,6결합이 최적화된 생성물을 제조하기 위하여 반응평형이 일어나기에 충분한 CgTG세포 양 및 반응시간의 조합에 의해 생성됨을 알 수 있다.As a result, it was confirmed that the lipolytic properties of L-IMO prepared using Cg TG were a product composition with enhanced effectiveness, and the amount of Cg TG cells sufficient for reaction equilibrium to occur in order to produce a product with optimized α-1,6 bond was used. It can be seen that it is produced by a combination of and reaction time.
Claims (17)
Transyltransferase ( Tt TG) for producing highly polymerized isomaltooligosaccharides, codon-optimized for expression in C. glutamicum .
The sugar transferase ( Tt TG) for producing highly polymerized isomaltooligosaccharide according to claim 1, wherein the codon optimized sequence consists of the DNA sequence of SEQ ID NO: 4.
pHCP vector containing glycosyltransferase ( Tt TG) gene for production of highly polymerized isomaltooligosaccharide
The pHCP vector according to claim 3, wherein the pHCP vector has the H36 promoter of SEQ ID NO: 1.
The pHCP vector according to claim 3, wherein the pHCP vector has a T7 Ribosome binding site (T7 RBS) of SEQ ID NO: 2.
The pHCP vector according to claim 3, wherein the pHCP vector contains the 6X his histidine tag sequence of SEQ ID NO: 3.
The pHCP vector according to claim 6, wherein the pHCP vector contains the 6
The pHCP vector according to claim 6, wherein the pHCP vector contains the 6
A transformant ( C. glutamicum ) transformed with the pHCP vector of any one of claims 3 to 8.
2) 상기 형질전환체를 기질과 세포반응 시키는 단계;
를 포함하는 고중합 이소말토올리고당(L-IMO)을 제조하는 방법.
1) Culturing the transformant of paragraph 9; and
2) allowing the transformant to react with the substrate;
A method for producing highly polymerized isomaltooligosaccharide (L-IMO) comprising.
The method of claim 10, wherein 1) the transformant is cultured at 25°C to 30°C.
The method of claim 10, wherein 1) the transformant is cultured at 30°C.
The method of claim 10, wherein the cell reaction time in step 2) is 72 hours.
The method of claim 10, wherein the product of step 2) has an α-1,4:α-1,6-glycosidic linkage ratio of 1:2.84. method.
Highly polymerized isomaltooligosaccharide (L-IMO), characterized in that it is manufactured according to the manufacturing method of claim 10.
The highly polymerized isomaltooligosaccharide (L-IMO) according to claim 15, wherein the α-1,4:α-1,6-glycosidic linkage ratio is 1:2.84.
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