A sensitive, accurate, and high-throughput gluco- oligosaccharide oxidase based HRP colorimetric method for lytic polysaccharide monooxygenase activity assay


 Background

The AA9 (auxiliary activities) family of lytic polysaccharide monooxygenases (AA9 LPMOs) are ubiquitous and diverse group of enzymes amongst the fungal kingdom. They catalyze the oxidative cleavage of glycosidic bonds in lignocellulose and exhibit great potential for secondary biorefinery applications. Screening of AA9 LPMOs for desirable properties is crucial for biorefinery industrial applications. However, robust, high-throughput and direct method for AA9 LPMO activity assay, which is prerequisite for screening of LPMOs with excellent properties, is still lacking. Here, we have described a gluco-oligosaccharide oxidase (GOOX) based horseradish peroxidase (HRP) colorimetric method for AA9 LPMO activity assay.
Results

We cloned and expressed a GOOX gene from Sarocladium strictum in Trichoderma reesei, purified the recombinant SsGOOX, validated its properties, and set up a SsGOOX based HRP colorimetric method for cellobiose concentration assay. Then we expressed two AA9 LPMOs from Thielavia terrestris, TtAA9F and TtAA9G in T. reesei, purified the recombinant proteins, and analyzed their product profiles and regioselectivity towards phosphoric acid swollen cellulose (PASC). TtAA9F was characterized as a C1 type (class 1) LPMO, while TtAA9G was characterized as a C4 type (class 2) LPMO. Finally, the SsGOOX based HRP colorimetric method was used to quantify the total concentration of reducing lytic products from LPMO reaction, and consequently, the activities of both C1 and C4 types of LPMOs were analyzed. These LPMOs could be effectively analyzed with limits of detection (LoDs) lower than 30 nmol/L, and standard curves between A515 and LPMO concentrations with determination coefficients greater than 0.994 were obtained.
Conclusions

A novel, sensitive and accurate assay method that directly targets the main activity of both C1 and C4 type of AA9 LPMOs was established. This method is easy to use and could be performed on a microtiter plate ready for high-throughput screening of AA9 LPMOs with high properties.

