瑞氏木霉表达黑曲霉葡萄糖氧化酶

利用高表达分泌纤维素酶的真菌瑞氏木霉表达重组的黑曲霉葡萄糖氧化酶。在大肠杆菌DH5α中构建瑞氏木霉纤维素酶CBHI启动子和CBHI信号肽基因黑曲霉葡萄糖氧化酶基因瑞氏木霉纤维素酶CBHI终止子构巢曲霉的甘油醛3磷酸脱氢酶启动子大肠杆菌抗潮霉素B磷酸转移酶基因构巢曲霉色氨酸C终止子pUC19(命名为pCBHGOD)质粒,线性化后用瑞氏木霉纤维素酶CBHI启动子和CBHI信号肽基因黑曲霉葡萄糖氧化酶基因瑞氏木霉纤维素酶CBHI终止子构巢曲霉的甘油醛3磷酸脱氢酶启动子大肠杆菌抗潮霉素B磷酸转移酶基因构巢曲霉色氨酸C终止子(命名为CBHGOD)核酸片段转化瑞氏木霉QM9414原生质体。用PCR扩增方法筛选出同源重组葡萄糖氧化酶基因的瑞士木霉突变株。用麦杆诱导瑞氏木霉突变株,生产黑曲霉葡萄糖氧化酶,Westernblot分析重组的葡萄糖氧化酶分子量与Sigma公司的天然黑曲霉葡萄糖氧化酶一致,生产的重组酶活性25umL,相当于Sigma公司葡萄糖氧化酶标准品的产量为0.5gL。瑞氏木霉可用于生产黑曲霉葡萄糖氧化酶。     

黑曲霉葡萄糖氧化酶基因的克隆及其在酵母中的高效表达

将黑曲霉葡萄糖氧化酶(GOD)基因重组进大肠杆菌酵母穿梭质粒Ppic9,转化甲基营养酵母Pichia pastoris GS115,构建出GOD的高产酵母工程菌株。在酵母αFactor及AOX1基因启动子和终止信号的调控下,黑曲霉GOD在甲基酵母中大量表达并分泌至胞外,经甲醇诱导3～4d,发酵液中的GOD活力可达30～40u/mL。SDS-PAGE证实GOD在培养物上清中的含量显著高于其它杂蛋白,约占胞外蛋白总量的60%～70%,经Q SepharoseTMFast Flow离子交换柱一步纯化即达电泳纯。重组酵母GOD比活达426.63u/mg蛋白,是商品黑曲霉GOD的1.6倍。动力学性质分析表明,重组酵母GOD的K_m和K_cat分别为38.25mmol/L和3492.66s-1,与商品黑曲霉GOD相比,具有更高的催化效率。重组酵母GOD的高活力特性可有效提高葡萄糖传感器的线性检测范围。     

The Metabolism of Glucose and Acetate in Aspergillus niger

1. The metabolism of a citric-acid-forming strain of A. nigerwhengrown on a glucose-acetate medium has been investigated.
2. Acetate greatly accelerated the rate of utilization of glucose.
3. Citric acid production from glucose was much increased bythepresence of acetate.
4. The formation of oxalate from glucose-acetatecultures was muchless than from acetate alone.
5. In some cultureslarge amounts of glucose and acetate were consumedbut no acidicproducts were formed.

     

Identification and Structural Analysis of Amino Acid Substitutions that Increase the Stability and Activity of Aspergillus niger Glucose Oxidase

Glucose oxidase is one of the most conspicuous commercial enzymes due to its many different applications in diverse industries such as food, chemical, energy and textile. Among these applications, the most remarkable is the manufacture of glucose biosensors and in particular sensor strips used to measure glucose levels in serum. The generation of ameliorated versions of glucose oxidase is therefore a significant biotechnological objective. We have used a strategy that combined random and rational approaches to isolate uncharacterized mutations of Aspergillus niger glucose oxidase with improved properties. As a result, we have identified two changes that increase significantly the enzyme''s thermal stability. One (T554M) generates a sulfur-pi interaction and the other (Q90R/Y509E) introduces a new salt bridge near the interphase of the dimeric protein structure. An additional double substitution (Q124R/L569E) has no significant effect on stability but causes a twofold increase of the enzyme''s specific activity. Our results disclose structural motifs of the protein which are critical for its stability. The combination of mutations in the Q90R/Y509E/T554M triple mutant yielded a version of A. niger glucose oxidase with higher stability than those previously described.     

