Starch is a polymeric carbohydrate composed of glucose. As a source of energy, starch can be degraded by various amylolytic enzymes, including &#x3b1;-amylase. In a large-scale industry, starch processing cost is still expensive due to the requirement of high temperature during the gelatinization step. Therefore, &#x3b1;-amylase with raw starch digesting ability could decrease the energy cost by avoiding the high gelatinization temperature. It is known that the carbohydrate-binding module (CBM) and the surface-binding site (SBS) of &#x3b1;-amylase could facilitate the substrate binding to the enzyme&#x2019;s active site to enhance the starch digestion. These sites are a noncatalytic module, which could interact with a lengthy substrate such as insoluble starch. The major interaction between these sites and the substrate is the CH/pi-stacking interaction with the glucose ring. Several mutation studies on the Halothermothrix orenii, SusG Bacteroides thetaiotamicron, Barley, Aspergillus niger, and Saccharomycopsis fibuligera &#x3b1;-amylases have revealed that the stacking interaction through the aromatic residues at the SBS is essential to the starch adsorption. In this review, the SBS in various &#x3b1;-amylases is also presented. Therefore, based on the structural point of view, SBS is suggested as an essential site in &#x3b1;-amylase to increase its catalytic activity, especially towards the insoluble starch.

Umi Baroroh

Muhammad Yusuf

Saadah Diana Rachman

Safri Ishmayana

Mas Rizky A. A. Syamsunarno

Jutti Levita

Toto Subroto

The Importance of Surface-Binding Site towards Starch-Adsorptivity Level in &#x3b1;-Amylase: A Review on Structural Point of View

Phytases are used for feeding monogastric animals, because they hydrolyze phytic acid generating inorganic phosphate. Aspergillus niger 3-phytase A (PDB: 3K4Q) and 3-phytase B (PDB: 1QFX) were characterized using bioinformatic tools. Results showed that both enzymes have highly conserved catalytic pockets, supporting their classification as histidine acid phosphatases. 2D structures consist of 43&#x25; alpha-helix, 12&#x25; beta-sheet, and 45&#x25; others and 38&#x25; alpha-helix, 12&#x25; beta-sheet, and 50&#x25; others, respectively, and pI 4.94 and 4.60, aliphatic index 72.25 and 70.26 and average hydrophobicity of &#x2212;0,304 and &#x2212;0.330, respectively, suggesting aqueous media interaction. Glycosylation and glycation sites allowed detecting zones that can affect folding and biological activity, suggesting fragmentation. Docking showed that H59 and H63 act as nucleophiles and that D339 and D319 are proton donor residues. MW of 3K4Q (48.84&#x2009;kDa) and 1QFX (50.78&#x2009;kDa) is similar; 1QFX forms homodimers which will originate homotetramers with several catalytic center accessible to the ligand. 3K4Q is less stable (instability index 45.41) than 1QFX (instability index 33.66), but the estimated lifespan for 3K4Q is superior. Van der Waals interactions generate hydrogen bonds between the active center and O2 or H of the phytic acid phosphate groups, providing greater stability to these temporal molecular interactions.

Doris C. Ni&#xf1;o-G&#xf3;mez

Claudia M. Rivera-Hoyos

Edwin D. Morales-&#xc1;lvarez

Edgar A. Reyes-Monta&#xf1;o

Nury E. Vargas-Alejo

Ingrid N. Ram&#xed;rez-Casallas

K&#xfc;bra Erkan T&#xfc;rkmen

Homero S&#xe1;enz-Su&#xe1;rez

Jos&#xe9; A. S&#xe1;enz-Moreno

Ra&#xfa;l A. Poutou-Pi&#xf1;ales

Janneth Gonz&#xe1;lez-Santos

Azucena Ar&#xe9;valo-Galvis

&#x201c;In Silico&#x201d; Characterization of 3-Phytase A and 3-Phytase B from Aspergillus niger

