Heterologous Expression And Molecular Engineering Of Alkaline Amylase

Alkaline amylase is a kind of hydrolase that is stable at alkaline condition (pH9.0~11.0)and can hydrolyze starch by cleaving α-1,4-glucosidic linkages. It is being widely used intextile and detergents industries. It was reported that the application of alkaline amylase indesizing of cotton fiber can save time, decrease environmental pollution, and minimize thedamage to textile fabric, and this can greatly improve the efficiency of the textilepre-treatment to achieve maximum economical benefits. Overall, alkaline amylase is a keyenzyme that can promote the rapid development of textile fabric pre-treatment process.The current studies mainly focus on strain selection, purification and characterization ofalkaline amylase. When alkaline amylase is used in textile and detergents industries, itshould possess the characteristics of anti-oxidation and high catalytic efficiency and so on.However, the native alkaline amylases have some disadvantages, such as easy oxidation,low catalytic efficiency and low yield and so on. So far, there is no report about themolecular engineering of alkaline amylases to improve oxidative stability and catalyticefficiency.In this thesis, we successfully expressed the alkaline amylase gene (Accession No.AY268953) in Escherichia coli, Bacillus subtilis, and Pichia pastoris, respectively. On thebasis of this, the anti-oxidation and catalytic efficiency of alkaline amylase were improved bymolecular engineering. The main results are listed as follows:1. Based on the different preferred codons of different expression hosts, the codons ofalkaline amylase was firstly optimized and expressed in E. coli, B. subtilis and P. pastoris,respectively. After purification, the kinetic parameters of alkaline amylase weredetermined. It was found that its catalytic efficiency was higher than those reported andcan be used for further study. The relative activity was only18%after incubation with500mM H2O2for30min. The optimum pH was9.5, and the stable pH range was8.0~11.0.The optimum temperature was50oC, and Eawas36.1kJ/mol. The half-life (t1/2) at50and60oC was15.5and3.2min, respectively, and the melting temperature (Tm) was64.3oC.1mM Na+could enhance the activity of alkaline amylase, but the other ions could notimprove the activity. Non-ions surfactants had little influence on the stability of alkalineamylase, but anion surfactant SDS (sodium dodecyl sulfate) could completely inhibit theenzyme activity. The activity of alkaline amylase was inhibited by the liquid detergentsand solid soaps, while solid washing powders had little influence on the enzyme activity.2. The3-D structure of alkaline amylase was modeled by online software Swiss Model. Byanalyzing the3-D structure, it was found that five key amino acids (Met145, Met214,Met229, Met247and Met317) around the active sites were important for theanti-oxidation of the enzyme. When these five key amino acids were reperatively replacedby leucine, the anti-oxidation ability of mutants was enhanced. But only the catalyticefficiency of M247L was improved. The pH and thermal stabilities of M247L were alsoenhanced. The single mutation did not affect the stability of enzyme on the surfactants.The compatibility of M247L with commercial detergents (washing powder) was improved.After compositive mutation, the anti-oxidation of mutants was significantly enhanced, especially mutants with Met247. The substrate binding ability of compositive mutants(M145-214L, M145-214-229L, M145-229-247L, M214-229-247L andM145-214-229-317L) was improved.3. Based on the3-D structure, five key amino acids were replaced with threonine, alanine,serine or isoleucine by single mutation, respectively. After mutation, the anti-oxidation ofmutants was significantly enhanced. The kcat/Kmvalues of M247T, M145I and M229Twere increased by1.3-,1.5-and2.1-fold, respectively. Based on the comprehensiveanalysis about the anti-oxidation and catalytic efficiency of single mutants, eight positivesingle mutations (M145A, M145I, M214A, M229A, M229T, M247T, M247L and M317I)were determined for the next step of compositive mutation. After compositive mutation,eight five-point mutants were obtained. Among all the mutants, the characteristics ofM145I-214A-229T-247T-317I were most significantly enhanced: kcat/Kmvalue increasedby3.2-fold; anti-oxidation, pH stability, thermal stability, anti-surfactants andcompatibility with solid detergents were also improved.4. After fusion with six oligopeptides of different characteristics at N-terminal domain ofalkaline amylase, recombinant enzymes were expressed in E. coli BL21(DE3). Afterfusion with oligopeptide1(AEAEAKAKAEAEAKAK), the specific activity ofAmyK::OP1was increased by2.4-fold compared to that before fusion. The Kmvalues ofall fused enzymes decreased, and the ability of substrate binding was enhanced. The kcatvalue of AmyK::OP1was increased by3.4-fold compared to that before fusion. Thekcat/Kmvalue of AmyK::OP1was increased by5.4-fold compared to that before fusion.After fusion, the alkaline stability of fused enzymes was enhanced. After fusion witholigopeptide1,3,4or5, the optimum temperature of fusion proteins was improved. Thethermal stability and anti-oxidation of AmyK::OP1were enhanced. The activity ofAmyK::OP1could be activated by washing powder, and the compatibility of other fusionproteins was also high. But fusion proteins had low stability under the incubation withsolid soaps and liquid detergents.5. Based on the analysis of the domains and amino acid sequence of alkaline amylase, twelverandom truncations at C-or N-terminus were done, and twelve truncated mutants wereobtained. And then, after fusion with oligopeptide1at N-terminus of mutants, anothertwelve truncation-fusion mutants were obtained. After truncation, the specific activity ofmutants AmyKΔC500-587and AmyKΔC492-587was increased by3.2-and2.7-fold,respectively. The kcatvalues of AmyKΔC500-587and AmyKΔC492-587were increasedby2-fold. The kcat/Kmvalues of AmyKΔC500-587and AmyKΔC492-587were increasedby3.4-and2.6-fold, respectively. The kcat/Kmvalues of AmyKΔC500-587::SOP andAmyKΔC492-587::SOP were increased by29.7-and22.5-fold, respectively. The starchbinding domain at C-terminus was important for hydrolyzing corn starch, but N-terminaltruncation did not affect hydrolysis of corn starch. Fusion with oligopeptide1couldenhance hydrolysis of trunctation mutants at C-terminus of corn starch. However, fusionwith oligopeptide1did not affect the hydrolysis of corn starch. The pH and thermalstabilities of mutants were not changed compared to those before trunctation and fusion.The anti-oxidation of AmyKΔC500-587, AmyKΔC492-587, AmyKΔC500-587::SOP and AmyKΔC492-587::SOP was enhanced. The stability against surfactants of mutants wasnot changed. The compatibility of AmyKΔC500-587, AmyKΔC492-587,AmyKΔC500-587::SOP and AmyKΔC492-587::SOP with solid detergents (washingpowder) was enhaned.