Transfer of high-mannose-type oligosaccharides to disaccharides by endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae

Endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) has transglycosylation activity, and high-mannose-type oligosaccharides are transferred to suitable glycosides as acceptor substrates. The acceptor specificity of Endo-A-catalyzed transglycosylation toward various disaccharides was investigated. To identify an effective acceptor for the transglycosylation by Endo-A, the reaction was carried out using various disaccharides. Endo-A transferred high-mannose-type oligosaccharides more efficiently to beta-linked disaccharides (cellobiose, gentiobiose, sophorose, and laminaribiose) than to alpha-linked disaccharides (isomaltose, maltose, nigerose, kojibiose, and trehalose) as acceptor substrates. The transglycosylation products, (Man)6GlcNAc-Glc-beta-Glc, were more rapidly hydrolyzed than (Man)6GlcNAc-Glc-alpha-Glc. These results indicate that Endo-A recognizes the anomeric configuration of the acceptor substrates, and beta-linked glycosides are suitable for the synthesis of transglycosylation products.     

Some distinguishable properties between acid-stable and neutral types of alpha-amylases from acid-producing koji

The highly humid climate of Japan facilitates the growth of various molds. Among these molds, Aspergillus oryzae is the most important and popular in Japan, and has been used as yellow-koji in producing many traditional fermented beverages and foods, such as Japanese sake, and soy sauce. Taka-amylase A (TAA), a major enzyme produced by the mold, is well known worldwide to be a leading enzyme for industrial utilization and academic study, since many extensive studies have been carried out with TAA. In southern Kyushu, the other koji's of citric acid-producing molds have often been used, such as in the production of a traditional distilled liquor of shochu. The koji molds black-koji and white-koji produce two types of alpha-amylase, namely, acid-stable (AA) and common neutral (NA). The latter enzyme is enzymatically and genetically similar to TAA. In this review, we investigate AA from three molds, Aspergillus niger, A. kawachii and A. awamori, and the yeast Cryptococcus sp. regarding the distinguishable properties between AA and NA. (i) The N-terminus amino acid sequences of AA determined by molecular cloning started with the sequence of L-S-A-, whereas those of NA started with A-T-P-. (ii) Most of the full sequences of AA were composed of, besides a core catalytic domain, an extra domain of a hinge region and a carbohydrate binding domain, which could be responsible for raw-starch-digestibility. The AA from A. niger has no exceptionally extra domain, similarly to NA. (iii) Simple methods for distinguishing AA from NA using CNP-alpha-G3 and G5 as substrates were developed by our group. (iv) The number of subsite in AA on the basis of its cleavage pattern of maltooligosaccharides was estimated to be five, which differs from that of TAA, 7-9. AA has many advantages in industrial applications, such as its acid-stability, thermostability, and raw-starch digesting properties.     

Enhancement of C2C12 differentiation by perfluorocarbon-mediated oxygen delivery

We have studied the effect of enhanced oxygen delivery by perfluorocarbons on the differentiation of C2C12 cells. The extent of differentiation was assessed by means of phase contrast/fluorescence microscopy, active tension measurement and the glucose consumption/lactate production rates. We found that enhanced oxygen delivery is suitable for full differentiation of C2C12 cells.     

Formation of stable foam by the cells and culture supernatant of Gordonia (Nocardia) amarae

Gordonia amarae is the cause of foaming activated sludge. In this study, the mechanism of foam formation by G. amarae SC1 was investigated. A liquid culture of SC1 cells generated a stable foam when shaken reciprocally. This foam formation was dependent on the presence of both bacterial cells and culture supernatant. A high-molecular-weight fraction (Mw>10000) of the supernatant was capable of emulsifying n-hexadecane in addition to exhibiting foaming activity, indicating that it contains a surface-active substance(s). The bacterial cells showed a high affinity to hexadecane. This hydrophobic cell surface property might be involved in the attachment of cells to air bubbles to generate a stable foam. The results demonstrated the participation of cells and the extracellular biosurfactant in the formation and stabilization of foam in G. amarae SC1 culture.     

