生淀粉糖化酶高产菌的选育

从土壤及霉变淀粉质等样品中分离出对生淀粉具有降解作用的菌种约 6 0株 ,其中生淀粉糖化酶最高的一株根霉OR-1 ,其酶活为 90U/mL ,通过一次紫外和亚硝基胍诱变 ,酶活分别达到 200U/mL、325U/mL ,RDA值分别为 70 %、 65%。诱变株是一株产生淀粉糖化酶较高的根霉。     

Efficiency of genetically engineered yeast in the production of ethanol from dextrinized cassava starch

Summary The ability of a polyploid/aneuploidSaccharomyces diastaticus spheroplast fusion product and a diploidSaccharomyces diastaticus hybridization product, to produce ethanol from dextrinized cassava starch with varying amounts of supplemented glucoamylase (amyloglucosidase), was investigated. It was found that the added glucoamylase could be reduced by over 50% using these glucoamylase producing strains as compared to a commercially availableSaccharomyces cerevisiae strain commonly used in ethanol producing industries.     

TiO2膜吸附固定糖化酶特性的研究

分别以醋酸纤维素TiO2膜(AC.TiO2膜)、羧甲基纤维TiO2膜(CMC.TiO2膜)和聚丙烯TiO2膜(PP.TiO2膜)为载体吸附固定糖化酶,并与醋酸纤维素、羧甲基纤维素和聚丙烯固定糖化酶的性能进行了比较,得出以AC.TiO2膜和PP.TiO2膜对糖化酶的吸附性能及稳定性能均较好,PP.TiO2膜固定的糖化酶使用8次后其剩余酶活仍能保持在72%.     

Raw cassava starch-digestive glucoamylase of Aspergillus sp. N-2 isolated from cassava chips

One hundred and eighty strains of black aspergilli isolated from cassava fields and factories in Thailand were screened for the activity of raw cassava starch-digestive glucoamylase. Aspergillus sp. N-2 was selected as the best producer and its extracellular glucoamylase production was investigated. Conditions for the production were optimized for both liquid and solid cultures, and solid culture was found to be approximately three times more efficient than liquid culture. The culture filtrate showed strong glucoamylase activity at low pH (pH 2.0) and high temperature (55°C), and could digest high concentration raw cassava starch. The glucoamylase activity was separated to four fractions (A, B, C and D) by DEAE-Sephacel column chromatography. Fraction C was obtained in a homogeneous state with a molecular weight of 92,000. Each fraction was characterized in terms of the properties of the glucoamylase activity and the efficiency of digestion of cooked and raw cassava starch.     

Poly(N-acryloyl-4- and -5-aminosalicylic acids). 3. Uses as their titanium complexes for the insolubilisation of enzymes

The titanous and titanic complexes of the water-insoluble poly(N-acryloyl-4- and -5-aminosalicylic acids) have been prepared by several methods, and alpha-amylase, glucoamylase, and polygalacturonase (pectinase) have been coupled to the various preparations. The products from alpha-amylase and glucoamylase were enzymically active, but the alpha-amylase was washed off after only one use. With glucoamylase, the derivative withstood extensive washing and could be used continuously in a column. Particular advantages of the glucoamylase preparation were that maximal coupling of the enzyme was achieved in one hour and that a very high specific activity towards a macromolecular substrate was achieved. The polygalacturonase derivative was inactive, possibly because the polysalicylic acid acts as an inhibitor of the enzyme.     

Temperature-Triggered Enzyme Immobilization and Release Based on Cross-Linked Gelatin Nanoparticles

A glucoamylase-immobilized system based on cross-linked gelatin nanoparticles (CLGNs) was prepared by coacervation method. This system exhibited characteristics of temperature-triggered phase transition, which could be used for enzyme immobilization and release. Their morphology and size distribution were examined by transmission electron microscopy and dynamic light scattering particle size analyzer. Their temperature-triggered glucoamylase immobilization and release features were also further investigated under different temperatures. Results showed that the CLGNs were regularly spherical with diameters of 155±5 nm. The loading efficiencies of glucoamylase immobilized by entrapment and adsorption methods were 59.9% and 24.7%, respectively. The immobilized enzyme was released when the system temperature was above 40°C and performed high activity similar to free enzyme due to the optimum temperature range for glucoamylase. On the other hand, there was no enzyme release that could be found when the system temperature was below 40°C. The efficiency of temperature-triggered release was as high as 99.3% for adsorption method, while the release of enzyme from the entrapment method was not detected. These results indicate that CLGNs are promising matrix for temperature-triggered glucoamylase immobilization and release by adsorption immobilization method.     