The demand for enzymes in the global market is projected to rise at a fast pace in recent years. There has been a great increase in industrial applications of pectinase owing to their significant biotechnological uses. For applying enzymes at industrial scale primary it is important to know the features of the enzyme. Thus, this study was undertaken with aims of characterizing the pectinase enzyme from Bacillus subtilis strain Btk27 and proving its potential application in demucilisation of coffee. In this study, the maximum pectinase activity was achieved at pH 7.5 and 50°C. Also, the enzyme activity was found stimulated with Mg2+ and Ca2+ metal ions. Moreover, it was stable on EDTA, Trixton-100, Tween 80, and Tween 20. Since Bacillus subtilis strain Btk27 was stable in most surfactants and inhibitors it could be applicable in various industries whenever pectin degradation is needed. The enzyme <svg xmlns:xlink="http://www.w3.org/1999/xlink" xmlns="http://www.w3.org/2000/svg" style="vertical-align:-0.0498209pt" id="M1" height="8.68572pt" version="1.1" viewBox="-0.0498162 -8.6359 9.95144 8.68572" width="9.95144pt"><g transform="matrix(.013,0,0,-0.013,0,0)"><path id="g113-76" d="M743 650H503L496 622L527 618C563 613 564 603 532 573C449 495 371 431 323 392C301 374 272 355 246 346L280 522C297 609 300 614 379 622L385 650H135L129 622C209 614 215 609 198 522L124 133C106 39 99 35 23 28L17 0H271L277 28C193 35 192 39 208 133L239 316C264 328 280 325 303 288C368 183 435 90 502 0H652L659 28C602 34 584 43 543 94C495 154 403 283 347 369L574 554C634 603 659 612 735 624L743 650Z"/></g></svg>m and <svg xmlns:xlink="http://www.w3.org/1999/xlink" xmlns="http://www.w3.org/2000/svg" style="vertical-align:-0.2064009pt" id="M2" height="8.8423pt" version="1.1" viewBox="-0.0498162 -8.6359 9.35121 8.8423" width="9.35121pt"><g transform="matrix(.013,0,0,-0.013,0,0)"><path id="g113-87" d="M697 650H468L461 623L492 619C539 613 547 605 518 546C481 471 367 264 278 116H276C239 278 197 500 186 567C180 604 185 613 226 619L252 623L260 650H24L17 623C78 617 92 613 108 533L216 -11H247C365 200 515 462 560 529C616 612 624 615 689 623L697 650Z"/></g></svg>max values were identified as 1.879 mg/ml and 149.6 U, respectively. The potential application of the enzyme for coffee processing was studied, and it is found that complete removal of mucilage from coffee beans within 24 hours of treatment indicates the potential application in coffee processing.

Oliyad Jeilu Oumer

Dawit Abate

Characterization of Pectinase from Bacillus subtilis Strain Btk 27 and Its Potential Application in Removal of Mucilage from Coffee Beans

Cellulase is a family of at least three groups of enzymes that participate in the sequential hydrolysis of cellulose. Recombinant expression of cellulases might allow reducing their production times and increasing the low proteins concentrations obtained with filamentous fungi. In this study, we describe the production of Trichoderma reesei cellobiohydrolase II (CBHII) in a native strain of Wickerhamomyces anomalus. Recombinant CBHII was expressed in W. anomalus 54-A reaching enzyme activity values of up to 14.5 U L−1. The enzyme extract showed optimum pH and temperature of 5.0–6.0 and 40°C, respectively. Enzyme kinetic parameters (<svg xmlns:xlink="http://www.w3.org/1999/xlink" xmlns="http://www.w3.org/2000/svg" style="vertical-align:-3.1815pt" id="M1" height="11.8174pt" version="1.1" viewBox="-0.0498162 -8.6359 18.9508 11.8174" width="18.9508pt"><g transform="matrix(.013,0,0,-0.013,0,0)"><path id="g113-76" d="M743 650H503L496 622L527 618C563 613 564 603 532 573C449 495 371 431 323 392C301 374 272 355 246 346L280 522C297 609 300 614 379 622L385 650H135L129 622C209 614 215 609 198 522L124 133C106 39 99 35 23 28L17 0H271L277 28C193 35 192 39 208 133L239 316C264 328 280 325 303 288C368 183 435 90 502 0H652L659 28C602 34 584 43 543 94C495 154 403 283 347 369L574 554C634 603 659 612 735 624L743 650Z"/></g><g transform="matrix(.0091,0,0,-0.0091,9.23,3.132)"><path id="g50-78" d="M1006 650H829L492 147L357 650H174L166 618C270 609 275 601 247 502L194 315C158 189 136 127 122 96C104 56 80 37 18 32L12 0H256L263 32C181 40 176 51 183 98C188 132 197 179 226 281L299 541H301L433 6H458L809 551H811L753 141C739 41 734 39 650 32L642 0H917L923 32C836 39 827 41 839 142L887 535C896 610 901 612 997 618L1006 650Z"/></g></svg> of 2.73 mM and Vmax of 23.1 µM min−1) were between the ranges of values reported for other CBHII enzymes. Finally, the results showed that an enzymatic extract of W. anomalus 54-A carrying the recombinant T. reesei CBHII allows production of reducing sugars similar to that of a crude extract from cellulolytic fungi. These results show the first report on the use of W. anomalus as a host to produce recombinant proteins. In addition, recombinant T. reesei CBHII enzyme could potentially be used in the degradation of lignocellulosic residues to produce bioethanol, based on its pH and temperature activity profile.

