Preparation Of Cross-Linked Amylase Aggregates And Its Application In Starch Fermentation For Ethanol Production

There is an upsurge of interest in searching for renewable biomass. The production of alternate fuels such as bioethanol, has several advantages in environmental protection. Starch is one of the most widely used material for producing bioethanol. However, incomplete pretreatments of starch material during manufacturing process cause the low utilization rate of fermentation, resulting in increase of cost.Based on the approach of immobilized enzymes to overcome the inefficient pretreatment of starch fermentation, corss-linked alpha-amylase aggregates (CLEA) and cross-linked glucoamylase aggregates (CLGA) were prepared in this study. Robust and high activity of CLEA and CLGA were obtained when the protecting agent added during the crosslinking process. Then, the experiment of Simultaneous Saccharification and Fermentation was performed using immobilized enzymes. Finally, the surface display system of glucoamylase on Pichia pastorris was studied, these works laid the groundwork for the display of glucoamylase and alpha-amylase on the surface of yeast.Main results were gained in this study as follows:1. Preparation of cross-linked α-amylase aggregates (CLEA) and their characters were investigated in this article. To ensure the optimal final concentration of glutaraldehyde was0.2-0.3%and0.5%during2h crosslinking process. The cross-linked enzyme protectant was dextrin and starch with concentration being2%and3%(w/v), the related activities were137and108%. The suitable temperature range50-70℃or40-55℃after immobilized. Compare with the protectant BAS, it was showed that CLEA-S and CLEA-BSA contain the higher affinity of substance proved by the lower Km and the429min about t1/2at60℃, The other side, the affinity of CLGA-D to dextrin was decreased, the t1/2was991.7min at55℃, The recovery activity of CLEA-BSA and CLEA-S were73.2%and69.4%left respectively after20-times runs. The residual activity of CLGA-D and CLGA-BSA were58.6%and69.4%respectively after2months stored at4℃. The recovery activity of CLGA-D were58.6%still reserved after 25-times runs.It showed that Na-, Mg2+, K+, Ca2+, Mn2+and Fe2+could activate CLEA and free enzyme. The affinity of CLEA-BSA (with BSA) and CLEA-S was increased and the activity of CLEA-S was66%at60℃for390mins. The activity of immobilized and free glucoamylase was inhibited by Na+, K+,Mn2+and Mg2+slightly, but Cu2+, Zn2+and Fe2+were seriously inhibit the activity of the enzyme.2. The hydrolysis of starch using CLGA-D and CLEA-S with S. cerevisiae1912simultaneously was studied. The hydrolysis and fermentation efficiency of free and immobilized enzyme were basically the same. The free enzyme was4-6hours earlier than the immobilized enzyme in the utilization of reducing sugar. As a result,8.3%(v/v) ethanol was produced by starch.3. Glucoamylase gene (GLA) and anchor gene (FL) from Aspergillus niger Jland Saccharomyces cerevisiae S288c have been cloned. Then linked these fragments with pET28a and pPIC9K plasmid and transformed them into DH5α, pET-GLA, pPIC9K-GLA and pET-NS-GLA has been constructed. These works laid the groundwork for the display of glucoamylase on the surface of Pichia pastorris.