Screening,Engineering And Immobilizing A Novel Thermophilic Pullulanase

Pullulanase?EC 3.2.1.41?is a starch-debranching enzyme in the?-amylase family and specifically cleaves?-1,6-glycosidic linkages in starch-type polysaccharides,such as pullulan,?-limited dextrin,glycogen,and amylopectin.It plays a key role in debranching and hydrolyzing starch completely,leading to improved product quality,increased productivity,and reduced production costs in producing resistant starch,sugar syrup,and beer.The acidic pullulanases that matched well with fungal glucoamylases,such as Klebseilla pneumonia,Bacillus acidopullulyticus,Bacillus naganoensis,and Bacillus deramificans pullulanases,have been commercially produced and widely applied in starch processing.Under the protection of the patent rights for commercial pullulanases,the high costs of pullulanases impede the continuous and healthy development of starch processing industry in China.Furthermore,the developing starch processing needs various pullulanases suitable for different processing conditions.Thus,new pullulanases with high catalytic performance should be developed to meet diversified demands for processing starch.In this study,to obtain new thermo-and acido-stable pullulanases,a pullulan-degrading thermophile was isolated and identified as Anoxybacillus sp.WB42 for the first time.Its pullulanase gene,pulWB42,was cloned and expressed in Escherichia coli,and the catalytic properties of the recombinant protein demonstrate that PulWB42 is a novel thermophilic amylopullulanase.Furthermore,PulWB42 was engineered by semi-rational mutagenesis to improve enzyme catalysis and alter its substrate selectivity.Lastly,to reduce enzyme costs,PulWB42 was immobilized onto magnetic nanoparticles?MNPs?which were modified with various functional groups.The main results are as follows:?1?A new pullulan-degrading strain was isolated from apple pomace with red pullulan agar plates at 50?,and it was identified as Anoxybacillus sp.WB42 which grew optimally at pH range from 7.0 to 7.5 and temperature range from 50 to 55?.Anoxybacillus sp.WB42grew and produced extracellular pullulanases in oligotrophic media,and the pullulanase production was inhibited as pullulan concentration in media exceeded 2.0 g�L^-1.The crude pullulanase from Anoxybacillus sp.WB42 exhibited the optimum pullulytic activity at pH 5.5and temperature ranging from 55 to 65?,and it remained its initial activity completely after incubation at 60? for 24 h.The pullulanase gene,pulWB42,was cloned from Anoxybacillus sp.WB42 by homology cloning strategy and genome walking.The protein encoded by pulWB42,PulWB42,shares 71%pairwise identity with the known type I pullulanases?PULIs?and thus is assigned to glycoside hydrolase family 13 subfamily 14?GH13₁4?PULI.The secretory recombinant protein from E.coli harboring pET22b-pulWB42 cleaved both the?-1,6 glycosidic bonds in pullulan and the?-1,4 glycosidic bonds in amylose,demonstrating an amylopullulanase that is type II pullulanase with single active center?PULII_I?,and it hydrolyzed pullulan and amylose in a Ca²⁺-independent manner and showed the optimal pullulytic activity at a temperature range of 55 to 65? and pH 5.8 as well as high thermostability at 60? and pH 5.5.?2?The homology model of PulWB42 constructed by SWISS-MODEL reveals that the fold of PulWB42 is a typical GH13₁4 PULI structure which consists of carbohydrate-binding module?CBM?68,CBM48,?-amylase catalytic domain?Aamy?,and?