Dennis J. D&#xed;az-Rinc&#xf3;n

Ivonne Duque

Erika Osorio

Alexander Rodr&#xed;guez-L&#xf3;pez

Angela Espejo-Mojica

Claudia M. Parra-Giraldo

Carlos J. Alm&#xe9;ciga-D&#xed;az

Balkys Quevedo-Hidalgo

Production of Recombinant Trichoderma reesei Cellobiohydrolase II in a New Expression System Based on Wickerhamomyces anomalus

The aim of the present study was to compare the in vitro inhibitory effects on the salivary and pancreatic &#x3b1;-amylases and the in vivo hypoglycemic actions of the hydrolysable tannin from Chinese natural gall and the condensed tannin from Acacia mearnsii. The human salivary &#x3b1;-amylase was more strongly inhibited by the hydrolysable than by the condensed tannin, with the concentrations for 50&#x25; inhibition (IC50) being 47.0 and 285.4&#x2009;&#x3bc;M, respectively. The inhibitory capacities of both tannins on the pancreatic &#x3b1;-amylase were also different, with IC50 values being 141.1&#x2009;&#x3bc;M for the hydrolysable tannin and 248.1&#x2009;&#x3bc;M for the condensed tannin. The kinetics of the inhibition presented complex patterns in that for both inhibitors more than one molecule can bind simultaneously to either the free enzyme of the substrate-complexed enzyme (parabolic mixed inhibition). Both tannins were able to inhibit the intestinal starch absorption. Inhibition by the hydrolysable tannin was concentration-dependent, with 53&#x25; inhibition at the dose of 58.8&#x2009;&#x3bc;mol/kg and 88&#x25; inhibition at the dose of 294&#x2009;&#x3bc;mol/kg. For the condensed tannin, inhibition was not substantially different for doses between 124.4&#x2009;&#x3bc;mol/kg (49&#x25;) and 620&#x2009;&#x3bc;mol/kg (57&#x25;). It can be concluded that both tannins, but especially the hydrolysable one, could be useful in controlling the postprandial glycemic levels in diabetes.

Camila Gabriel Kato

Geferson de Almeida Gon&#xe7;alves

Rosely Aparecida Peralta

Flavio Augusto Vicente Seixas

Anacharis Babeto de S&#xe1;-Nakanishi

L&#xed;via Bracht

Jurandir Fernando Comar

Adelar Bracht

Rosane Marina Peralta

Inhibition of &#x3b1;-Amylases by Condensed and Hydrolysable Tannins: Focus on Kinetics and Hypoglycemic Actions

