Characterization of amylase, lipase and xylanase produced by actinobacteria cultivated in licuri [ Syagrus coronata - (Martius) Beccari] residues

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INTRODUCTION
The solid state fermentation (SSF) of fungi has been vastly proposed in the literature, mostly due to the simplicity (for example, agitation is not necessary and the cellular biomass is retained in the solid fraction which facilitates the downstream process) and to shorter time of cultivation (when compared to bacteria which have more complex cultivation conditions).However, the cultivation of actinobacteria is also capable of producing equally efficient multi-enzymatic extracts for multiple purposes and a great variety of metabolites, for example: acids, amino acids, vitamins, pigments (COSTA-GUTIERREZ et al., 2021), antibiotics and anticâncer drugs (JOSE, Aguiar et al. Revista de Biotecnologia & Ciência, v.12, e13183, 20232 MAHARSHI & JHA, 2021) and enzymes, such as, β-galactosidase (XU et al., 2011), cellulases (JOHN J. et al., 2022), chitinases (WANG et al., 2023), proteases (BOUBEKRI et al., 2022), xylanases (SOUZA et al., 2022) and others.Actinobacteria are, more commonly, identified as soil bacteria (ARAÚJO et al., 2020) and have been proposed, for example, for soil biorremediation (APARICIO et al. 2018) but they also have been cultivated in different agro-industrial residues -sugarcane bagasse and soil (RAIMONDO et al., 2020), corn straw (ZHAO et al., 2017), wheat bran, rice husk, rice bran and bagassae (KHANDEPARKAR & BHOSLE, 2006), wheat straw (BERROCAL et al., 2000), shrimp shell (WANG et al., 2023) and others -in order to obtain different metabolites.
It is important to mention that the number of published papers with actinobacteria is still smaller compared to other bacteria and fungi, but it has been improving with the knowledge that they can be valuable producers of metabolites besides some unique characteristics of biotechnological interest (JAIN et al., 2022).In this present work, two actinobacteria, previously isolated from a cave in the Northeast of Brazil, identified as: Arthrobacter polychromogenes CDPI-30 and Streptomyces violaceoruber CDPA-32 (BISPO, 2010) were studied for the production of amylase, lipase and xylanase.Recently, it was also reported their ability to produce lipase and pectinase by SSF with licuri residues and wheat bran as substrate (RODRIGUES et al., 2022).
Licuri [Syagrus coronata -(Martius) Beccari] is a palm tree native to the Caatinga biome located in the semiarid region of Brazil (SOUZA et al., 2020) and, in this work, two residues from the processing of its fruits to obtain oil (fruit shells and pressed cake) were used as substrate for solid state fermentation (SSF) of the two above mentioned actinobacteria.Licuri cake can contain, for example: 230 g/kg of protein, 174 g/kg of cellulose, 229 g/kg of hemicellulose, 58.5 % (w/w FAME) of polyunsaturated fatty acids and 65.1 g/kg of ashes (SILVA et al., 2022); considering the composition of the fruit shells it has been reported, for example: 30.0 g/kg of protein, 218.9 g/kg of cellulose, 327.6 g/kg of hemicellulose, 382.0 g/kg of lignin, 4.7 g/kg of lipids and 2.3 g/kg of ashes (MENEZES et al., 2016).These data confirms that the two residues are rich substrates for SSF.The proposal for the valorization of residues/waste into other products (such as enzymatic solutions) has scientific and industrial recognition of its importance and is in line with circular economy concept (LIU et al., 2021).
Therefore, this present work reports the obtainance of crude multi-enzymatic extracts (CME) containing activities of three enzymes of great industrial interest: amylases, used for example to obtain different types of starch for the food industry (TESTER, QI & KARKALAS, 2006); lipases, which can be applied, for example, in detergentes and soaps (BHARATHI & RAJALAKSHMI, 2009) and xylanases, applyied, for example, in the hydrolisys of lignocelulosic material for bioethanol production (JUTUTU & WU, 2012).A basic characterization of theses three enzymatic activities identified in the obtained CMEs -in relation to pH, temperature, kinetic parameters and stability -was also performed and it can contribute to the scarce data about these enzymes obtained from SSF with licuri residues and actinobacteria.In view of the diversity of enzymes produced by actinobacteria, it is important to highlight that they can also be a valid choice for SSF with agro-industrial residues along with fungi.Furthermore, CME can be an effective choice for enzyme application when it is not necessary to use pure solutions which requires extensive and expensive downstream steps.

