Characteristics and production of thermostable α-amylase

Thermostable α-amylases have application in a variety of industrial processes and enzymes from a substantial number of thermophilic bacteria and fungi have been screened and characterized to varying degrees. The characteristics of these enzymes are summarized in this review. The genetics of α-amylase production inBacillus subtilis is reviewed and classical and recombinant DNA approaches to increasing α-amylase production are discussed.     

Stainless steel capillary columns for high temperature gas chromatography

A layer of elemental silicon has been deposited on the surface of stainless steel tubing by means of chemical vapor deposition (CVD). Two kinds of capillary column were prepared from the deactivated tubing: cross-linked, silanol-terminated polydime-thylsiloxane wall coated open tubular (WCOT) columns and molecular sieve 13X porous layer open tubular (PLOT) columns. Unlike fused silica capillary columns, stainless steel WCOT and PLOT columns can be operated at temperatures in excess of 400°C. High temperature simulated distillation has been performed successfully with a macro bore WCOT column and rapid PNA (paraffin, naphthene, and aromatic) analysis with a multidimensional gas solid chromatographic (GSC) system using PLOT columns.     

High temperature gas chromatography: The development of new aluminum clad flexible fused silica glass capillary columns coated with thermostable nonpolar phases: Part 1

With the simultaneous development of blank aluminum clad flexible fused silica glass capillary tubing capable of withstanding temperatures up to 500°C, coincident with a series of special high temperature methyl polysiloxane polymers, it was possible to produce for the first time, long lived fused silica capillary columns containing thin films of thermostable stationary phases which could be maintained isothermally at 400425°C and temperature programmed to 425–440°C. The “bleed rate” here for a well conditioned column was 5 picoamperes or less. Under these circumstances, alkanes with carbon numbers in the C-90 to C-100 area were rapidly and efficiently eluted from these columns. By extrapolation here, one can easily detect certain compounds with boiling points in the 750°C range. Since this type of capillary column was found to possess certain favorable properties, it was thought that it will soon replace the packed column and will probably be more popular than the borosilicate capillary column for many high temperature applications. Moreover, evidence has now accumulated which leads us to further believe that the majority of analyses of “high molecular weight” compounds performed by Supercritical Fluid Chromatography (SFC), utilizing very narrow bore fused silica capillary columns at several hundred atmospheres, can be much more simply, much more rapidly, much more economically, and much more efficiently accomplished by gas chromatography utilizing this new generation of high temperature capillary columns.     

一类新型杂环聚芳酰胺的合成与性能研究

采用二氮杂萘类双酚 4 (4 羟基苯基 ) 2 ,3 二氮杂萘 1 酮 (DHPZ)与对氯苯腈反应制得二腈化合物 ,进一步水解为二酸 .以此新型二酸与不同二胺聚合制得的聚芳酰胺均具有较高的耐热性能 ,其Tg 在 2 91～318℃ ,且易溶于极性非质子溶剂中 .聚合物的特性粘数为 1 17～ 1 5 0dL g(2 5℃ ,0 5 %inNMP) ,对所得聚酰胺进行性能研究 ,其拉伸强度为 71～ 86MPa ,断裂伸长率为 8%～ 13% ,拉伸模量为 0 86～ 1 0 2GPa ,电阻系数数量级为 10 1 4～ 10     

Optimization and characterization of immobilized E. coli for engineered thermostable xylanase excretion and cell viability

There are many parameters that may have influenced the properties of cell during immobilization process. Particularly, the immobilization methods, carrier materials, and enzyme loading amount that have been proved to be important for immobilization process. The physiological responses of microorganisms are depending on the immobilization technique used. Typical alterations to the micro-environment of the immobilized cell involved the altered water activity, presence of ionic charges, cell confinement and modified surface tension. In this study, the graphene oxide was selected as a suitable carrier for immobilization process of recombinant E.coli and adsorption was chosen as an appropriate method to improve the production of engineered thermostable xylanase. High level production of thermostable xylanase by immobilized recombinant cell in the 5 L bioreactor was studied by using optimum research surface methodology (RSM) conditions was studied. The immobilization of E. coli onto nanoparticle matrix manages to improve the cell performance by improving the protein expression, reduced the occurrences of cell lysis as well as improved the plasmid stability of the host cell. Thus, immobilization contributes a physical support for both whole cells as well as enzymes to develop a better operative achievement system for industrialized fields and give rise to the biological advancement existing enzyme for instance xylanase.     

