Cell-surface Display Of β-glucosidase From Cassava In Pichia Pastoris And The Study On The Synthesis Of Alkyl Polyglycoside

APG is a "world class", "green" non-ionic surfactant. It has many advantages, such as small surface tension, excellent cleaning, foaming, dispersing, compatibility performance ability. It is non-toxic, skin irritation, biodegradable and can be obtained from recycled material. It is commonly used in chemical industry, agriculture, printing and dyeing, textile, oil and other fields. Currently, it is synthesized by chemical methods in the industry. But there are some flaws in chemical methods, such as not selective, no specific reaction. There will also have α and β anomer products at the same time. And the reaction process requires high temperatures and pressures. This leads to the purity and safety of APG can not be guaranteed, which limits its application in the field of food and medicine. Enzymatic synthesis method has many advantages compared to chemical method, such as selectivity and mild reaction condition, simple synthesis process.The whole cell catalyst GS115/pKMEBGL-GCWn(n = 12,19,21,51,61) were prepared based on the yeast cell surface display technology. This method has many advantages, such as the preparation of immobilized enzyme powder is simple and low cost. And the whole cell catalyst can be recycled is very convenient. This method overcomes the difficult of separating and recycling free enzyme. It also overcames the high cost of free enzyme. And then, this promotes the process of industrialization of enzymatic synthesis.In this study, I synthesize the whole codon optimized gene of cassava β- glucosidase. The synthesized mebgl gene was amplified by PCR. The target gene mebgl and extracted plasmid pKCALB-GCWn(n = 12,19,21,51,61) were digestion by EcoR I and Mlu I. With the help of T4 DNA ligase, the digestion products were ligated and constructed recombinant plasmid pKMEBGL-GCWn(n = 12,19,21,51,61). The successfully constructed recombinant plasmids were transformed into Pichia pastoris GS115. By MD plate screening, colony PCR validation and fermentation screening, I screened higher activity of recombinant Pichia GS115/pKMEBGL-GCWn(n=12,19,21,51,61). The above-mentioned recombinant yeasts were cultured in BMMY. The 2%(V/V) methanol was added into the BMMY every 24 h to induce the production of enzyme. The pNPG was used as the substrate to analyze the hydrolase activity of recombinant yeasts. The hydrolase activity was measured on the optimum conditions, the temperature was 35 ℃, pH 6.0. The highest hydrolase activity GS115/pKDCBGL-GCWn(n=12,19,21,51,61) is reached after cultured by 168 h, the maxmim is 0.56U/g(GCW21). At the same time, the recombinant yeasts GS115/pKDCBGL- GCWn(n = 12,19,21,51,61) were analysized by immunofluorescence. The green fluorescence can be observed under the fluorescence microscope. The FCM peaks of the recombinant yeasts in the X-axis drift obviously compared to the negative control yeast. They all show that the mebgl had demonstrated in yeast surface successfully.It is known from the literature, the cassava β- glucosidase is a weak hydrolase and strong synthetic glycosidases. In the synthesis of APG, the GS115/pKMEBGL-GCW21, which had the highest synthesis ability, was used to the synthesis of APG. At first, I assess the enzymatic properties. The total reaction volume is 5mL, 10%(V/V) p H 5.0 0.2M acetate buffer(V/V), 0.1g GS115/pKMEBGL-GCW21 lyophilized enzyme powder to a final concentration of 10 mM substrate, 40 ℃, 200 rpm. For the five kinds of glycosyl donors, which were pNPG, methyl-β-D-glucoside(MG), glucose, cellobiose and gentiobiose. The highest conversion rate was pNPG, up to 52%. MG had lower the conversion rate, which was only 10%. The enzyme completely cannot be able to use glucose, cellobiose and gentiobiose in the synthesis of APG. As for receptor fatty alcohols, the conversion rate decreases rapidly with the increasing carbon chain length of primary alcohol. N-hexanol highest conversion rate was 70%. Nbutanol conversion rate was slightly lower than the n-hexyl alcohol, which was 52%. For dec alcohol and dodecanol, since the carbon chains were too long, the conversion rates were very low, 18% and 2%.Then, GS115/pKMEBGL-GCW21 was used as the whole cell catalyst to synthesize OG through transglycosylation reaction, which pMPG was used as the substrate. The effects of temperature, pH, amount of enzyme powder, water content and substrate concentration were analysized at the transglycosylation reactions. In 2mL reaction system, the single factor optimization final reaction conditions were: temperature 40 ℃, pH5.0, add the amount of enzyme powder 0.05 g, 50% moisture content, substrate concentration of 30 mM, the conversion rate of nearly 100%. Based on the above results, the substrate concentration was increased to 50 mM, 70 mM and the moisture content was alos increased from 35% to 70%. When the substrate concentration was 50 mM and the moisture content was 50%, the conversion rate was 74%. The result was higher than the single factor experiment(conversion rate was 55% when using the water content reached 25%), indicating a high water content help to improve the conversion rate. However, with further increase in the substrate concentration, the conversion rate was gradually reduced. When the substrate concentration was 70 mM, the highest conversion rate could reach 37%. Based on the above experimental results, I conducted three factors response surface experiments. The three factors were added amount of enzyme powder, water content, substrates addition. The total reaction volume was 2ml. Enzyme dosage was: 0.03 g, 0.05 g, 0.07 g. Water content was: 40%, 45%, 50%. Substrate concentrations were: 30 Mm, 50 Mm, 70 mM. Design-Expert.8.05 b was used to design and fitting data of the response surface experimental. Based on the good fit model, the predict optimal reaction conditions for optimal enzyme dosage is 0.05 g, the optimum substrate concentration 30 mM, the optimum moisture content of 50%.