-amylase C-terminal all-?domain?AamyC?.The CBM68-deleted PulWB42 acted as an amylopullulanase with lowered pullulytic activity,amylose selectivity,and thermostability as compared to PulWB42.The replacement of Ala90 or Arg93 in CBM68 significantly changed the substrate specificity and catalytic efficiency of PulWB42,such as A90P,A90D,R93T,R93E,and R93K acting as PULI.Moreover,H5A/R6A/T7A,Q87A,L173D,Q87A/L173D,and M88D showed improvements in thermostability and changes in catalytic kinetics,and among these mutants,L173D enhanced catalytic efficiency by 39%,whereas the others reduced it to an extent.Therefore,the N-domain of PulWB42,CBM68,is not essential for catalysis,but it does modulate enzyme catalysis,especially with respect to substrate specificity.The modulation was achieved mainly by the Leu86�Arg93 segment adjacent to CBM48 and Aamy in the model of PulWB42,and the enzyme thermostability was enhanced by rigidifying the coil Met1-Phe8 or by strengthening the interactions between CBM48 and the random coil Leu86-Ala89 in CBM68.Additionally,N-terminal sequence significantly affects enzyme catalysis.The N-terminus-truncated mutants MR6E and MT7 were still amylopullulanases with enhanced thermostability,whereas MR93 and MR93/H362D transformed into PULIs,and MR93/H362D enhanced thermostability,pullulan affinity,and catalytic efficiency.By contrast,MT94 remained weakly amylolytic ability while its pullulytic efficiency was enhanced by 96%as compared to PulWB42.?3?His223?Glu271?Leu273?Asn291?Val374?Leu435,and Ser519 are near residues for substrate binding and catalysis in the catalytic domain of PulWB42?Aamy?,and these residues have significant effect on enzyme catalysis.With regard to the pH-dependent pullulytic activity,the replacement of Glu271 by Gln shifted the acidic limb of the pH-activity profile to acidity,such as the optimal pHs for E271Q/S519C and E271Q/L273M/S519C were5.0,and that for E271Q/L273Q/S519C was 4.5.While the replacement of His223,Asn291,Val374,Leu435,or Ser519 shifted the pH-activity curve towards basicity,such as replacing His223 with Tyr,Phe,or Asp,replacing Val374 with Asp,Thr,or Cys,or replacing Asn291 by Glu led the optimal pHs for these substituted mutants of E271Q/L273M/L435Y/S519C to increase or extend to 6.5.Moreover,the pullulytic efficiency(k_cat/K_m)is modulated by His223,Asn291,Val374,Leu435,or Ser519.The k_cat/K_m value for S519C increased by 2.3 folds,that forH223Q/E271Q/L273M/L435Y/S519Cby4.1folds,thatfor N291D/E271Q/L273M/L435Y/S519Cby3.7folds,andthatfor V374D/E271Q/L273M/L435Y/S519C by 6.6 folds as compared to the wild-type PulWB42.Additionally,Ser519 plays a key role in the enzyme thermostability,and the thermostability was enhanced significantly by replacing Ser519 with Cys.?4?The recombinant PulWB42 was immobilized onto MNPs grafted with various functional groups.Both the immobilized enzyme catalysis and its activity recovery are dependent on the nature and strength of the interfacial reaction between nanoparticle and enzyme.With regard to the activity recovery of the immobilized enzyme,it was in the range of 63%to 70%as the enzyme attached covalently to nanoparticle,whereas it was more than90%when the enzyme non-covalently immobilized on MNPs.The pH-dependent activity,thermostability,substrate affinity,and reusability of the immobilized enzyme are dependent on the surface chemical properties of MNPs which electrostatically or covalently combine with enzymes.Fe₃O₄@CMC/PDA-PEI-GTA/PUL-His6 showed enhanced pullulan affinity and reduced thermostability as compared to free enzyme,while it exhibited the highest reusability among the electrostatically immobilized enzymes,remaining 69%of the initial activity after 13 consecutive cycles with a total reaction time of 390 min.By contrast,the catalytic properties of the immobilized His6-tagged PulWB42 are mainly dependent on the orientation of enzymes chelated to nickel ions of MNPs.When His6-PulWB42 chelates nickel via N-terminal His6,the immobilized His6-PulWB42 showed a basic shift in the pH-activity profile,and decreases in the thermostability and pullulan affinity as compared to free His6-PulWB42,and it exhibited higher reusability than that of PulWB42-His6 chelated to MNPs via C-terminal His6.Fe₃O₄@PEI-BDDE-PEA400-IDA-Ni/His6-PUL displayed the highest reusability among the affinity-immobilized His6-tagged PulWB42,remaining 80%of the initial activity after 13-cycle pullulytic reactions with a total reaction time of 390 min.