Biochemical characterisation of truncated amylopullulanase from Thermoanaerobacter brockii brockii

Abstract

The last point reached as a result of the chemical transformation carried out by nature is life. Enzymes are responsible biomolecules for the chemical transformation that makes life possible. People had benefited from the reactions carried out by enzymes to obtain various products in many fields throughout history, from the times when they did not know the existence of these catalysts. Over time, people had understood how great potential these biocatalysts have with the structure and working principles. Since then the characterisation of novel enzymes and the usage area of these enzymes have been expanded day by day. Today, more than 3.000 enzymes are used in biotechnological and industrial fields and it is reported that the size of this enzyme market has reached 6 billion USD in 2021. The enzymes that dominate this market with a share of 30 % are those used in the food and beverage industry and the ones that have the upper hand in this group are the enzymes used in starch processing applications. Starch, the main component of many agricultural products and the primary source of carbohydrates for most people, is the main source of carbon in the world. In addition to starch, oligosaccharides, disaccharides and monosaccharides obtained as a result of hydrolysis are also used in various fields such as food, pharmaceuticals and biofuels. To obtain these hydrolysis products, starch is subjected to a three-step process: gelatinisation, liquefaction and saccharification. The viscous solution which is obtained by hydrolysis of α-1,4- and α-1,6- glycosidic bonds in the starch structure and gelatinisation is achieved by using α-amylase, β-amylase, glucoamylase and pullulanase enzymes during the liquefaction. During all these processes to remain the enzymes stable for a long time and maintain their activity, pH adjustment and the addition of Ca2+ ionsare required . Remove both the formed salt as a result of pH adjustments and excess Ca2+ ions causes additional steps and all of these lead to an increase in the cost of production. To eliminate these steps and perform a one-step liquefaction-saccharification process can be possible with an alternative enzyme. Amylopullulanases (E.C. 3.2.1.1/41) are enzymes capable of hydrolysing both α-1,4- and α-1,6- glycosidic bonds, while those obtained from extremophilic organisms can maintain their activities under harsh industrial conditions. In this study, the amylopullulanase (TbbApu) enzyme belonging to the Thermoanaerobium brockii brockii organism, is one of the strong candidates for starch hydrolysis processes, is examined. Previously, for the recombinant production of TbbApu, the whole apu gene had been obtained by using the primary walking method by our group and the optimisation of expression studies was in progress. As a result of the studies, pET-28 a (+) vector and E. coli BL21 (DE3) were chosen as the appropriate expression vector and and E. coli BL21 (DE3) the appropriate host, respectively. Additionally, the variants TbbApuΔSH3 without SH3 domain and TbbApuΔCBM20 without CBM20 domain were constructed to investigate the effect of SH3 and CBM20 domains. In the scope of this thesis, apart from TbbApuΔSH3 and TbbApuΔCBM20 variants, four additional truncated constructs namely TbbApuΔX25-1-SH3, TbbApuΔX25-2-SH3, TbbApuΔX25-1 and TbbApuΔX25-2-CBM20 were also obtained to disclose the effects of X25 domain. The biochemical characterisation, raw starch binding ability and kinetic studies of TbbApu and its six variants have been accomplished. The function of the X25 domain on substrate binding, activity and stability of the enzyme has been revealed for the first time with this study. The genes belonging to each construct were amplified by PCR with specific forward and reverse primers that have SacI and NotI restriction sites at their 5' ends and using the whole apu gene as a template. As a result of the amplification, tbbApuΔX25-1-SH3, tbbApuΔX25-2-SH3, tbbApuΔX25-1-CBM20 and tbbApuΔX25-2-CBM20 genes with a length of 4065, 3750, 3375 and 3060 base pairs, respectively, were obtained. Then, the obtained genes and the pET-28 a (+) expression vector were cut with SacI and NotI enzymes to form sticky ends and the ligation reactions were set up for the insertion of the genes into the pET-28 a (+) expression vector. Then, transformation was performed using competent E. coli BL21 (DE3) host cells. For the determination of positive colonies from the colonies obtained as a result of the transformation, half of the colonies were inoculated on agar plates containing red pullulan. 