Use of mutagenic agents in improvement of α-amylase production by bacillus subtilis

Various mutagenic agents were employed in succession to produce mutant strains of Bacillus subtilis with improved yields of α-amylase. The parent strain had previously been selected as a good producer of α-amylase. Highly productive mutant strains were selected throughout the work as the basis for further treatment. In shake cultures the yields of α-amylase of the best strains were double that of the parent strain, and in fermentor cultivation the improvement was even greater.     

Stabilisation in bacillus stearothermophilus of a recombinant plasmid carrying the homologous α-amylase gene

The phenotypic stability of the recombinant plasmid pAT9 (11.5 MD), which contained the cloned α-amylase gene from B. stearothermophilus, was studied in batch and continuous culture. Irrespective of the type of culture (batch or continuous; or using glucose or maltose as carbon source), deletion plasmids of the phenotype Km^rAmy^? the same size (7.3 MD) emerged with time. DNA sequencing analysis and Southern hybridization of a region adjacent to both ends of the pAT9 HindIII fragment containing the α-amylase gene showed that almost the entire part of the HindIII fragment was lost. Homology between fragments contiguous to the pAT9 HindIII terminals allowed exclusion of a fragment that did not encode the α-amylase gene and a new recombinant plasmid, pATHP9 (Km^rAmy⁺; 7.5 MD), was constructed which had enhanced phenotypic stability in B. stearothermophilus.     

Growth rate dependence of enzyme synthesis in chemostat cultures: α-amylase, β-galactosidase,acid phosphatase and β-fructosidase

Specific rate of enzyme synthesis in microbial cells was correlated to specific growth rate in chemostat culture using three strains of microorganism. α-Amylase production by Bacillus subtilis was linearly associated with growth rate in a glucose-limited chemostat culture. β-Galactosidase production by Saccharomyces fragilis was rather invariable with growth rate in a galactose medium. β-Fructosidase production by Saccharomyces carlsbergensis in a phosphate-poor glucose medium was independent on growth rate, except at low dilution rates where it increased with growth rate. Production of acid phosphatase by the same microbe showed a maximum at a particular intermediate dilution rate. These relationships were summarised with an equation of induction—repression model. Comparison of the chemostat results with those of batch fermentation provided some enhancement factors of a continuous fermentation: 1.23–9.82 regarding enzyme productivity and 0.53–2.88 regarding enzyme concentration. The production of β-fructosidase was much improved among the enzymes tested when continuous culture methods were used.     

Structure and properties of polyethylene produced by γ-radiation polymerization in flow system

The radiation-induced polymerization of ethylene was carried out by use of a benchscale plant with a flow-type reactor of 1 liter capacity under the following conditions: pressure, 200–400 kg/cm²; temperature, 30–90°C; irradiation intensity, 3.8 × 10⁵ rad/hr; and ethylene flow rate, 300–3000 nl/hr. The molecular weight of polymer formed was shown to decrease with increasing reaction temperature and to increase with increasing pressure. When the ethylene flow rate increases, the molecular weight decreases in the polymerization at 30–60°C, but it does not change in the polymerization at 75–90°C. Methyl group content, which is a measure of short-chain branching of the polymer, increases with increasing reaction temperature, i.e., ca. 1 CH₃/1000 CH₂ at 30°C and ca. 9 CH₃/1000 CH₂ at 90°C. Methyl content is independent of the ethylene flow rate. The changes in the melt index of polymer with reaction conditions corresponds to the change of the molecular weight. The density, crystallinity, and melting point of polymer decrease with the reaction temperature as the short-chain branching increases, and they are almost independent of ethylene flow rate and pressure.     

Characterization of a chemically modified β-amylase immobilized on porous silica

β-Amylase was coupled to a periodate oxidized dextran by reductive alkylation in the presence of sodium cyanoborohydride. The loss of activity (57%) during the cross-linking of the enzyme was the result of steric hindrance near the catalytic site. In order to verify this hypothesis, the residual activity was determined with substrates of variable molecular size. The residual activity was inversely proportional to the particulate size of the substrate. Increases in residual activity, of up to 53% were obtained using an orientated chemical modification in the presence of a substrate which protects the catalytic site. Native and dextran-conjugated β-amylase were immobilized on an amino activated silica by a classical method using glutaraldehyde for the native enzyme and by reductive alkylation for the modified enzyme. The relative activity of the enzymes obtained after this insolubilization was very high for the modified amylase, 45% for a medium enzyme concentration, compared with 4% at the same concentration for the native enzyme. This difference can be attributed to the variation of the length of the space arm between the silica and the enzyme. The soluble β-amylase dextran conjugate had a superior thermoresistance to that of the native enzyme; its optimal temperature of activity was 5°C higher than that of the native enzyme. This stabilization may be attributable to a rigidification of the protein structure. Immobilization of both native and modified enzymes on a amino silica resulted in thermostabilization of the enzymes. The optimal temperature of activity was 70°C for the native immobilized β-amylase (some 10°C higher than that of the native enzyme) and 75°C for the chemically modified, and subsequently immobilized, β-amylase. The immobilized forms of the enzymes were used for 14 days at 55°C in continuous substrate processing. The greater eflciency of the chemically modified β-amylase was demonstrated by a four-fold increase in maltose production compared to the classical method of immobilization. A kinetic study confirmed the stabilization of the β-amylase by a reduction of the rate constant of inactivation of the different modified enzymes.     

