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1.
聚丙烯酰胺固定化糖化酶特性的研究 总被引:5,自引:0,他引:5
本研究以丙烯酰胺单体通过反向悬浮聚合技术合成聚丙烯酰胺作为载体材料,采用包埋—交联法固定化葡萄糖淀粉酶,并对其特性进行了研究.结果表明,该固定化酶最适pH值为5.0,最适温度为55~58℃,而且具有较好的贮存稳定性和操作稳定性,8个月后该固定化酶的残余活力仍保持在94%左右,可重复使用43批次,此固定化酶酶活回收率达到56%.实验表明丙烯酰胺悬浮聚合固定化糖化酶的方法是简便可行的. 相似文献
2.
Among physical and nutritional parameters optimized by “one variable at a time” approach, four cultural variables (sucrose,
MgSO4
.7H2O, inoculum size, and incubation period) significantly affected glucoamylase production. These variables were, therefore,
selected for optimization using response surface methodology. The p-values of the coefficients for linear effect of sucrose and inoculum size were less than 0.0001, suggesting them to be the
key experimental variables in glucoamylase production. The enzyme production (34 U/ml) attained under optimized conditions
(sucrose, 2%; MgSO4
.7H2O, 0.13%; yeast extract, 0.1%; inoculum size, 5 × 106 spores per 50 ml production medium; incubation time, 48 h; temperature, 40°C; and pH 7.0) was comparable with the value predicted
by polynomial model (34.2 U/ml). An over all 3.1-fold higher enzyme titers were attained due to response surface optimization.
The experimental model was validated by carrying out glucoamylase production in shake flasks of increasing capacity (0.25–2.0 l)
and 22-l laboratory bioreactors (stirred tank and airlift), where the enzyme production was sustainable. Furthermore, the
fermentation time was reduced from 48 h in shake flasks to 32 h in bioreactors. 相似文献
3.
Summary Several mobile phase parameters were investigated for controlling enantioselective retention and resolution on a chiral stationary
phase made in-house. The chiral selector was the enzyme amyloglucosidase, which was immobilized onto a silica support via
reductive amination. The influences of the mobile phase pH, concentration and type of uncharged organic modifier, ionic strength
and column temperature on enantios-electivity were studied. The analysis time for resolving enantiomers could be adjusted
with only a minor decrease in enantioselectivity by using a high ionic strength mobile phase buffer. This indicated a retention
mechanism involving ion-exchange interactions. It was further confirmed by the decreasing enantioselectivity of amines when
using a mobile phase pH below the isoelectric point of the native protein. Interesting effects were observed when the organic
modifier concentration was increased and also when the column temperature was raised. Both retention and enantioselectivity
increased with increasing concentration of 2-propanol in the mobile phase. Examples are given where both enantioselectivity
and retention increased with increasing column temperature. Thermodynamic studies were performed to calculate the entropy
and enthalpy constants. The results showed that, depending on mobile phase composition, the enantioselective retention may
be caused by differences in entropy or enthalpy. 相似文献
4.
S.H. Imam S.H. Gordon R. Harry-O'kuru G.M. Glenn 《Polymer Degradation and Stability》2006,91(12):2894-2900
Granular cornstarch was treated with microbial glucoamylase (50 mM sodium acetate buffer at pH 5.5 at 30 °C, 150 rpm) for up to 8 h. Treated starch was recovered and evaluated for changes in granular morphology, chemical properties, thermal properties, crystallinity and impact on its biodegradability. As the enzyme treatment progressed, reducing sugars began to accumulate in the liquid culture media (total of 6% in 8 h) and the granule suffered roughly 6% weight loss within 8 h of incubation. While the granules appeared intact morphologically, numerous small pits developed throughout the surface of the granules as a result of the enzyme treatment. Even after 8 h of enzyme treatment, the pitted granules were not disrupted and remained intact. X-ray diffraction indicated no loss of crystallinity in the enzyme treated granules but rather an increase in relative crystallinity, suggesting that the enzyme preferentially catalyzed the anhydroglucose units in amorphous regions of the granule. These findings were further supported by FTIR data suggesting that granules become more resistant to enzyme attack as amorphous amylose is hydrolyzed faster than the crystalline amylopectin domains. These results also suggest that variations in the crystallinity of different types of starches have the potential to affect their rates of biodegradation. Enzyme treated starch granules exhibited resistance to biodegradation, and the degree of resistance was related to the length of enzyme treatment. Granules treated with enzyme for a total of 7 h and subjected to biodegradation in soil produced 40-50% less CO2 in a closed circuit respirometer compared to the untreated samples. Differential scanning calorimetry (DSC) thermograms showed an endothermic reaction with little change in the onset and peak temperatures indicating that glucoamylase started by degrading the starch granules from the surface. 相似文献
5.