Localization of growth and secretion of proteins in Aspergillus niger

Hyphal growth and secretion of proteins in Aspergillus niger were studied using a new method of culturing the fungus between perforated membranes which allows visualization of both parameters. At the colony level the sites of occurrence of growth and general protein secretion were correlated. In 4-d-old colonies both growth and secretion were localized at the periphery of the colony, whereas in a 5-d-old colony growth and secretion also occurred in a more central zone of the colony where conidiophore differentiation was observed. However, in both cases glucoamylase secretion was mainly detected at the periphery of the colonies. At the hyphal level immunogold labelling showed glucoamylase secretion at the tips of leading hyphae only. Microautoradiography after labelling with N-acetylglucosamine showed that these hyphae were probably all growing. Glucoamylase secretion could not be demonstrated immediately after a temperature shock which stopped growth. These results indicate that glucoamylase secretion is located at the tips of growing hyphae only.     

Glucose oxidase biosynthesis using immobilized mycelium of Aspergillus niger

A simple method is described for the immobilization of Aspergillus niger GIV-10 which produces an extracellular glucose oxidase. A. niger conidia were immobilized on sintered glass Raschig rings, pumice stones or polyurethane foam. Mycella growing out from the spores produced extracellular glucose oxidase: the highest production was with the pumice stone carrlers. This technique facilitates the growth of the filamentous cultures in the spongy structure of a support with continuous accumulation of biomass. After 24 to 36 h, a culture liquid with 2.7 to 3.1 U of glucose oxidase/ml was obtained. This procedure also made possible repeated batch enzyme production and as many as 25 subsequent 24-h batches could be fermented by using the same carrier with only a small loss of glucose oxidase activity.The authors are with the Institute of Microbiology, M. Curie-Sklodowska University, Akademicka 19, 20-003 Lublin, Poland.     

Glucose oxidase biosynthesis in relation to biochemical mutations in Aspergillus niger

The production of extracellular glucose oxidase in a submerged culture by a number of auxotrophic, 2-deoxy-D-glucose resistant and protease-less mutants of Aspergillus niger was evaluated. Among the auxotrophic strains, no evident dependence was found between the kind of the nutritional requirements and the level of the glucose oxidase activity. However, the majority of auxotrophs, requiring serine or niacin, showed a higher enzyme activity (from 16 to 680%) than the parent strain. The dynamics of the glucose oxidase synthesis by the free and immobilized mycelium of the most active niacin^? mutant of A. niger was also investigated.     

Cloning,Heterologous Expression,Purification and Characterization of M12 Mutant of Aspergillus niger Glucose Oxidase in Yeast Pichia pastoris KM71H

Aspergillus niger glucose oxidase (GOx) genes for wild-type (GenBank accession no. X16061, swiss-Prot; P13006) and M12 mutant (N2Y, K13E, T30 V, I94 V, K152R) were cloned into pPICZαA vector for expression in Pichia pastoris KM71H strain. The highest expression level of 17.5 U/mL of fermentation media was obtained in 0.5 % (v/v) methanol after 9 days of fermentation. The recombinant GOx was purified by cross-flow ultrafiltration using membranes of 30 kDa molecular cutoff and DEAE ion-exchange chromatography at pH 6.0. Purified wt GOx had k _cat of 189.4 s^?1 and K _m of 28.26 mM while M12 GOx had k _cat of 352.0 s^?1 and K _m of 13.33 mM for glucose at pH 5.5. Specificity constants k _cat/K _m of wt (6.70 mM^?1 s^?1) and M12 GOx (26.7 mM^?1 s^?1) expressed in P. pastoris KM71H were around three times higher than for the same enzymes previously expressed in Saccharomyces cerevisiae InvSc1 strain. The pH optimum and sugar specificity of M12 mutant of GOx remained similar to the wild-type form of the enzyme, while thermostability was slightly decreased. M12 GOx expressed in P. pastoris showed three times higher activity compared to the wt GOx toward redox mediators like N,N-dimethyl-nitroso-aniline used for glucose strips manufacturing. M12 mutant of GOx produced in P. pastoris KM71H could be useful for manufacturing of glucose biosensors and biofuel cells.     

Glucose oxidase synthesis by Aspergillus niger GIV-10 on starch.