The prerequisite for large scale screening of LPMOs with high enzymatic activities is to establish 74 a convenient, sensitive, and high-throughput assay method. AA9 LPMOs catalyze the cleavage of 75 crystalline cellulose, releasing a small amount of reducing sugars which although detected by 76 traditional methods such as 3,5-dinitrosalicylic acid (DNS) method, yet not considered sensitive 77 enough. In order to solve this problem, advanced quantitative analyzing methods based on 78 ingenious equipment, such as high performance anion exchange chromatography (HPAEC), 79 Ultra-high performance liquid chromatography (UHPLC), or chromatography equipment 80 combined with a mass spectrum, have been established [8][9][10]. Though, these methods are 81 accurate and can reveal detailed insights of the product formation, but require expensive 82 instruments, are generally time-consuming and labor-intensive [11]. In this context, some new 83 methods have been set up to determine the activity of LPMOs. When LPMOs are incubated with 84 an external electron donor such as ascorbic acid in the absence of cellulose substrate, hydrogen 85 peroxide is produced linearly depending upon the concentration of LPMOs. Thus, a fast and easy 86 method for LPMO activity assay was established by quantifying the formation of hydrogen 87 peroxide with Amplex Red/horseradish peroxidase reaction [12]. In another study, 88 2,6-dimethoxyphenol (2,6-DMP) or hydrocoerulignone were used as chromogenic substrates to 89 assay LPMO activity [11,13]. 2,6-DMP or hydrocoerulignone are converted to colored product 90 HPR colorimetric assay was applied to monitor the production of H2O2, with 4-amino-antipyrine 156 (4-AAP) and 3,5-dichloro-2-hydroxybenzenesulfonic acid (DCHBS) as the chromogenic 157 substrates. Upon oxidation of cellobiose via SsGOOX, a stoichiometric amount of H2O2 was 158 produced, which was then used by HRP to convert AAP and DCHBS into a pink substance with 159 maximum absorbance at 515 nm (A515). Concentrations of the chromogenic substrate 4-AAP and 160 DCHBS used in the HRP colorimetric assay were determined as 0.1 mM and 2.0 mM, respectively, 161 based on the experiments of A515 dependence on 4-AAP and DCBHS concentrations (Fig. S1).
The initial parameters were set referring to the parameters determined by Vuong    SsGOOX still exhibits about 70% of the maximal activity at 30℃, for ease of handling, in the 197 following assays, the reaction was carried out at room temperature. However, the recombinant 198 SsGOOX has an optimum pH of 9.0 as compared to native GOOX of Acremonium strictum T1 199 which has an optimum pH of 10.0 [16]. Moreover, the activity of the recombinant SsGOOX is less 200 affected. As we found that at pH values 8.0 and 7.0, the recombinant SsGOOX still retains 97% 201 and 85% activity, respectively, while at pH10.0 it has only 71% activity. We propose that 202 difference between pH effects in our experiment and that of Lin et al might be due to different glycosylation pattern of proteins in T. reesei and Acremonium strictum T1. As, SsGOOX is 204 coupled with HRP in the detection of cello-oligosaccharides, therefore, pH value in the reaction 205 system was controlled at 7.0 with phosphate buffer in the following assays. With respect to 206 thermo-stability, SsGOOX is rather stable at 50 and 55℃, which means that it retains almost 207 100% activity when incubated for an hour at these temperatures. However, higher temperature, i.e., 208 60℃ can result in its quick deactivation (Fig. S3). 209 Detection of reducing sugar 210 We propose that SsGOOX is highly effective in oxidizing oligosaccharides under appropriate 211 reaction conditions. Indeed, in our reaction condition, up to 0.32 mM cellobiose has been 212 completely oxidized within 25 min, as shown in Fig. 3. Initially, when cellobiose concentration 213 was low, A515 increased gradually during the first 25 min and reached to a maximum value which 214 remained constant thereafter, indicating complete exhaustion of cellobiose. Therefore, the duration 215 of assay based on the concentrations of cellobiose or oligosaccharides was determined as 25 min. 216 When the initial cellobiose concentration was high (for example, 0.32 mM), A515 value decreased 217 over time slightly after reaching the peak value possibly due to further oxidation of the pink 218 substance by excessive H2O2. To observe this phenomenon, we added H2O2 manually and tested 219 its effect on the stability of the pink substance and witnessed slight fading of the color (data not 220 shown).  We determined the detection range of cellobiose concentration for SsGOOX based assay. At low 230 cellobiose concentrations (0-0.08 mM), A515 value was proportional to cellobiose concentration 231 and a standard cure was obtained with a R 2 value of 0.996 (Fig. 4). In a broader cellobiose 232 concentration range, the slope of the curve decreased at high cellobiose concentration region, 233 which might be due to the absorbance property of the pink substance, substrate inhibition, or 234 further oxidation of the pink substance by excessive H2O2. 235 abundance of peak 1011 exceeds 20% of that of peak 1013 in the product of TtAA9G, while this 283 peak is negligible in the product of TtAA9F. Therefore, peak 1011 most probably corresponds to 284 sodium adduct of ketoaldose derivate of hexasaccharide rather than sodium adduct of lactone 285 derivate of hexasaccharide, because the hydrated gemdiol derivates generated by C4 oxidation are 286 more easily dehydrated as a result of sample preparation for MALDI-TOF MS analysis than the 287 aldonic acid derivates generated by C1 oxidation. In addition, peak 1029 and peak 1051 that 288 probably correspond to sodium adducts of aldonic acid derivates are very small. Therefore, 289 recombinant TtAA9G is most probably a C4 LPMO which oxidatively cleaved PASC, releasing 290 native cello-oligosaccharides and cello-oligosaccharide gemdiols.