多孔球状淀粉接枝共聚物的合成及其性质研究 总被引:13,自引:0,他引:13
报道了一种多孔球状淀粉接枝共聚物的合成。此球有很强的机械性能,良好的稳定性和较高的致孔率,平均孔径约为19.5nm,以其为载体固定化糖化酶活力为1462IU/g干胶,比活为29.31IU/mg蛋白。 相似文献
6.
Glucoamylase was immobilized onto novel porous polymer supports. The properties of immobilized glucoamylase and the relationship
between the activity of immobilized enzyme and the properties of porous polymer supports were investigated. Compared with
the native enzyme, the temperature profile of immobilized glucoamylase was widened, and the optimum pH was also changed. The
optimum substrate concentration of immobilized glucoamylase was higher than that of native enzyme. After storage for 23 d,
the immobilized glucoamylase still maintained about 84% of its initial activity, whereas the native enzyme only maintained
about 58% of the initial activity. Moreover, after using repeatedly seven times, the immobilized enzyme maintained about 85%
of its initial activity. Furthermore, the properties of porous polymer supports had an effect on the activity of the immobilized
glucoamylase. 相似文献
7.
pH and temperature play critical roles in multistep enzymatic conversions. In such conversions, the optimal pH for individual
steps differs greatly. In this article, we describe the production of glucoamylase (from Aspergillus oryzae MTCC152 in solid-state fermentation) and glucose isomerase (from Streptomyces griseus NCIM2020 in submerged fermentation), used in industries for producing high-fructose syrup. Optimum pH for glucoamylase was
found to be 5.0. For glucose isomerase, the optimum pH ranged between 7.0 and 8.5, depending on the type of buffer used. Optimum
temperature for glucoamylase and glucose isomerase was 50 and 60°C, respectively. When both the enzymatic conversions were
performed simultaneously at a compromised pH of 6.5, both the enzymes showed lowered activity. We also studied the kinetics
at different pHs, which allows the two-step reaction to take place simultaneously. This was done by separating two steps by
a thin layer of urease. Ammonia generated by the hydrolysis of urea consumed the hydrogen ions, thereby allowing optimal activity
of glucose isomerase at an acidic pH of 5.0. 相似文献
8.
Use of immobilized amyloglucosidase as chiral selector in chromatography. Immobilization and performance in liquid chromatography 总被引:1,自引:0,他引:1
Summary The enzyme amyloglucosidase was immobilized on oxidized DIOL silica and used to separate enantiomers of amino alcohols. The
influence of pore size on enantioselectivity was studied and an optimum in the separation factors was found using 500 ? DIOL
silica as the starting material. About twice the amount of the protein could be immobilized on the 500 ? DIOL silica material
as on the 300 and 1000 ? materials. The immobilization procedure was easy to reproduce and no significant difference in the
chromatographic behavior was observed between two amyloglucosidase columns produced in-house. The effect of solute structure
on enantioselective retention was studied using a set of 10 closely related amino alcohols. High separation factors (α>2)
were obtained and the efficiency of the amyloglucosidase columns was greater than 25 000 plates/m based on the last eluted
enantiomer. 相似文献
9.
10.
Rey MW Brown KM Golightly EJ Fuglsang CC Nielsen BR Hendriksen HV Butterworth A Xu F 《Applied biochemistry and biotechnology》2003,111(3):153-166
Thielavia terrestris is a soil-borne thermophilic fungus whose molecular/cellular biology is poorly understood. Only a few genes have been cloned
from the Thielavia genus. We detected an extracellular glucoamylase in culture filtrates of T. terrestris and cloned the corresponding glaA gene. The coding region contains five introns. Based on the amino acid sequence, the glucoamylase was 65% identical to Neurospora crassa glucoamylase. Sequence comparisons suggested that the enzyme belongs to the glycosyl hydrolase family 15. The T. terrestris glaA gene was expressed in Aspergillus oryzae under the control of an A. oryzae α-amylase promoter and an Aspergillus niger glucoamylase terminator. The 75-kDa recombinant glucoamylase showed a specific activity of 2.8 μmol/(min·mg) with maltose
as substrate. With maltotriose as a substrate, the enzyme had an optimum pH of 4.0 and an optimum temperature of 60°C. The
enzyme was stable at 60°C for 30 min. The K
m
and k
cat
of the enzyme for maltotriose were determined at various pHs and temperatures. At 20°C and pH 4.0, the enzyme had a K
m
of 0.33±0.07 mM and a k
cat
of (5.5±0.5)×103 min−1 for maltotriose. The temperature dependence of k
cat
/K
m
indicated an activation free energy of 2.8 kJ/mol across the range of 20–70°C. Overall, the enzyme derived from the thermophilic
fungus exhibited properties comparable with that of its homolog derived from mesophilic fungi. 相似文献