Inhibition of dextran synthesis by glucoamylase and endodextranase

In a model experiment, glucoamylase was shown to inhibit α-D-glucan synthesis as catalyzed by potato phosphorylase. Both glucoamylase and endodextranase inhibited dextran synthesis with dextransucrases of Leuconostoc mesenteroides. The inhibition could be ascribed to competition between glucoamylase and dextransucrase for the glucosyl groups at the non-reducing end of dextran. The inhibition caused by endodextranase may result from rapid and random hydrolysis of acceptor dextrans. Moreover, significantly low units of glucoamylase, as compared with endodextranase, effectively inhibited dextran synthesis. These results thus present evidence that bio-synthesis of dextran occurs by the addition of glucosyl groups at the non-reducing end of the growing dextran. The measurement of initial velocity suggested that the ping-pong Bi-Bi mechanism proposed for the levansucrase of Bacillus subtilis is also applicable to dextransucrase.     

Fermentation of starch to ethanol by an amylolytic yeast Saccharomyces diastaticus SM-10

A total of fifteen yeast strains were isolated from natural sources including fruits, soil, molasses, honey and a variety of indigeneous fermented foods. Screening of these strains for growth, ethanol production and glucoamylase activity led to selection of a yeast strain SM-10 identified as S. diastaticus having maximum glucoamylase activity (80 units ml(-1)) and ethanol production from starch (3.5%). Ethanol production from wheat flour was found to be 1.75% which could be increased to 5.2% after treatment of wheat flour with pepsin, diastase and glucoamylase.     

Construction of a starch-utilizing yeast by cell surface engineering.

We have engineered the cell surface of the yeast Saccharomyces cerevisiae by anchoring active glucoamylase protein on the cell wall, and we have endowed the yeast cells with the ability to utilize starch directly as the sole carbon source. The gene encoding Rhizopus oryzae glucoamylase with its secretion signal peptide was fused with the gene encoding the C-terminal half (320 amino acid residues from the C terminus) of yeast alpha-agglutinin, a protein involved in mating and covalently anchored to the cell wall. The constructed plasmid containing this fusion gene was introduced into S. cerevisiae and expressed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter from S. cerevisiae. The glucoamylase activity as not detected in the culture medium, but it was detected in the cell pellet fraction. The glucoamylase protein transferred to the soluble fraction from the cell wall fraction after glucanase treatment but not after sodium dodecyl sulfate treatment, indicating the covalent binding of the fusion protein to the cell wall. Display of the fused protein was further confirmed by immunofluorescence microscopy and immunoelectron microscopy. The transformant cells could surely grow on starch as the sole carbon source. These results showed that the glucoamylase was anchored on the cell wall and displayed as its active form. This is the first example of an application of cell surface engineering to utilize and improve the metabolic ability of cells.     

Measurement of α-amylase and glucoamylase activities produced during fermentation

A method for the automatic measurement of α-amylase and glucoamylase activities during fermentation has been developed. Soluble starch dyed with Remazol Brilliant Orange was used as the substrate for α-amylase and 4-nitrophenyl α-d-glucopyranoside for glucoamylase. The same automatic analysis system could be used for both of these enzymes because the reaction products were measured at the same wavelength. Simultaneous pick-up of enzyme and the respective substrate was enabled by using two samplers. The presence of α-amylase did not interfere with the glucoamylase determination. Absolute values for α-amylase activity were obtained using a mathematical correction. Monitoring of these enzymes was accomplished during microbial fermentation.     