Starch hydrolysis kinetics of Bacillus licheniformis α-amylase

The reaction kinetics for thermostable Bacillus licheniformis α-amylase was determined under two sets of conditions: an initial rate study over a range of low starch concentrations (1–8%, w/w) in a batch reactor, and a continuous rate study at a high starch concentration of 40% during which a twin-screw extruder was used as a bioreactor for the first 600 s and the reaction was continued to equilibrium in a batch reactor. The Michaelis–Menten model was found to be sufficient for describing the kinetics at low starch concentrations; however, a modified first-order model was required at high concentration. There was no evidence of substrate inhibition at the high starch concentration. The rate of product formation, calculated in terms of dextrose equivalent (DE) values, was found to be proportional to the square root of the rate of starch consumption.     

Characterization of mechanical properties of γAl2O3 dispersed epoxy resin cured by γ‐ray radiation

Epoxy resins are widely utilized as high performance thermosetting resins for many industrial applications, but they are characterized by relatively low toughness. Incorporation of rigid inorganics is suggested to improve the mechanical properties of epoxy resins. An attempt is made to disperse nanosized γ‐Al₂O₃ particles into diglycidyl ether of bisphenol A epoxy resins for the improvement of the mechanical properties. These hybrid epoxy–alumina composites are prepared using by the γ‐ray curing technique conducted at 100 kGy under nitrogen at room temperature. The composites are characterized by determining the gel content, flexural strength, Youngis modulus, and toughness at room temperature using scanning electron microscopy and FTIR studies. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 91: 1898–1903, 2004     

Design Strategies for Integrated β‐Galactosidase Purification Processes

The operational conditions for an aqueous two‐phase system (ATPS) for β‐galactosidase purification were optimized and applied to the design of a purification strategy as an alternative to the primary purification steps. The ATPS proved to be suitable for the recovery and primary enzyme purification. The purification process design developed by ATPS, diafiltration, ion exchange, and diafiltration/ultrafiltration was successful, yielding a more than tenfold purification. The purification strategy design resulted in a powerful integrated purification and recovery process, an evidence of the potential for a scale‐up of the β‐galactosidase purification process.     

氧化石墨烯固定化α-淀粉酶

以石墨粉为原料,使用改进Hummer法制备氧化石墨烯,采用沉积法固定化α-淀粉酶,并对固定条件进行优化。分别采用NaOH-CH₂ClCOOH法和HNO₃-H₂SO₄法制备羧基化氧化石墨烯,并将其用于固定化α-淀粉酶。结果表明,沉积法固定化酶的最适宜温度为65 ℃,最适宜pH=7.0。连续催化反应9次后,固定化酶活力仍能保持初始固定化酶活力的47.81%。经比较发现,NaOH-CH₂ClCOOH法更有利于羧基化氧化石墨烯的制备,该方法制备的羧基化氧化石墨烯产率为HNO₃-H₂SO₄法的1.2倍。     

Radiation-induced copolymerization of α,β,β-trifluoroacrylonitrile with α-olefin

Homopolymerization and copolymerization of α,β,β-trifluoroacrylonitrile (FAN) with γ-olefins were carried out in bulk by γ-ray irradiation at 25°C. FAN gives very small quantities of brown and greasy low molecular weight polymer. Cyano groups in FAN polymer were found to be readily hydrolyzed to acid amide groups in the atmosphere. FAN was found to copolymerize with ethylene, propylene, and isobutylene via a radical mechanism to form equimolar copolymers in a wide range of monomer compositions. The polymerization rate increases linearly with FAN fraction in the monomer mixture. These copolymers are also hydrolyzed in the atmosphere, and the hydrolysis proceeds with more difficulty for the copolymer with higher α-olefin. The reactivity ratios r₁ (FAN) and r₂ (α-olefin) were determined to be 0.01 and 0.12 for the FAN/ethylene copolymerization and 0.01 and 0.07 for the FAN/propylene copolymerization. These results confirm that an alternating copolymerization takes place in the FAN/α-olefin system.     