Licuri residues
The residues of licuri (Syagrus coronata -(Martius) Beccari): shells (external part of the fruits which is removed to obtain the seeds) and pressed cake (obtained after pressing of the seeds to obtain vegetable oil), were purchased from the Cooperativa de Produção da Região de Piemonte da Diamantina (COOPES) (Capim Grosso, Bahia, Brazil).The procedures for pre-treatment of these residues (drying and oil removing with hexane) were described in detail elsewhere (AGUIAR et al., 2022).

Solid state fermentation and multi-enzymatic crude extract obtention
The protocol for solid state fermentation of A. polychromogenes CDPI-30 and S. violaceoruber CDPA-32 in 20 g of a mixture (substrate) 7:3 (w/w) of licuri cake and licuri shells was detailed described in Aguiar et al. (2022), humidity was set as 70 % (v/w) with water and nutrient solution.Incubation took place in a B.O.D. incubator (Eletrolab, EL101/1) at 28 °C / 12 days.After fermentation, the crude multi-enzymatic extracts (CME) of each strain were obtained by water extraction and centrifugation, as described previously (AGUIAR et al., 2022;RODRIGUES et al., 2022).The total protein content of each CME was determined by Bradford (1976) methodology with bovine albumin as a standard.

Determination of the enzymatic activities
The activities of amylase, CMCase, FPase, laccase, lipase, pectinase, tannase and xylanase were determined in triplicate by colorimetric methodologies and a spectrophotometer (2000UV, BEL) based on: Bonine (2001), Ghose and Bisaria (1987), Lu et al. (2013), Sharma et al. (2000), Umsza-Guez et al. (2011) andVasconcelos et al. (2013).For xylanase, pectinase, CMCase, FPase and amylase, the activities were based on the release of reducing sugars (expressed as xylose, D-galacturonic acid and glucose) determined by the DNS (3,5dinitrosalicyclic acid) methodology with the absorbance measured at 540 nm.For xylanase, the reaction occurred with 10 mg/mL of xylan from birchwood in sodium acetate buffer (50 mM / pH 5) at 50 °C / 10 min; for CMCase, the reaction occurred with CMC (carboxymethyl cellulose) in sodium citrate buffer (50 mM / pH 4.8) at 50 °C / 10 min; for FPase a piece of Whatman filter paper n° 1 measuring 0.5 cm x 3.0 cm in sodium citrate buffer (50 mM / pH 4.8) at 50 °C / 60 min was applied; for general amylases 10 mg/mL of soluble starch in potassium phosphate buffer (100 mM / pH 7.0) at 50 °C / 30 min were necessary, and, for pectinase, citric pectin in sodium acetate buffer (200 mM / pH 5) at 60 °C / 10 min was employed.For laccase, ABTS [2,] in sodium acetate buffer (0.05 M / pH 4) at 40 °C / 5 min was used and absorbance was measured at 420 nm.Tannase activity employed methyl gallate in sodium citrate buffer (50 mM / pH 5) at 33 °C / 5 min and the release of gallic acid was determined at 520 nm.For lipase, 7.95 µmol/mL of pNPP (4-nitrophenyl palmitate) in potassium phosphate buffer (50 mM / pH 7) was applied and the reaction occurred at 37 °C / 5 min, with the release of 4-nitrophenol measured at 410 nm.The enzymatic activities were expressed as unit of enzymatic activity per gram of solid substrate used in the fermentation (U/g).

Basic characterization of enzymes
The obtained CMEs were applied to the basic characterization of enzymes.Based on previous studies from literature regarding enzyme characterization (CASTILHA et al., 2017;SANJIVKUMAR et al., 2017;SANCHEZ et al., 2019), the activities of amylases, lipase and xylanase were selected to estimate the optimum pH and temperature, kinetic parameters and pH and temperature stability described as follows.The activities were expressed as relative activities: [(U/Uo)•100%] with Uo being equivalent to the highest activity value detected in the range evaluated.The activities initially detected in the CMEs (Tab. 1) were equivalent to the basic conditions of pH, T and substrate concentration.
To obtain different pH ranges, different buffer solutions were used: sodium citrate (50 mM) and sodium acetate (100 mM) at pH 3 -6, sodium phosphate (50 and 100 mM) at pH 7 and 8 and glycine-NaOH (100 mM) at pH 10.To determine the optimal temperatures, the range of 20 to 100 °C was selected.The substrate concentrations varied from 1.0 to 15 mg/mL of soluble starch and xylan and from 3.95 to 9.95 µmol/mL of pNPP.Additionally, for the estimative of the kinetic constants, Km and Vmax, the enzymatic activities obtained (V, U/g) were plotted against the substrate concentrations (S, mg/mL or µmol/mL), considering the linearization of Lineweaver-Burk -(1/v) x (1/S) -with at least four or five points.
The stability of each enzyme at different temperatures (35 -75 °C) and pHs (3.0 -10.0), was evaluated by estimating the deactivation constant (kd, 1/min) and the half-life (t1/2, min).For each enzyme, in triplicate, 0.6 mL of CME were added in 2.4 mL of standard buffer and incubation occurred in different temperatures for 120 min in a thermostatic bath (CE-160 -CINELAB).Aliquots of 400 µL were collected every 20 min and kept refrigerated (4 -5 °C) until the end of the incubation time to determine activity.Triplicates of each activity in the collected samples (U) presented deviations bellow 2,2 % of the mean values (data not presented).The residual activities [ln(U/Uo)], using the mean values of activities, were plotted against the incubation time (t, min), and the kd value was estimated by the slope of the linear adjustment.The t1/2 was calculated based on the adjusted linear regression when: U/Uo = 0.5 and expresses the time necessary under the evaluated conditions to reduce the activity to half of its initial value.