利用PiggyBac转座子系统快速控制GPI锚定蛋白的表达

GPI锚定蛋白的合成是一个发生在内质网中多步骤、多基因参与的过程。PIGK是GPI锚定蛋白转酰胺酶复合物的一个催化亚基,负责把GPI前体转移到蛋白质上。作者在人体胚胎肾细胞(HEK 293)中获得了PIGK敲除的细胞株,敲除PIGK的细胞不能表达GPI锚定蛋白。利用piggyBac(PB)转座子系统,在细胞中重新插入PIGK基因,细胞膜表面的GPI锚定蛋白恢复表达。实验成功构建了HEK293pB-PIGK细胞株并命名为G36,通过引入PB转座酶或FLP重组酶,可以控制GPI锚定蛋白的表达。本系统可以快速调控细胞表面蛋白质的表达并能够用于基础研究和工业应用。     

HEK 293细胞中重组型抗体表达和分泌的监测系统的构建

免疫球蛋白G(Immunoglobulin G,IgG)作为一种十分重要的生物药物,已被广泛应用于多种医学诊断和治疗。为构建高效表达重组抗体的哺乳动物生产菌株,运用可以灵活检测抗体分泌运输的表达细胞株,从而充分理解抗体的折叠、转运及分泌尤为重要。作者在哺乳动物细胞株(HEK 293)中构建了一个稳定表达绿色荧光蛋白(GFP)融合型抗体E-F-HyHEL10的表达系统,可通过流式细胞术检测抗体的分泌运输。此哺乳动物细胞表达系统具有良好的抗体表达和分泌水平,且稳定维持了抗体的功能活性。使用布雷菲德菌素A(brefeldin A)和放线菌酮处理细胞后,来自胞内E-F-HyHEL10的GFP荧光信号与抗体分泌水平呈正相关。结果表明,本研究中构建的抗体表达系统可实现抗体表达、累积和分泌水平的灵活准确监测,未来可应用于哺乳动物细胞中抗体分泌相关因子的筛选。     

Degradation of cyanuric acid in soil by Pseudomonas sp. NRRL B-12227 using bioremediation with self-immobilization system

The rates of degradation of cyanuric acid, a key intermediate in a metabolic pathway of s-triazine herbicides, were measured for Pseudomonas sp. NRRL B-12227. The rate of degradation was affected by the rate of cyanuric acid transport through cell membranes and the activity of cyanuric acid amidohydrolase inside the cells. At low concentrations of cyanuric acid, the acclimation of cells to cyanuric acid and/or added nutrients effectively enhanced the degradation rate. The strain was also applied to bioremediation using a Bioremediation with Self-Immobilization System (BSIS), in which Pseudomonas sp. NRRL B-12227 cells were co-immobilized with Bacillus subtilis, the latter of which secretes a viscous polymer, in a shallow layer of soil packed in a column. More than 70% of the Pseudomonas sp. NRRL B-12227 cells were co-immobilized with the B. subtilis in a 7.5 cm layer of the packed soil by self-aggregation. More than 60% of the 1 mM cyanuric acid supplied to the packed soil was degraded in this layer during a 72 h period.     

Production and properties of phytase and acid phosphatase from a sake koji mold, Aspergillus oryzae

We identified three types of acid phosphatase (ACP-I, ACP-II, and ACP-III) produced by Aspergillus oryzae in a submerged culture using only phytic acid as the phosphorous substrate. The optimum pH for the activities of the three enzymes was in the range of 4.5 to 5.5. Analysis of the substrate specificities of these enzymes revealed that ACP-I and ACP-III were acid phosphatases, and ACP-II was a phytase. These enzymes were produced during different periods of mycelial growth: ACP-II was produced during the early phase of cultivation (around 24 h), and ACP-I was produced between 24 to 72 h. ACP-III was detected after the production of ACP-I and ACP-II had ceased. The release of phosphate from phytic acid was expected to be due to the cooperative hydrolysis of these enzymes.