Tensile properties of microbial β‐glucan films

β‐glucan films with different plasticizer concentrations (glycerol and water) were deformed in tension and various mechanical characteristics (modulus of elasticity, yield stress, strength, and strain at rupture point) were evaluated. The influence of both plasticizers was of additive nature describable by the total plasticizer content. The influence is reflected by an exponential decrease of either modulus of elasticity or yield stress with increasing total plasticizer content. The film strength decreased linearly with plasticizer concentration, whereas the decline in strain at the rupture point followed a power function. It was also found that the stress parameters can be expressed via modulus of elasticity: the strength as linear function of the square root of the elasticity modulus and the yield stress as a linear function of the latter parameter. On the basis of such relations a quadratic function between strength and yield stress was proposed. This relation well describes the character of the deformation curves that were obtained at different deformation conditions. POLYM. ENG. SCI., 2011. © 2011 Society of Plastics Engineers     

Kinetic Resolution of Aliphatic β‐Amino Acid Amides by β‐Aminopeptidases

Access to enantiopure β‐amino acids : β‐Aminopeptidases are hydrolases that possess the unique ability to cleave N‐terminal β‐amino acids from peptides and amides. Hydrolysis of racemic β‐amino acid amides catalyzed by these enzymes displays enantioselectivity with strong preference for substrates with the L ‐configuration, and gives access to various aliphatic β‐amino acids of high enantiopurity.

     

Immobilization of α-amylase on polymeric carriers having different structures

α-Amylase was immobilized onto several polymeric carriers having carboxyl groups by the Woodward's reagent K better than by other methods. The amount of immobilized α-amylase mainly depended on the surface area of carriers, while the enzymic activity depended on the texture of carrier surface. Flat surface was favorable for making the enzyme exhibit high activity and for keeping it active in wide pH range. The durability of immobilized enzyme was excellent at 20°C and pH 5.5.     

β-nucleators and β-crystalline form of isotactic polypropylene

Different levels of β-form crystallinity in isotactic polypropylene (iPP) were produced by blending a series of additives. X-ray diffraction analyses showed that the Kx values varied from 0 to 0.05. Indigosol Brown IRRD, Indigosol Red Violet IRH, Cibantine Orange HR, Indigosol Pink IR, Cibantine Blue 2B, Indigosol Golden Yellow IGK, and Indigosol Grey IBL were found to be effective β-nucleators. The respective Kx values of the iPP samples containing the above seven β-nucleators of 0.05 wt % are 0.54, 0.68, 0.82, 0.82, 0.86, 0.92, and 0.95. All the β-nucleators possess fused benzene rings or heterocycles in their molecular structures. X-ray diffraction studies on them revealed a mutual character in that the strongest or the second-strongest reflections almost locate at the d-spacing of 2.83 Å. The magnitude is about half of that of the β-spherulites possess a bright color and that their Maltese Crosses display some concentric banding of a rather spiky, jagged character. Their lamellae exhibit a correlated and dendritic structure seen by a scanning electron microscope. © 1995 John Wiley & Sons, Inc.     

Functional monomers and polymers, 64. Synthesis of poly-β-alanine from β-alanine, β-alanyl-β-alanine,and β-alanyl-β-alanyl-β-alanine 4-dodecanoyl-2-nitrophenyl esters

Active 4-dodecanoyl-2-nitrophenyl esters of β-alanine, β-alanyl-β-alanine, and β-alanyl-β-alanyl-β-alanine were prepared, and tried to polymerize in various solvents. Nonpolar solvents were found to be convenient for the polycondensation reaction. The yield of the polycondensation was high for the monopeptide ester, and less for the dipeptide and tripeptide esters. The effect of temperature on the polycondensation reaction was also studied.     

Asymmetric α‐Hydroxylation of β‐Indanone Esters and β‐Indanone Amides Catalyzed by C‐2′ Substituted Cinchona Alkaloid Derivatives

The highly catalytic asymmetric α‐hydroxylation of β‐indanone esters and β‐indanone amides using peroxide as the oxidant was realized with a new C‐2′ substituted Cinchona alkaloid derivatives. The two enantiomers of α‐hydroxy‐β‐indanone esters could be obtained by simply changing the oxidant. This protocol allows a convenient access to the corresponding α‐hydroxy‐β‐indanone esters and α‐hydroxy‐β‐indanone amides with up to 99% yield and 98% ee.