Enzymatic processes for the purification of trehalose

A dual‐enzyme process aiming at facilitating the purification of trehalose from maltose is reported in this study. Enzymatic conversion of maltose to trehalose usually leads to the presence of significant amount of glucose, by‐product of the reaction, and unreacted maltose. To facilitate the separation of trehalose from glucose and unreacted maltose, sequential conversion of maltose to glucose and glucose to gluconic acid under the catalysis of glucoamylase and glucose oxidase, respectively, is studied. This study focuses on the hydrolysis of maltose with immobilized glucoamylase on Eupergit® C and CM Sepharose. CM Sepharose exhibited a higher protein adsorption capacity, 49.35 ± 1.43 mg/g, and was thus selected as carrier for the immobilization of glucoamylase. The optimal reaction temperature and reaction pH of the immobilized glucoamylase for maltose hydrolysis were identified as 40°C and 4.0, respectively. Under such conditions, the unreacted maltose in the product stream of trehalose synthase‐catalyzed reaction was completely converted to glucose within 35 min, without detectable trehalose degradation. The conversion of maltose to glucose could be maintained at 0.92 even after 80 cycles in repeated‐batch operations. It was also demonstrated that glucose thus generated could be readily oxidized into gluconic acid, which can be easily separated from trehalose. We thus believe the proposed process of maltose hydrolysis with immobilized glucoamylase, in conjunction with trehalose synthase‐catalyzed isomerization and glucose oxidase‐catalyzed oxidation, is promising for the production and purification of trehalose on industrial scales. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Characteristics of raw-starch-digesting glucoamylase from thermophilic Rhizomucor pusillus

A newly isolated thermophilic fungus, NH-139, identified as Rhizumucor pusillus (Lindt) Schipper produced only a single form of raw-starch-absorbable, raw-starch-digesting glucoamylase on solid wheat bran medium at 45°C. The electrophoretically homogenous preparation of glucoamylase, molecular weight 68,000, had its optimal temperature on gelatinized starch at 65°C and on raw corn starch at 50°C. However, this raw-starch-digesting glucoamylase, unlike other glucoamylases, could not completely hydrolyze glycogen but hydrolyzed it to the extent of 80% as glucose, and is classified as type B. The subtilisin-modified glucoamylase of this strain, molecular weight 60,000, still belonged to type B in the hydrolysis curve on glycogen and lost the ability to digest and adsorb onto raw starch.     

Sago starch as a low-cost carbon source for exopolysaccharide production by Lactobacillus kefiranofaciens

Low-cost sago starch was used as a carbon source for production of the exopolysaccharide kefiran by Lactobacillus kefiranofaciens. A simultaneous saccharification and fermentation process of sago starch for kefiran production was evaluated. Factors affecting the process such as an initial pH, temperature, starch concentration, including a mixture of α-amylase and glucoamylase were determined. The highest kefiran concentration of 0.85 g/l was obtained at the initial pH of 5.5, temperature of 30 °C, starch concentration of 4% and mixed-enzymes with activity of 100 U/g-starch. The use of a mixture of α-amylase and glucoamylase could enhance the productivity compared to the use of α-amylase alone. The optimal ratio of α-amylase to glucoamylase of 60:40 gave the highest kefiran production rate of 11.83 mg/l/h. This study showed that sago starch could serve as a low-cost substrate for kefiran production.     

Effect of carbon and nitrogen sources on glucoamylase production inLactobacillus brevis

A strain ofLactobacillus brevis produced extracellular glucoamylase. Induction of the glucoamylase occurred when saccharides such as starch, dextrin, maltose, mannitol and sucrose were employed as sole carbon sources. Synthesis of the amylase also occurred when soybean extract and peptone were used as sole nitrogen sources. The organism could be employed as a starter culture for local food fermentation.     

Reversible immobilization of glucoamylase onto magnetic carbon nanotubes functionalized with dendrimer