Enzymatic screening
The crude multi-enzymatic extracts (CMEs) obtained for the actinobacteria CDPI-30 and CDPA-32 were investigated for some enzymes of biotechnological importance as an initial screening step of the project (AGUIAR, 2020).According to the results (Table 1), the obtained CMEs presented a promising enzymatic profile for: amylase, CMCase, lipase, pectinase and xylanase.However, for FPase, laccase and tannase, the obtained values (< 1 U/g) were very close to the control conditions of the reactions, thus indicating that for these enzymes, it would be more appropriate to quantify their activities by another methodology of higher precision (e.g.chromatography).Based only on the highest activity values obtained (Tab.1), amylase, lipase and xylanase were selected for basic characterization as presented in the next topics.

Amlyase
Amylases from both strains, CDPI-30 and CDPA-32, showed optimum activities at pH 7 (Fig. 2a) and, within the pH range of 3.0 -6.0, the residual activities (U/Uo) were around 80 %; for pH 8, however, the profiles showed an abrupt decay.The temperature profiles obtained (Fig. 2b) were more differentiated between the strains, and for CDPI-30 the optimal activity was obtained at 50 °C and for CDPA-32 at 60 °C.It is also important to note that for CDPI-30, the residual activities were up to 80 % in the range of 40 -100 °C, and with CDPA-32 this range was restricted to 50 -80 °C. Figure 3 presents the estimated values for the deactivation constant (kd) and half-life (t1/2).For both strains, at pH 4, the loss of activity showed a simple linear decay, but above pH 5.0, activation (U/Uo > 100 %) was observed in the first 20 min, followed by a linear decay (Fig. 3.a and 3.c).Activation was maximal at pH 7.0 (optimum pH) with an increase in activities over the initial time of about 1.6 times for CDPI-30 and 2 times for CDPA-32.In addition, regarding t1/2, pH 8 stood out for CDPI-30, but for both strains, pH 6.0 and 7.0 also resulted in t1/2 above 300 min (Figs.3.a and 3.c).Considering thermal stability, activation was also observed in the first 20 min of incubation (except at 30 °C) with activity values up to 30 % higher.For CDPI-30, the highest t1/2 values (> 400 min) were estimated at 60 and 75 °C (Fig. 3.c) and for CDPA-32 at 45 and 75 °C (Fig. 3.d).
The substrate profiles of both strains were similar considering the evaluated range of starch concentration (Fig. 2.c), consequently, the estimated values for the kinetic parameters were also similar, as demonstrated in Table 2.

Lipases
For lipase activity, pH 8.0 was identified as optimal for both strains, but residual activities between 80 to 90 % were obtained at pH 10.0 for both strains and at pH 7.0 for CDPI-30 (Fig. 4.a).Between 20 to 40 °C it was possible to observe the best activities (U/Uo ≥ 80 %) for both strains, with optimum at 40 °C for CDPI-30 and 30 °C for CDPA-32 (Fig. 4b).
The substrate profiles for lipases were differentiated between the two strains analyzed (Fig. 4.c) and the estimated Vmax for CDPI-30 was about five times higher than with CDPA-32, with similar Km values (Tab.2).Regarding the stability of lipases, at pHs 8.0 and 10.0 (Fig. 5.a and 5.c) -consistent with the optimal pH conditions -the best stabilities were observed and, consequently, higher t1/2 values.However, as discussed before for amylases, only at pH 7.0, an effect of activation during the 120 min of incubation was observed with a linear increase up to 62 % for CDPI-30 and a smaller increase (47 %) for CDPA-32.For this reason, it was not possible to estimate the kd values at pH 7.0.Activations were also observed at pH 8 only during the first 20 min of incubation reaching 97 % for CDPI-30 and 34 % for CDPA-32.Considering the temperature, the highest t1/2 were estimated at 45 °C (Fig. 5.b and 5.d), a higher temperature than the optimum described above.