The effect of various kinds of starch, as the sole source of organic carbon, on the biosynthesis of glucose oxidase by A. niger GIV-10 was examined. A. niger grown on 6% wheat starch medium provided extracellular and intracellular glucose oxidase with the highest enzymatic activities. A new method of intracellular glucose oxidase extraction (without disruption of mycelium), developed and discussed in this paper, increased 2 to 3.8-times glucose oxidase yield, as compared to that described earlier.     

超声波对酵母过氧化氢酶及多酚氧化酶活性的影响

用微超声处理酵母过氧化氢酶(CAT)和多酚氧化酶(PPO),结果表明,在一定参数范围内CAT和PPO活性都升高。对CAT来说,其最适当的超声参数是135kHz、40Wcm2、处理10min。对PPO来说,其最适当参数是135kHz、25Wcm2、处理10min。经光谱分析,PPO超声处理后紫外差示光谱在一定的波长下呈现出明显的正峰和负峰 。     

Yellow Pigments of Aspergillus niger and Aspergillus awamori

Alkaline degradation of Aurasperone A, C₃₂H₂₆O₁₀, gave a binaphthyl (IIa), m.p. 255°C and acetone. (IIa) afforded a tetraacetate (IIb), C₃₂H₃₀O₁₂ m.p. 219°C and a tetramethyl ether (IId), C₂₈H₃₀O₈, m.p. 188°C. These facts along with the NMR spectra of aurasperone A and (IIb) confirm that aurasperone A is a dimeric 2-methyl-5-hydroxy-6,8-dimethoxy-4H-naphtho[2,3-b]pyran-4-one with asymmetric C-C linkage (7-10′ or 9-10′). The ether (IId) is not identical with 1,1′ ,3,3′ ?6,6′ ,8,8′-octamethoxy-4,4′-binaphthyl. Thus, it follows that (IId) is a 2,4′-binaphthyl and hence aurasperone A is 2,2′-dimethyl-5,5′- dihydroxy-6,6′,8,8′-tetrahydroxy-7,10′-bi[4H-naphtho[2,3-b]pyran-4-one] (I).     

Glucoamylase of Aspergillus niger

Aspergillus niger produces two extracellular glucoamylases (GAI of Mr 85 300 and GAII of Mr 77 600) separable on DEAE-cellulose. The enzymes differes in electrophoretic mobility, thermostability and substrate specificity. The GAI/GAII ratio depends on the concentration and form of nitrogen (nitrate or ammonium) in the culture medium. Proteinase VIII from Bacillus subtilis converts GAI to a form showing properties similar to those of GAII. Possible proteolytic degradation of GAI to GAII by Asp. niger endogenous proteinase(s) is suggested.     

Microbial Production of Glucose Oxidase

Productivity of extracellular glucose oxidase was examined for various microorganisms and it was found in strains belonging to genus Penicillium except one species of Tallalomyces.

As the best glucose oxidase producer, Penicillium purpurogenum No. 778 was isolated from natural source. This microorganism produced 32,000 units per ml broth of glucose oxidase in a simple medium containing beet molasses, NaNO³ and KH²PO⁴ by submerged culture for 3 days. That value was about 10-times of that of Penicillium amagasakiense which has been known as an excellent glucose oxidase producer.

Culture conditions for glucose oxidase production were examined, which were extremely different among microbial species. In the case of Penicillium chrysogenum AJ 7007 and Penicillium purpurogenum No. 778, the effects of aeration and carbon sources were remarkably different from each other.

Penicillium purpurogenum No. 778 produces catalase sufficiently in a culture broth for glucose oxidase application in food industry.

Glucose oxidase was purified about 25-fold from culture supernatants of Penicillium purpurogenum No. 778, and some properties of the enzyme were examined. The optimum temperature and pH for the activity were 35°C and 5.0, respectively. The enzyme was stable at pH 5.0 to 7.0 when it was incubated at 40°C for 2 hr, while it was stable at temperature lower than 50°C when incubated at pH 5.6 for 15 min. The enzyme was specific for d-glucose and apparent Michaelis constant for d-glucose was 12.5 mm. The enzyme was inhibited by 1 mm of HgCl², CuSO⁴, NaHSO⁴ and phenylhydrazine, but not inhibited by 1 mm of p-hydroxy-mercuribenzoate, EDTA, hydroxylamine and dimedone. Four percents NaCl inhibited the activity about 50%, while the addition of ethanol (from 0 to 16%) increased oxygen uptake more than that expected from the peroxidase activity of catalase.