Magnetic carbon nanotubes (MCNTs) with necklace-like nanostructures was prepared via hydrothermal method, and hyperbranched poly(amidoamine) (PAMAM) was grafted on the surface of MCNTs on the basis of the Michael addition of methyl acrylate and the amidation of the resulting ester with a large excess of ethylenediamine (EDA), which could achieve generational growth under such uniform stepwise reactions. The terminal –NH₂ groups from the dendritic PAMAM were reacted with differently functionalized groups to form functionalized MCNTs. Subsequently, enzyme was immobilized on the functionalized MCNTs through adsorption, covalent bond, and metal-ion affinity interactions. The immobilization of glucoamylase, hereby chosen as model enzyme, onto the differently functionalized MCNTs is further demonstrated and assessed based on its activity, thermal stability, as well as reusability. Besides ease in recovery by magnetic separation, the immobilized glucoamylase on functionalized MCNTs offers superior stability and reusability, without compromising the substrate specificity of free glucoamylase. Furthermore, the results indicate that the metal-chelate dendrimer offers an efficient route to immobilize enzymes via metal-ion affinity interactions. The applicability of the regenerated supports in the current study is relevant for the conjugation of other enzymes beyond glucoamylase.     

Enzyme-entrapping behaviors in alginate fibers and their papers

Enzyme immobilization in the form of fiber and paper was easily achieved by wet spinning of aqueous admixture of sodium alginate and enzymes into divalent metallic ion solution as a coagulating bath, followed by paper making of resultant shortly cut fibers. Entrapment yields of enzymes used, e.g., glucoamylase, cyclodextrin glucanotransferase, endo-polygalacturonase, and protease, were always higher in calcium alginate fibers and their papers than those in corresponding beads. It was found that the yields increased with an increase of the discharge rate through the spinning nozzle because the higher discharge rate could provide more highly oriented metal-chelate linear polymer molecules along the fiber axis for preventing leakage of entrapped enzymes. Divalent metallic ions affected greatly the entrapment of glucoamylase in alginate fibers, the order of which followed roughly the ionotropic series of Thiele. Entrapment of glucoamylase in bicomponent systems comprising alginate and other water-soluble polymers was also investigated.     

Isolation and Identification of Novel Terpene Lactones from Quince Fruit (Cydonia oblonga Mill., Marmelo)

A glucoamylase gene has been cloned from a Rhizopus genomic DNA library using synthetic oligonucleotides corresponding to the amino acid sequence of the glucoamylase. Since this glucoamylase gene was not expressed in yeast cells, we have cloned a glucoamylase gene from a cDNA library prepared from Rhizopus mRNA. Sequence analysis of both glucoamylase genes revealed that the genomic gene contained 4 intervening sequences and the cDNA gene lacked 145 nucleotides corresponding to the N-terminal region. The glucoamylase consists of 604 amino acids including a putative signal peptide and its molecular weight was calculated to be 65,000. The glucoamylase gene to be expressed in yeast cells was constructed by recombination of both genes. The yeast cells containing this constructed glucoamylase gene secreted the glucoamylase into the culture fluid and grew at almost the normal rate on a medium containing starch as the sole carbon source.     

产糖化酶黑曲霉固定化方法比较的研究

采用海藻酸钙凝胶电埋法、以沸石、多孔聚酯等材料为固定化载体的吸附法固定黑曲霉（Aspergillus niger AS3.4309)菌丝细胞,以游离菌丝体作为对照,进行发酵产糖化酶的比较,结果表明：以聚酯泡沫作为固定化载体吸附固定化菌丝细胞产糖化酶活力最高。在产糖化酶的发酵过程中,与游离菌丝体细胞相比,固定化黑曲霉持续产酶时间有一定程度的延长。     

Identification and characterization of glucoamylase from the fungus Thermomyces lanuginosus

The glucoamylase from the thermophilic fungus Thermomyces lanuginosus has a molecular weight of 66 kDa and was characterized with isoelectric point, pH and temperature optimum of 3.8-4.0, 5.0 and 70 degrees C, respectively. In addition, the activation energy is 60.4 kJ/mol, Km is 3.5 mM and kcat is 25.3 s(-1). The glucoamylase was partially sequenced on the protein level, and the complete glucoamylase gene including its promoter (but excluding its terminator region) was cloned and sequenced. The glucoamylase protein comprises 617 amino acid residues and shows 60% identity with the glucoamylase from the thermophilic fungus Talaromyces emersonii. cDNA encoding Thermomyces lanuginosus glucoamylase was expression cloned into Pichia pastoris, producing approximately 7.4 U/ml. It was concluded that alternative mRNA splicing as it might occur in Aspergillus niger glucoamylase is not responsible for the occurrence of different glucoamylase isoforms in Thermomyces lanuginosus.