Production of N-Acetylglucosamine Using Recombinant Chitinolytic Enzymes

Abstract

The pharmaceutically important compound Nacetylglucosamine (NAG), is used in various therapeutic formulations, skin care products and dietary supplements. Currently, NAG is being produced by an environmentunfriendly chemical process using chitin, a polysaccharide present in abundance in the exoskeleton of crustaceans, as a substrate. In the present study, we report the potential of an eco-friendly biological process for the production of NAG using recombinant bacterial enzymes, chitinase (CHI) and chitobiase (CHB). The treatment of chitin with recombinant CHI alone produced 8% NAG and 72% chitobiose, a homodimer of NAG. However, supplementation of the reaction mixture with another recombinant enzyme, CHB, resulted in approximately six fold increase in NAG production. The product, NAG, was confirmed by HPLC, TLC and ESI-MS studies. Conditions are being optimized for increased production of NAG from chitin.

Recombinant expression of insoluble enzymes in Escherichia coli: a systematic review of experimental design and its manufacturing implications

“…The remaining 12% utilized K12 derivates. Commercially available K12 strains used include JM109 [ 25 , 51 , 55 ], DH5α [ 30 , 56 ], NovaBlue [ 57 ], XL1 Blue [ 58 ], M15 [ 31 , 59 , 60 ], and Top10 [ 61 , 62 ]; alternatively non-commercial K12 strains W3110 [ 63 ] and MG1655 [ 62 , 64 ] were used in a limited number of cases. K12 strains serve as useful tools when plasmid instability is encountered resulting in plasmid loss from the host [ 54 ]; this may explain its use in limited cases for expression.…”

“…High transformation efficiency [ 78 ] Fructose-1,6-bisphosphate aldolase DH5α (K12) Various Suppliers (Thermo Fisher Scientific, New England Biolabs, Gold Biotechnology Ltd.) Generally, a strain used for cloning and blue/white screening. (recA) mutation allows for better insert stability [ 30 ] Chitobiase M15 (K12) Qiagen Generally used in conjunction with plasmid (pQE) found within a expression kit from Qiagen [ 59 ] Cellobiose phosphorylase JM109 (K12) Promega Corporation Generally, a strain used for cloning and blue/white screening [ 55 ] Quinoprotein glucose dehydrogenase B NovaBlue (DE3) (K12) Merck KGaA Generally, a strain used for cloning and blue/white screening [ 57 ] Fuculose-1-phosphate aldolase XL-1 Blue (K12) Promega Corporation Generally, a strain used for cloning and blue/white screening [ 58 ] Trehalose transferase TOP10 (K12) Thermo Fisher Scientific Generally, a strain used for cloning and plasmid propagation [ 61 ] …”

“…Tight basal expression control of proteins appeared to be a key factor that was prevalent among the literature. In most instances, this control was managed through lower concentrations of inducer compounds such as 0.1–0.5 mM IPTG for inducible promoters such as T7/lac [ 6 , 59 , 65 , 72 , 104 – 106 ]. Soluble expression of Bacillus acidopullulyticus pullulanase was highly dependent on the control of basal gene expression that was only achieved in pET22b/pET28a harboring an inducible T7/lac promoter as opposed to pET20b that contained a constitutive T7 promoter region [ 104 ].…”

Draft Genome Sequence of Amantichitinum ursilacus IGB-41, a New Chitin-Degrading Bacterium

“…Chitin is the second most abundant biopolymer on earth and can be found as a structural polysaccharide in a variety of organisms, including fungi, crustaceans, and insects ( 1 ). The polymer consists of N -acetylglucosamine and glucosamine, two amino sugars that are of increasing economic interest, e.g., as food additives, cosmetic ingredients, and platform chemicals ( 2 , 3 ). Compared to cellulose, chitin is much more resistant to biodegradation, and only the concerted action of different classes of chitinases is able to cleave the molecule efficiently ( 4 ).…”

Transcriptional and Translational Characterization of Rice Chitinase Genes

“…作 物 学 报 第 40 卷 量往往在 1000 kD 以上。它在生物体中主要是作为 身体骨架且具自身保护作用。在植物与病原物的互 作过程中, 几丁质的降解产物 N-乙酰葡萄糖胺可以 发挥病原物相关的分子模式(microbe/pathogen associated molecular pattern, MAMP)的作用激发植物的 防 卫 反 应 [1] 。 拟 南 芥 受 体 激 酶 AtCERK1 (chitin elicitor receptor kinase 1 of Arabidopsis)的胞外区有 3 个赖氨酸基序(lysine motif, LysM), 它们可以与几丁 质寡糖结合从而激活植物免疫反应。几丁质诱发的 AtCERK1 的二聚化可进一步激发 AtCERK1 介导的 信号途径 [2] 。在水稻中, 第一个报道的几丁质酶寡糖 受体是 CEBiP (chitin elicitor binding protein), 它带 有 2 个 LysM 的糖蛋白, 与几丁质寡糖具有很高的亲 和力, CEBiP 和激酶 OsCERK1 共同作用, 可使植物 对来自几丁质寡糖的信号做出反应 [3][4] 。最近又有报 道表明, LYP4 (lysine motif-containing protein 4)和 LYP6 与 CEBiP 具有同源性, 它们对水稻识别几丁质 信号也发挥着作用 [2] 。将 CEBiP 与水稻白叶枯病抗 性基因 XA21 或稻瘟病抗性基因 PI-D2 重组形成嵌合 基因, 可以增强水稻对稻瘟病菌的抗性 [5][6] 。 几丁质酶(EC 3.2.1.14)的作用是将几丁质分解为 N-乙酰葡萄糖胺, 在自然界中, 几丁质酶在细菌、真 菌、植物甚至动物中都存在。根据作用于几丁质的方 式不同, 几丁质酶可分为内切和外切几丁质酶二大 类 [7] , 根据氨基酸序列的相似性和几丁质结合结构域 的有无可把几丁质酶分为 Class I~Class VII [8][9] 。 有大量报道表明, 几丁质酶在植物中的表达受 生物或非生物胁迫的诱导。在拟南芥中, 非亲和病 原物的侵染使 Class IV 几丁质酶基因的转录更快地 积累 [10] , 而用亲和病原物侵染可使 Class III 几丁质 酶的转录升高 [11] 。在甜菜中也观察到亲和病原物的 侵染可使 Class IV 几丁质酶转录增强的现象 [12] 。从 稻瘟病菌侵染后的叶片 cDNA 中克隆出一个属于…”