| Owing to cleaving the a-1,6-glucosidic linkages specifically in amylopectin, pullulanase(EC 3.2.1.41) can be widely used in industrial production of glucose. In the starch saccharification process, it can increase the yield of glucose, improve the quality of glucose, save cost and time by using pullulanase and glucoamylase. In this study, the recombinant Bacillus naganoensis pullulanase(PUL) with excellent enzymatic properties was adopted, and it nearly reached the performance of commercial pullulanase by means of analyzing saccharification products, optimizing saccharification process, investigating the application stability of recombinant pullulanase, which laid the foundation for industrial production and application. The main results are as follows:After the saccharification from maltodextrin substrate by a combination of glucoamylase and pullulanase, the resulting solution mainly contained glucose, maltose and isomaltose. And in saccharification stage, the glucose content(DX) was ascendant firstly and started to decline after 48 h, while isomaltose content(IM) maintained mounting trend, indicating the reverse oligomerization from glucose to isomaltose. Under the conditions of glucoamylase loading 50 U·g–1 dry maltodextrin, saccharification temperature 55°C, p H 4.5 and time 72 h, the DE and DX value of the experimental group with PUL(0.5 U·g–1 dry maltodextrin) was significantly higher than the control group, and the IM value was lower than the control group. The DE value of the experimental group with PUL was extremely approaching to the experimental group within OPTIMAX L-1000. However, the DX value was lower than the experimental group within OPTIMAX L-1000, which indicating that there was a certain gap between PUL and OPTIMAX L-1000 in terms of saccharification effect.The optimal ratio of glucoamylase to pullulanase for mixed enzymatic saccharification(MES) was fixed on 50:1 by comparing PUL and OPTIMAX L-1000. Then, the effects of different d.m. values on DX value, IM value and space-time yield were investigated, repectively. When the optimum concentration of maltodextrin was set at 20%, the highest DX value and space-time yield were 98.87 % and 5.49 mg·g–1·h–1 respectively. By means of the single factor test, orthogonal design and response surface methodology analysis, the optimum conditions were determined. Consequently, the actual DX value of 99.35% was achieved with multi enzyme loading 75 U·g–1 at 55°C and p H 4.5 for 36 h.The effects of different substances on thermostability of the pullulanase were investigated. The results indicated that the thermostability of PUL could be significantly improved by adding in gelatin, glycerin, Tween 20 and Ca2+. Based on the results of single factor experiments, the optimal combination additive(Tween 20 2 m L·L–1, gelatin 6 g·L–1, glycerin 5%, Ca2+ 0.1 mol·L–1) was obtained using the orthogonal experimental design. The residual activity of PUL reached 94.68% in the presence of combination additive after incubating 48 h at 40°C. Then the influence of combination additive on pullulanase thermostability was investigated. The half-life of PUL in the presence of combination additive was 47 h at 55°C, 3.27 times higher compared with control(11 h). Furthermore, the effect of combination additive on the MES was also studied. After adding in combination additive, glucose content represented an upward trend in general, but isomaltose content expressed an opposite tendency. Therefore, the combination additive made a significant effect on the enhancement of the pullulanase thermostability.To inhibit bacteria contamination and improve the storage stability of the enzyme solution, preservatives were added in the liquid enzyme. Finally, the residual activity of PUL containing combination additive and preservatives(0.1% potassium sorbate, 0.1% sodium benzoate) reached up to 85.25% after storing 90 days at room temperature, meeting the requirements of industrialization. |
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