295
We further identified native cello-oligosaccharides and their oxidative derivates released from the 296 reactions of TtAA9F and TtAA9G towards PASC by H PAE C -PAD (Fig. 7). The samples for 297 H PAE C -PAD assay were prepared in three different modes: i) sample prepared with normal 298 LPMO reaction using Asc as electron donor (the middle blue line); ii) sample prepared with 299 LPMO reaction without Asc addition (the lower green line); iii) sample prepared with normal 300 LPMO reaction using Asc as electron donor, and the lytic products further oxidized by SsGOOX 301 (the upper purple line). The products generated by TtAA9F were detected as a series of native 302 cello-oligosaccharides with degree of polymerization (DP) from 4 to 6 and aldonic acid derivates 303 of cello-oligosaccharides with DP from 1 to 6. The cello-oligosaccharides were eluted at retention 304 times ranging from 7.6 min to 9.5 min, while the aldonic acid derivates were eluted at retention 305 times ranging from 7.3 min to 13.4 min. In control reaction which lacked Asc addition, there was 306 no cello-oligosaccharide or oxidized derivate released. When the cello-oligosaccharides and 307 oxidized derivates generated by LPMO reaction were further oxidized by SsGOOX, the peaks 308 corresponding to native cello-oligosaccharides disappeared, while those corresponding to 309 C1-oxidized derivate of cello-oligosaccharide remained and slightly increased in peak area (Fig.  310 7A). Therefore, the peak profile of HPAEC-PAD analysis, along with the alteration of the peak 311 profile after SsGOOX oxidation strongly supported that TtAA9F was a C1 type LPMO. 312 As for the products generated by TtAA9G, the peaks corresponding to native 313 cello-oligosaccharides from DP1-DP6 were eluted at retention times ranging from 3.6 min to 9.6 min, while the peaks corresponding to C4-oxidized oligosaccharide derivates emerged at retention 315 times ranging from 21.3 min to 23.3 min. There was no obvious formation of aldonic acid derivate 316 of cello-oligosaccharide. When the products generated by TtAA9G were further oxidized by 317 SsGOOX, the native cello-oligosaccharides were transformed into aldonic acid derivates, as 318 indicated by the disappearance or emergence of the corresponding peaks, while the C4-oxidized 319 cello-oligosaccharide derivates were transformed into 4-ketoaldonic acids or gemidiol aldonic 320 acids and the retention time of the corresponding peaks increased significantly (Fig. 7B).

Removal of residual Asc with ascorbic acid oxidase 332
Asc is a commonly used external electron donor for LPMO reaction. However, as previously 333 reported, reducing agents such as Asc interfere with the HRP colorimetric assay [22]. In our 334 LPMO activity assay method, the HRP colorimetric assay was applied to detect H2O2 generated 335 along with the oxidation of cello-oligosaccharides or derivates by SsGOOX. Therefore, we first evaluated the effect of Asc on SsGOOX based HRP colorimetric assay and found a way to 337 eliminate this effect. As shown in Fig. 8, the existence of 1 mM Asc in the cellobiose detection 338 system indeed affected the detection results significantly. Fortunately, the interference of Asc 339 could be easily eliminated by ascorbic acid oxidase. With the addition of 5 U/ml or above ascorbic 340 acid oxidase and reaction for 1 min, Asc in the cellobiose detection system was efficiently 341 oxidized and the detection results were similar to that of control reaction without Asc addition (Fig.  342   8). To ensure the complete oxidization of residual Asc, in the following assays, more ascorbic acid 343 oxidase (20 U/ml) was used and the reaction time for Asc oxidization was prolonged to 5 min. respectively (Fig. 9). Activities of both the C1 type and C4 type LPMOs can be analyzed through 360 GOOX based assay HRP colorimetric method, and more importantly, this method directly targets 361 the main activity of LPMOs. 362 To determine the lower limit of detection (LoD), we assayed the activities of both LPMOs with a 363 series of low concentrations, and found the lowest detectable concentrations of 15 nM for TtAA9F 364 and TtAA9G (Fig. 9C and 9D). Therefore, the A515 readings with deduction of the blank average Where, LoB was calculated according to Eq. (4). 371 (4) 372 The LoBs (in A515 reading and with deduction of the average blank A515 reading) for TtAA9F and 373 TtAA9G were calculated as 0.000735 and 0.000713, respectively. The LoDs (in A515 reading) for 374 TtAA9F (C1 type) and TtAA9G were 0.00140 and 0.00162, respectively. The LoDs in LPMO 375 concentration for TtAA9F and TtAA9G were calculated as 28.6 nM 27.9 nM, respectively, 376 according to the standard curves for LPMO activity analysis ( Fig. 9A and 9B). The purified 377 TtAA9F and TtAA9G samples were respectively diluted to concentrations of 28.6 nM and 27.9 378 nM. Each dilution was independently repeated for 60 times, and the diluted samples were assayed 379 with our method and compared with the corresponding LoBs. The results revealed that all the 380 assayed values were higher than the LoBs (Fig. 9E and 9F). Therefore, the LoDs of C1 type 381 LPMO and C4 type LPMO in the SsGOOX based HRP colorimetric assay were verified as 28.6 382 nM and 27.9 nM, respectively. There is a noteworthy difference between the ratios of the total cello-oligosaccharides or the 394 derivates to the reducing cello-oligosaccharides or the derivates released by C1-type LPMOs and 395 C4-type LPMOs, i.e., both geminal diols and native cello-oligosaccharides generated by C4 396 LPMOs bear a reducing end, while only native cello-oligosaccharides generated by C1 LPMOs 397 bear a reducing end. Therefore, in principle, in C4 LPMO activity assay with our method, all the 398 soluble products released can be detected, while in C1 LPMO activity assay, only native 399 cello-oligosaccharides can be detected, leading to the underestimation of C1 LPMO activity. 400 However, the underestimation of C1 LPMO activity can be corrected with the ratio of the amount 401 of total soluble lytic products to the amount of native soluble cello-oligosaccharide released by C1-LPMO, which was designated as T/R ratio in this work and was estimated as 4.75 according to 403 calculation of the peak areas in Fig. 7A. Although this coefficient may not be applicable to other 404 C1 LPMOs, however it could provide a reference, or this coefficient may be independently 405 determined for other C1 LPMOs. 406 In a batch enzyme reaction, initially the reaction speed is highest and known as "initial reaction 407 speed" which remains constant for a considerable period and then the reaction speed starts 408 declining due to consumption of the substrate, deactivation of the enzyme, or accumulation of the 409 inhibitors. To obtain more accurate analysis, the "initial reaction time" for LPMOs during which 410 the reaction speed remains constant should be determined. For TtAA9F, when the amount of 411 enzyme used was 3.6 µM, the initial reaction time was 30 min during which the reducing 412 cello-oligosaccharide concentration increased linearly over time. As for TtAA9G, the initial 413 reaction time was also 30 min when the amount of enzyme used was 6.4 µM (Fig. 10). Therefore, 414 we assume that a 30 min reaction time is appropriate for LPMO activity assay. 415 Based on the analysis results, we provided a definition of LPMO activity, that is, one unit of 416 LPMO activity was defined as the amount of enzyme that released one nmol of 417 cello-oligosaccharides and derivates in one minute. According to this definition, the specific 418 activity of TtAA9G was determined as 121.6 U/mg, while the specific activity of TtAA9F was 419