1 µM IPTG and 40 µg/mL kanamycin were also included in the agar plate for induction and selection respectively. For the control of detected positive colonies from the PRR plate, the other half of the colonies were inoculated into Luria-Bertani (LB) medium and plasmid isolation was performed from these cells. Then, the isolated plasmids were cut with FastDigest SacI enzyme and linearised to determine the length of the plasmids. By linearisation, tbbApuΔX25-1-SH3 - pET-28 a (+) vector with a length of 9434 base pairs; 9119 base pairs long tbbApuΔX25-2-SH3 -pET-28 a (+) vector, 8744 base pairs tbbApuΔX25-1-CBM20- pET-28 a (+) vector and 8429 base pairs tbbApuΔX25-2-CBM20- pET-28 a (+) vector were obtained from all selected colonies. After the cloning, TbbApu, TbbApuΔSH3, TbbApuΔCBM20, TbbApuΔX25-1-SH3, TbbApuΔX25-2-SH3, TbbApuΔX25-1-CBM20 and TbbApuΔX25-2-CBM20 were over-expressed using magic media. To purify these recombinant enzymes, purification was achieved in three steps by using metal affinity chromatography, ion exchange chromatography and heat purification respectively. Then, the pullulanase and α-amylase activities of TbbApu and its six variants were checked by the red pullulan and starch-containing polyacrylamide gel. The biochemical characterisation of the enzymes was completed. As a result of the studies, the optimum reaction temperature for pullulanase activities was determined as 70 °C for TbbApu, TbbApuΔSH3 and TbbApuΔCBM20, 75 °C for TbbApuΔX25-1-SH3, TbbApuΔX25-2-SH3 and TbbApuΔX25-1-CBM20 and also 80 °C for TbbApuΔX25-2-CBM20 domain. The optimum reaction temperature for α-amylase activities was specified as 75 °C for TbbApu, TbbApuΔSH3 and TbbApuΔCBM20, and 80 °C for TbbApuΔX25-1-SH3, TbbApuΔX25-2-SH3, TbbApuΔX25-1-CBM20 and TbbApuΔX25-2-CBM20. The pure variants were incubated for 24 hours at variable temperatures to examine the effect of temperature on the stability of enzymes. It was observed that the most stable temperature of 60 °C for TbbApu, TbbApuΔSH3, TbbApuΔCBM20, TbbApuΔX25-1-SH3 and TbbApuΔX25-2-SH3, 50 °C for TbbApuΔX25-1-CBM20 and 40 °C for TbbApuΔX25-2-CBM20. Optimum pH values for both activities were found to be 6.5 for TbbApu, TbbApuΔX25-1-SH3 and TbbApuΔX25-1-CBM20, 6 for TbbApuΔSH3, TbbApuΔCBM20 and TbbApuΔX25-2-SH3, and 7 for TbbApuΔX25-2-CBM20. It has been determined that the enzymes reached 80 % of their α-amylase activities with pH 5 and protected it up to pH 8. When the effects of different pH values on the stability of the enzymes were examined, the most stable pH according to pullulanase activities was found as pH 4 for TbbApu, TbbApuΔSH3, TbbApuΔCBM20 and TbbApuΔX25-2-CBM20, pH 6 for TbbApuΔX25-2-SH3, pH 6.5 for TbbApuΔX25-1-SH3 and TbbApuΔX25-1-CBM20 after 24 hours of incubation. According to their α-amylase activities, the most stable pH was found as pH 3 for TbbApu, TbbApuΔSH3, TbbApuΔCBM20, pH 6 for TbbApuΔX25-2-SH3, TbbApuΔX25-1-CBM20 and TbbApuΔX25-2-CBM20, pH 7 for TbbApuΔX25-1-SH3. In addition, it is represented that TbbApu and its variants can maintain their stability between pH 3 and 8 and TbbApu can be used in starch hydrolysis processes without pH adjustment. When the effect of metal ions was examined, while Mn2+ and Co2+ ions increased both pullulanase and α-amylase activities of TbbApu and its variants whereas, Mg2+, Zn2+ and Al3+ ions decreased both activities of all enzymes except pullulanase activity of TbbApuΔCBM20 variant. Although α-amylase enzymes used in starch processing are Ca2+ ion-dependent, TbbApu and its variants are not dependent on Ca2+ ion for activity and stability, making the enzyme and its variants a strong candidate for starch hydrolsis process. Also, in the presence of 20 % and 50 % hexane and acetone, with some exceptions, both activities of TbbApu and its variants were increased and 20 % and 50 % butanol, DMF and DMSO presence strongly inhibited both activities of the enzymes. When the effects of organic solvents on the stability of enzymes were examined, it was observed that, with some exceptions, the stability of the enzymes increased in the presence of 20 % and 50 % acetone and hexane, compared to both pullulanase and α-amylase activities, after 24 hours of incubation. In the case of inhibitors and detergents, it was observed that inhibitors moderately inhibited pullulanase and α-amylase activities of TbbApu and its variants with some exceptions, whereas all inhibitors increased the pullulanase activity of TbbApuΔCBM20 and nonionic and anionic enzymes inhibited both activities moderately with. However, the activities of all enzymes were strongly inhibited in the presence of CTAB, which is a cationic detergent. Then, the kinetic parameters of the enzymes were elucidated. The results imply that, truncation SH3 domain improves both α-amylase and pullanase activities of the enzyme. However, truncation of CBM20 and X25 domains from TbbApu caused to loss of affinity and specificity of the enzyme to soluble starch and to shift in the specificity of the enzyme to pullulan. The removal of the SH3 domain and also CBM20 domain, the carbohydrate-binding module in the enzymes did not have any effect on the raw starch binding capacity of TbbApu. The removal of the SH3 domain and also CBM20 domain, the carbohydrate binding module in the enzymes did not have any effect on the raw starch binding capacity of TbbApu. However, sequential removal of X25 and SH3 domains resulted in 27.3 % and 58.4 % reduction in raw starch binding ability, while removal of CBM20 and X25 domains resulted in a 90 % decrease in raw starch binding ability. Thus, it was revealed that the X25 domain is involved in binding raw starch. The results of TLC analysis represented that TbbApu and its variants released maltotriose and maltose as a result of pullulan hydrolysis and maltotriose starch hydrolysis, respectively. The results of TLC analysis represented that TbbApu and its variants released maltotriose and maltose in pullulan hydrolysis and maltotriose in starch hydrolysis, respectively.