The immobilization of cellulases could be an economical alternative for cost reduction of enzyme application. The derivatives obtained in the immobilization derivatives were evaluated in recycles of paper filter hydrolysis. The immobilization process showed that the enzyme recycles were influenced by the shape (drop or sheet) and type of the mixture. The enzyme was recycled 28 times for sheets <svg xmlns:xlink="http://www.w3.org/1999/xlink" xmlns="http://www.w3.org/2000/svg" style="vertical-align:-0.04979992pt" id="M1" height="11.4859pt" version="1.1" viewBox="-0.0498162 -11.4361 11.3069 11.4859" width="11.3069pt"><g transform="matrix(.013,0,0,-0.013,0,0)"><path id="g121-67" d="M517 162C503 123 484 88 467 68C445 42 417 34 341 34C291 34 256 34 237 47C219 59 213 81 213 128V317H308C395 317 402 311 415 240H444V431H415C403 364 398 356 307 356H213V584C213 613 215 616 246 616H322C394 616 421 609 435 587C448 566 458 544 467 502L496 506C493 557 488 625 488 650H42V622C120 616 128 612 128 523V125C128 43 120 35 29 28V0H511C520 31 540 125 546 158L517 162Z"/></g><g transform="matrix(.0091,0,0,-0.0091,7.384,-5.741)"><path id="g50-31" d="M310 541L304 571C290 586 211 619 185 610L80 76L131 52L310 541Z"/></g></svg> and 13 times for drops <svg xmlns:xlink="http://www.w3.org/1999/xlink" xmlns="http://www.w3.org/2000/svg" style="vertical-align:-0.04979992pt" id="M2" height="11.4859pt" version="1.1" viewBox="-0.0498162 -11.4361 11.5678 11.4859" width="11.5678pt"><g transform="matrix(.013,0,0,-0.013,0,0)"><path id="g121-64" d="M372 352C427 368 507 407 507 502C507 548 486 592 451 616C420 638 376 650 301 650H44V622C124 616 128 610 128 527V123C128 40 122 34 36 28V0H257C336 0 402 12 453 40C512 72 548 124 548 191C548 291 471 335 372 350V352ZM213 362V558C213 591 216 602 226 608C235 614 258 618 282 618C377 618 415 558 415 490C415 406 369 362 260 362H213ZM213 328H258C380 328 450 283 450 181C450 76 377 35 296 34C231 34 213 53 213 125V328Z"/></g><g transform="matrix(.0091,0,0,-0.0091,7.644,-5.741)"><use xlink:href="#g50-31"/></g></svg>. The derivative <svg xmlns:xlink="http://www.w3.org/1999/xlink" xmlns="http://www.w3.org/2000/svg" style="vertical-align:-0.04979992pt" id="M3" height="11.4859pt" version="1.1" viewBox="-0.0498162 -11.4361 11.3069 11.4859" width="11.3069pt"><g transform="matrix(.013,0,0,-0.013,0,0)"><use xlink:href="#g121-67"/></g><g transform="matrix(.0091,0,0,-0.0091,7.384,-5.741)"><use xlink:href="#g50-31"/></g></svg> showed the highest stability in the recycle obtaining 0.05 FPU/g, RA of 10%, and FPU Yield of 1.64 times, higher than FPU spent or Net FPU Yield of 5.3 times, saving more active enzymes. The derivative B showed stability in recycles reaching 0.15 FPU/g of derivative, yield of Recovered Activity (RA) of 25%, and FPU Yield of 1.57 times, higher than FPU spent on immobilization or Net PFU Yield of 2.81 times. The latex increased stability and resistance of the drops but did not improve the FPU/gram of derivative.

D. F. Silva

A. F. A. Carvalho

T. Y. Shinya

G. S. Mazali

R. D. Herculano

P. Oliva-Neto

Recycle of Immobilized Endocellulases in Different Conditions for Cellulose Hydrolysis