425
LPMO activity assay protocol 426 Based on the above results, we suggest the following assay protocol for testing LPMO activity. 427 Step 1: Set up the LPMO reaction system of 196 µl in 1.5 ml Eppendorf tubes which consist of 428 100 µl PA SC suspension (with concentration of 90 g/L for C1 LPMO activity assay, or 30 g/L for 429 C4 LPMO activity assay), 10 µl LPMO solution, and 76 µl 100 mM phosphorate buffer (pH7.5). 430 Set a reaction system with 10 µl pure water substituting for 10 µl LPMO solution as control. 431 Pre-incubate the reaction mixture at 50℃ for 3 min and start the reaction by adding 10 µl 20 mM 432 freshly prepared Asc and incubate at 50℃ for 30 min. 433 Step 2: Place the reaction mixture immediately on ice after removing it from the water bath, add 4 434 µl ascorbate oxidase solution (1000 U/ml) into the reaction mixture, and vortex for 5 minute to 435 remove Asc. Centrifuge the reaction mixture on a mini centrifuge at 12,000 r/min for 5 min and 436 transfer the supernatant to fresh tubes. 437 Step 3: Prepare the SsGOOX based HRP colorimetric assay on a 96-well microtiter plate. The Step 4: Incubate the 96-well plate at room temperature for 25 min and then record A515 on a 443 microtiter plate reader. 444 Step 5: Calculate the total amount of reducing oligosaccharides or derivates released according to a pre-established A515-cellubiose concentration standard curve with the SsGOOX based HRP 446 colorimetric assay method (for example, the insert of Fig. 4 in this work). For C4 LPMO activity 447 assay, the amount of total oligosaccharides or derivates equals the total amount of reducing 448 oligosaccharides or derivates, while for C1 LPMO, the amount of total oligosaccharides or 449 derivates equals the amount of reducing oligosaccharides times the T/R ratio. This ratio was The second approach utilizes the side reaction activities which generate H2O2 or catalyze the 461 oxidation of chromogenic substrates such as 2,6-DMP or rPHP, and analyze the LPMO activity 462 using a colorimetric method [11,12,14]. Although, this approach is considered potentially 463 high-throughput but the assayed side reaction activities may not accurately correlate with the main 464 activity that oxidatively cleaves the cellulosic substrate. Our work sets up a GOOX based HRP 465 colorimetric method for LPMO activity assay, which is accurate, sensitive, potentially 466 high-throughput, and more importantly it directly targets the main activity of LPMOs. targeting the reducing sugars such as DNS method can be used for the assay of AA9 LPMO 484 activity. However, the amount of reducing sugars released by AA9 LPMO is relatively much 485 smaller than that released by cellulase, therefore the sensitivity of DNS method is inadequate for 486 an accurate assay. In contrast to the DNS method, the GOOX based HRP colorimetric method is 487 far more sensitive in the detection of reducing sugars, and thus exhibits great potential for accurate 488 assay of reducing sugars released by LPMOs. We expressed a S. strictum GOOX gene in T. reesei by using the strong constitutive pdc promoter, and obtained purified recombinant SsGOOX [29]. 490 Albeit with slight differences, the properties of recombinant SsGOOX are by and large similar to 491 those of native or P. pastoris expressed SsGOOX[18]. The purified recombinant SsGOOX was 492 used for cellobiose assay along with the HRP colorimetric system and an A515-cellobiose 493 concentration standard curve was obtained. The standard curve was comparable to that of Ferrari 494 et al [27]. In order to avoid complexity and keep the operational simplicity we used cellobiose to 495 establish the standard curve. Actually, SsGOOX has broad substrate specificity and catalyze the 496 oxidation of cello-oligosaccharides with different DP with kinetic parameters comparable to that 497 of cellobiose [17]. This broad substrate specificity could also be verified with HPAEC-PAD 498 analysis (as performed in this work, Figure 7). Therefore, we suggest that SsGOOX based assay 499 method can detect all the soluble cello-oligosaccharides or derivates which bear a reducing 500 aldehyde group and can be used for cellulases or AA9 LPMO activity assay, where the units of 501 activity are generally defined according to the rate of oligosaccharide releasing. 502 For AA9 LPMO activity assay, Asc is frequently used as the external electron dono [24,26,30]. 