Purification and properties of cyclodextrinase fromBacillus stearothermophilus HY-1

A thermostable cyclodextrinase (EC 3.2.1.54) fromBacillus stearothermophilus HY-1 was purified to homogeneity by disc-electrophoresis after sonication disruption, ammonium sulfate fractionation, DEAE-cellulose(DE32) column chromatography, hydroxyapatite chromatography, Sephadex G150 gel-filtration, and α-cyclodextrin-AH-Sepharose 4B affinity chromatography. The enzyme was purified 230-fold with 21.2% of activity recovery. The optimal substrates of the enzyme were α-, Β-, and γ-cyclodextrins and linear maltooligosaccharides, and the final product was mainly maltose. The enzyme could hydrolyze pullulan to produce panose. It could also hydrolyze soluble starch, amylose, and amylopectin, but not glycogen. The Km and Vmax for α-, Β-, and γ-cyclodextrins were 1.79, 1.67, and 2.50 mg/mL, and 336, 185, and 208 Μmol/mg/min, respectively. The molecular weight of the enzyme was 61,000 by SDS-gel-electrophoresis. The isoelectric point was pH 5.0. The enzyme was most active at pH 6.2 and 55‡C, and it was strongly inhibited by Cu²⁺, Hg²⁺, Zn²⁺, Pb²⁺, and slightly by Fe²⁺. The effect of some protein modification reagents on the activity of the enzyme suggested that tryptophan and histidine residue(s) may be located at the active site. The amino acid composition of the enzyme was also determined.     

新型可溶性聚芳酰胺及其共聚物的合成与性能研究

采用新型二胺 1,2 二氢 2 (4 氨基苯基 ) 4 [4 (4 氨基苯氧基 ) 苯基 ] 二氮杂萘 1 酮与对苯二甲酸、间苯二甲酸、萘二酸进行均聚或共聚 ,所得聚芳酰胺的Tg 均高于 30 0℃ ,且易溶于非质子极性溶剂中 ,聚合物的特性粘数为 1 2 6～ 1 5 1dL g(2 5℃ ,NMP) ,拉伸强度为 6 2～ 89MPa ,断裂伸长率为 5 %～ 9% ,拉伸模量为 2 2～ 2 9GPa ,电阻系数为 10 1 4 ～ 10 1 6     

Thermostable amperometric lactate biosensor with Clostridium thermocelluml-LDH for the measurement of blood lactate

The gene for Clostridium thermocelluml-lactate dehydrogenase enzyme was cloned into pGEX-4T-2 purification vector to supply a source for a thermostable enzyme in order to produce a stable lactate biosensor working at relatively high temperatures. The purified thermostable enzyme (t-LDH) was then immobilized on a gold electrode via polymerization of polygluteraldehyde and pyrrol resulting in a conductive co-polymer. t-LDH working electrode (t-LDHE) was used for determination of lactate in CHES buffer. Amperometric response of the produced electrodes was measured as a function of lactate concentration, at a fixed bias voltage of 200 mV in a three-electrode system. The linear range and sensitivity of the biosensor was investigated at various temperatures in the range of 25-60 °C. The sensitivity t-LDHE increased with increasing the temperature and reached its highest value at 60 °C. The calculated value was nearly 70 times higher as compared to the sensitivity value of the same electrode tested at 25 °C. The sensing parameters of t-LDHE were compared with the electrodes produced by commercially available rabbit muscle LDH (m-LDH). The sensitivity of t-LDHE was nearly 8 times higher than that of m-LDHE. t-LDHE was found to retain its activity for a week incubation at refrigerator (+5 °C), while m-LDHE lost its activity in this period. t-LDHE was also tested in the presence of human blood serum. The results showed that the current increased with increasing concentrations of lactate in the human blood serum and the biosensor is more sensitive to serum lactate as well as the commercial lactate dissolved in serum as compared to the commercial lactate dissolved in CHES buffer.     

Gene Clone and Characterization of a Novel Thermostable β-Galactosidase with Transglycosylation Activity from Thermotoga naphthophila RUK-10

We cloned and expressed a new recombinant β-galactosidase(TN0949) from Thermotoga naphthophila RKU-10 with the pET28a(+) vector system in Escherichia coli BL21(DE3), and determined its catalytic capability to synthesize alkyl glucosides. The recombinant enzyme was purified to a single band via heat treatment and Ni²⁺-NTA affinity chromatography. The molecular mass of the recombinant enzyme was estimated to be 79 kDa with sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE). TN0949 can hydrolyze o-nitrophenyl β-D- galactopyranoside at the optimum pH and temperature of 6.5 and 80 ℃, respectively. TN0949 can also hydrolyze lactose at the optimum pH and temperature of 5.2 and 80 ℃, respectively. The K_m values for the hydrolyses of o-nitrophenyl β-D-galactopyranoside and lactose were 0.82 and 83.65 mmol/L, respectively. TN0949 was stable over a wide range of pH(3.0 to 7.0) after 24 h of incubation. The half-lives of TN0949 at 75, 80 and 85 ℃ were 22, 6 and 1.33 h, respectively. The enzyme displayed the capability to use lactose as the transglycosylation substrate to synthesize butyl galactopyranoside and hexyl galactopyranoside, indicating its suitability as a candidate industrial biocatalyst.