Laccases are multicopper oxidases that catalyze aromatic and nonaromatic compounds with concomitant reduction of molecular oxygen to water. They are of great interest due to their potential biotechnological applications. In this work we statistically improved culture media for recombinant GILCC1 (rGILCC1) laccase production at low scale from Ganoderma lucidum containing the construct pGAPZαA-GlucPost-Stop in Pichia pastoris. Temperature, pH stability, and kinetic parameter characterizations were determined by monitoring concentrate enzyme oxidation at different ABTS substrate concentrations. Plackett-Burman Design allowed improving enzyme activity from previous work 36.08-fold, with a laccase activity of 4.69 ± 0.39 UL−1 at 168 h of culture in a 500 mL shake-flask. Concentrated rGILCC1 remained stable between 10 and 50°C and retained a residual enzymatic activity greater than 70% at 60°C and 50% at 70°C. In regard to pH stability, concentrated enzyme was more stable at pH 4.0 ± 0.2 with a residual activity greater than 90%. The lowest residual activity greater than 55% was obtained at pH 10.0 ± 0.2. Furthermore, calculated apparent enzyme kinetic parameters were a <svg xmlns:xlink="http://www.w3.org/1999/xlink" xmlns="http://www.w3.org/2000/svg" style="vertical-align:-3.2957pt" id="M1" height="11.9316pt" version="1.1" viewBox="-0.0498162 -8.6359 24.0515 11.9316" width="24.0515pt"><g transform="matrix(.013,0,0,-0.013,0,0)"><path id="g113-87" d="M697 650H468L461 623L492 619C539 613 547 605 518 546C481 471 367 264 278 116H276C239 278 197 500 186 567C180 604 185 613 226 619L252 623L260 650H24L17 623C78 617 92 613 108 533L216 -11H247C365 200 515 462 560 529C616 612 624 615 689 623L697 650Z"/></g><g transform="matrix(.0091,0,0,-0.0091,7.332,3.132)"><path id="g58-107" d="M810 0V30C751 35 744 40 744 106V296C744 396 694 451 614 451C583 451 558 438 537 425C512 409 485 391 454 368C434 420 390 451 342 451C309 451 285 439 260 424C228 405 206 387 185 372V453C141 434 90 421 43 413V386C102 378 105 373 105 309V106C105 41 96 35 29 30V0H243V30C194 35 185 41 185 106V335C216 360 256 389 297 389C357 389 384 348 384 275V106C384 41 374 35 312 30V0H530V30C475 35 465 41 465 104V294C465 313 464 324 463 336C502 367 539 389 576 389C637 389 663 346 663 274V109C663 41 653 35 593 30V0H810Z"/></g><g transform="matrix(.0091,0,0,-0.0091,14.894,3.132)"><path id="g58-95" d="M442 39L431 68C420 63 406 57 394 57C377 57 359 71 359 118V301C359 353 349 393 314 424C291 443 261 451 230 451C171 438 107 401 79 381C59 366 46 355 46 340C46 316 72 295 91 295C106 295 117 303 122 319C131 350 139 371 152 384C163 397 178 404 197 404C246 404 281 365 281 293V278C260 262 186 232 124 213C69 194 41 161 41 112C41 47 92 -12 163 -12C195 -12 243 25 283 51C288 35 293 22 308 7C319 -4 339 -12 354 -12L442 39ZM281 87C262 68 226 51 199 51C169 51 129 74 129 126C129 162 151 181 190 198C211 208 259 229 281 240V87Z"/></g><g transform="matrix(.0091,0,0,-0.0091,18.98,3.132)"><path id="g58-118" d="M482 0V30C420 38 407 48 370 103L273 248C308 299 331 332 352 357C387 397 401 404 463 409V439H277V409C318 405 324 400 307 369C290 337 270 307 251 279L194 367C175 396 180 402 220 409V439H18V409C74 401 87 395 117 349L204 213C173 168 147 130 119 95C78 46 67 38 5 30V0H195V30C144 38 143 47 162 79C181 112 202 149 225 181L297 70C314 43 307 35 264 30V0H482Z"/></g></svg> of 6.87 × 10−5 mM s−1, with an apparent <svg xmlns:xlink="http://www.w3.org/1999/xlink" xmlns="http://www.w3.org/2000/svg" style="vertical-align:-3.291101pt" id="M2" height="11.927pt" version="1.1" viewBox="-0.0498162 -8.6359 17.2153 11.927" width="17.2153pt"><g transform="matrix(.013,0,0,-0.013,0,0)"><path id="g113-76" d="M743 650H503L496 622L527 618C563 613 564 603 532 573C449 495 371 431 323 392C301 374 272 355 246 346L280 522C297 609 300 614 379 622L385 650H135L129 622C209 614 215 609 198 522L124 133C106 39 99 35 23 28L17 0H271L277 28C193 35 192 39 208 133L239 316C264 328 280 325 303 288C368 183 435 90 502 0H652L659 28C602 34 584 43 543 94C495 154 403 283 347 369L574 554C634 603 659 612 735 624L743 650Z"/></g><g transform="matrix(.0091,0,0,-0.0091,9.23,3.132)"><path id="g50-110" d="M781 91L765 118C733 86 702 70 693 70C686 70 685 77 692 106L738 297C772 439 736 451 712 451S675 445 647 431C605 410 526 356 452 257H450L459 294C491 426 459 451 429 451C403 451 386 445 361 431C312 404 239 343 170 253H168L188 333C208 411 205 451 175 451C148 451 85 417 24 346L39 318C57 338 100 374 112 374C120 374 122 372 115 339C91 226 62 112 28 -6L37 -12C59 -4 87 3 114 6C125 68 142 127 154 168C184 227 316 383 372 383C397 383 396 366 381 289C362 192 338 92 312 -6L317 -12C339 -5 367 3 396 6L434 170C468 234 598 383 650 383C668 383 676 369 664 319L609 90C593 23 600 -12 629 -12C654 -12 722 23 781 91Z"/></g></svg> of 5.36 × 10−2 mM. Collectively, these important stability findings open possibilities for applications involving a wide pH and temperature ranges.

&#xc1;ngela M. Cardozo-Bernal

Aura M. Pedroza-Rodr&#xed;guez

Claudia L. Cuervo-Pati&#xf1;o

Plackett-Burman Design for rGILCC1 Laccase Activity Enhancement in Pichia pastoris: Concentrated Enzyme Kinetic Characterization

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Enzyme Research publishes original research articles as well as review articles that advance our understanding of enzymes and the way in which they work, both in vivo and in vitro.

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Enzyme Research ceased publication in 2019 and is no longer accepting submissions.

description

Enzyme Research publishes original research articles as well as review articles that advance our understanding of enzymes and the way in which they work, both in vivo and in vitro.

Enzyme Research