503 However, as a reducing reagent, Asc interferes in the HRP colorimetric detection results. Thus, we 504 use ascobate oxidase to remove residual Asc, which perfectly performed i.e., with the addition of 505 small amount of ascobate oxidase (5 U/ml) and short reaction time (within 1 min), the interference 506 of Asc on HRP reaction is completely eliminated (Fig. 8). In addition, efficient removal of Asc by 507 ascorbic acid oxidase results in fast termination of the LPMO reaction. Therefore, ascorbate 508 oxidase has two functions in our SsGOOX based HRP colorimetric assay, elimination of the 509 interference of Asc and fast termination of the LPMO reaction. 510 Based on sequence alignment analysis, we selected two AA9 LPMOs from T. terrestris for enzyme activity assay. One is TtAA9F, which is determined as C1 type LPMO through 512 MALDI-TOF analysis and HPAEC-PAD analysis of the cleavage products released from PASC; 513 the other one is TtAA9G, which is determined as C4 type LPMO. It is noteworthy that GOOX can 514 also play an important role in regioselectivity analysis of LPMOs. The oxidative cleavage 515 products of LPMO reaction when further treated with SsGOOX, the peak profile of HPAEC-PAD 516 analysis changed significantly, and the changes were different for C1 oxidized products and C4 517 oxidized products (Fig. 7). By detecting the concentration of reducing cello-oligosaccharides and 518 derivates, the SsGOOX based HRP colorimetric method can detect as low as 28.6 nmol/L of C1 519 type LPMO (for TtAA9F, 28.6 nmol/L equals to 0.929 mg/L) and 27.9 nmol of C4 type LPMO 520 (for TtAA9G, 27.9 nmol/L equals to 0.726 mg/L). The standard curves of A515 versus LPMO 521 concentration possess coefficients of determination greater than 0.994. Thus, we suggest that the 522 SsGOOX based HRP colorimetric method for LPMO acitvity assay presented here has the 523 following advantages: i) after LPMO reaction, the whole assay process can be conveniently 524 operated conveniently on a microtiter plate and the assay results can be recorded with a microtiter 525 plate reader, therefore it is a high-throughput assay method; ii) the assay targets directly at the 526 main activity of LPMOs rather than the side activities; iii) activities of both the C1 and C4 type 527 LPMOs can be assayed; iv) it is sensitive and accurate; v) except a microplate reader, no other 528 expensive instrument is needed, which portrays it as a cheap and ready to use method. 529 In the assay process, three tool enzymes are used, HRP, ascorbate oxidase, and SsGOOX. HRP 530 and ascorbate oxidase are both commercially available, while SsGOOX is prepared by ourselves. 531 The HRP colorimetric system is a convenient and commonly used tool for the detection of H2O2. 532 Ascorbate oxidase is added right after the LPMOs reaction which removes Asc residue and simultaneously terminates the LPMOs reaction, thus the step of Asc removal does not add any 534 complexity to the assay process. SsGOOX is added along with the HRP colorimetric mixture, 535 therefore the application of SsGOOX also does not add any complexity. If SsGOOX or other 536 comparable GOOXs are commercially available, it will be convenient to use this GOOX based 537 LPMO activity assay method. 538 For C1 type LPMO activity assay, there might exist an underestimation of their activity because 539 the aldonic acids released cannot be detected by the SsGOOX based HRP colorimetric assay. 540 Therefore, a ratio of the amount of total soluble lytic products to the amount of native 541 cello-oligosaccharides (T/R ratio) is introduced to compensate this underestimation. Sampling the 542 product of LPMO reaction for activity assay is carried out in the initial speed region, i.e., within 543 30 min of the reaction (Fig. 10). In the initial speed region of an enzymatic reaction, the reaction 544 speed is generally assumed to be constant, i.e. the formation of total oligosaccharides and 545 derivates is linearly correlated with the reaction time. On the other hand, the formation of reducing 546 oligosaccharides also has linear correlation with the reaction time as reflected in Fig. 10. Therefore, 547 the ratio of the amount of total oligosaccharides and derivates to the amount of reducing 548 oligosaccharides (T/R ratio) should be constant in the initial speed region which was determined 549 as 4.75 according to the HPAEC-PAD analysis results. 550

551
We have established a sensitive, robust, and direct gluco-oligosaccharide oxidase based HRP 552 colorimetric assay for the detection of LPMO activity. This method can detect the activities of 553 both the C1 and C4 type LPMOs at concentrations as low as 28.6 nmol/L and 27.9 nmol/L, respectively, targeting their main activities with a standard curve of R 2 value greater than 0.994. 555 The assay process can be operated on microtiter plates ready to use for high-throughput screening, 556 and therefore has potential applications in large scale screening of LPMOs with high activity or in 557 screening procedures for directed evolution of LPMOs. Based on the assay method, we have 558 proposed a definition of LPMO activity unit that accurately reflects the main activities of LPMOs 559 and can be used for the comparison of LPMOs of different origins. 560

561
Strains, plasmids, and cultivation conditions 562 Escherichia coli DH5α' was used for plasmid construction and propagation, and was cultivated in 563 LB medium (1% tryptone, 0.5% yeast extract, 1% NaCl, supplemented with 100 μg/mL ampicillin 564 if necessary). Trichoderma reesei QM9414 strain (ATCC 26921) was used as a host for the 565 heterologous expression of the recombinant proteins. The cultivation conditions of T. reesei were 566 followed as described previously [29]. Thielavia terrestris (ATCC 38088) was cultivated on PDA 567 agar at 45℃. Plasmid pUC19 was used for the construction of vectors or expression cassettes.

Construction of expression vector and Protoplast transformation 582
The genomic DNA of T. reesei and T. terrestris was extracted using the fungal genomic DNA 583 extracting kit (Sangon Biotech, Shanghai). The promoter and the terminator sequences of the pdc 584 gene were PCR amplified from the genomic DNA of T. reesei QM9414 as template. The coding 585 sequence of GOOX from Sarocladium strictum was codon-optimized for expression in T. reesei 586 (Table S2)     The relationship between A515 and cellobiose concentration in SsGOOX based HRP colorimetric method for cellobiose concentration assay. The standard curve of low cellobiose concentration range is shown in the insert.    Elimination effect of ascorbate oxidase on residual Asc in SsGOOX based HRP colorimetric aasay. The reaction time for ascorbic acid oxidation was 1 min, the cellobiose concentration for detection was set at 0.1 mM, and the absorbance without ascorbic acid addition was de ned as 100%. Detection of the LPMO activity with the SsGOOX based HRP colorimetric assay method. AThe A515concentration curve of TtAA9F, a linear relation was found in the range 0-3.6 μM; BThe A515concentration curve of TtAA9G, the linear range was 0-6.4 μM; CComparison of the raw assay readings of 12 blanks and 12 independently diluted samples containing 15 nM TtAA9F; DComparison of the raw assay readings of the blanks and the 15 nM TtAA9G samples; EComparison of the LoB and 60 independently assayed results of 28.6 nM TtAA9F samples; FComparison of the LoB and 60 independently assayed results of 27.9 nM TtAA9G at samples.

Figure 10
Determination of the initial speed range for LPMO activity assay. A: TtAA9F; B: TtAA9G.

Supplementary Files
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