Xylanase
For the xylanase activities, the pH profiles (Fig. 6.a) were similar for both strains, with the pH range of 4.0 -6.0 resulting in the highest residual activities (U/Uo > 90 %) with pH 5.0 as the optimal pH, while pH 3.0 was less favorable for CDPI-30 xylanases.Concerning the temperature (Fig. 6.b), at 50 °C the maximum activities were observed, in addition, from 30 to 80 °C the relative activities were higher than 77 % for CDPI-30 and for CDPA-32 this range was reduced to 50 -70 °C.The substrate profiles, within the analyzed range, were similar for both strains (Fig. 6.c) and the estimated Km and Vmax were, consequently, also similar (Tab.3).
Regarding the pH stability (Fig. 7.a and 7.c), at pH 4, xylanase activities remained practically constant in the first 20 -40 min of incubation followed by a linear decay.However, at pH 5 the stability profiles presented a crescent linear behavior over 120 min of incubation, with final residual activities increased by about 23 -25 %; for this reason, it was not possible to determine kd for pH 5; this also suggests enzymatic activation which can be promoted at specific conditions.At temperatures of 45 and 60 °C (conditions around optimal temperatures), increases of 24 -35 % in the residual xylanase activities were also observed in the first 20 min of incubation in both extracts.

Enzymatic screening
In comparison to fungi, actinobacteria may require a longer period of incubation.In this present work, for example, incubation occurred for 12 days, however, different incubation period have been reported such as 6 days (AL-DHABI et al., 2020) and 21 days (BERROCAL et al.,200).To overcome a longer (possible) incubation time, the application of crude multienzymatic extracts (CMEs) obtained from actinobacteria (instead of fermentation or the application of purified enzyme solutions) can present an interesting and economic alternative.For that reason, it is necessary to obtain and characterize different CMEs since, as a crude form, enzymes may exhibit more complex behaviors than purified forms due to, for example, the presence of isoforms with different behaviors as observed for amlyases (GEBREMARIAM et al., 2013) or the presence of compounds capable of interfering in their thermostability (SILVA et al., 2018).MECs may have sufficient enzymatic activities for certain processes that do not require enzymatic solutions with a high degree of purity and should be better explored in order to value enzymatic solutions as potent alternatives to fermentation processes.Still, for some specific applications, it is necessary to provide purified enzymatic solutions, as mentioned in the safety evaluation of a phospholipase, produced by a genetically modified Streptomyces violaceoruber, for food application (LAMBRÉ et al, 2023).
Actinobacteria have been suggested and investigated less than fungi for solid state fermentations (SSF), but they also have been identified as good sources of several enzymes, as confirmed by the results presented in Cellulases (CMCase), exocellulases (FPase) and xylanases are important enzymes for the degradation of lignocellulosic material and, activities of 3.0 -8.5 U/g of CMCase and 1.5 -4.0 U/g of FPase, for example, have been reported in a study with four different actinobacteria (ZHAO et al., 2017).Another study reported activities around 400 to 5200 U/mL of xylanase by actinobacteria cultivated in synthetic medium and isolated from mangrove environment (SANJIVKUMAR et al., 2017).In addition, Sharma and Salwan (2018) suggested that actinobacterial xylanases may be more efficient than fungi xylanases due to the absence of co-secretion of cellulases.Laccase-like actinobacteria are also important because they participate in the specific degradation of lignin and Streptomyces cyaneus, for example, was able to reduce the lignin content of wheat straw by up to 19 % after 28 days (BERROCAL et al., 2000).Actinobacteria are also capable of degrading starch and pectic substances naturally found in different parts of plants.Amylase activity of 165.47 U/mL has been observed for Streptomyces sp.MBRC-82 (MANIVASAGAN et al., 2015) as well as a pectinase activity of 4857 U/g by Streptomyces sp.
RCK-SC cultivated in supplemented wheat bran (ANISHA & PREMA, 2008).In relation to tannases, which are necessary for tannin degradation, one study found that among 22 tannase-positive bacteria isolated from olive mil waste (a residue rich in polyphenols), six of them were actinobacteria (FEDERICI et al., 2011).Additionally, among other strains, Arthrobacter was identified by Li et al. (2016) with the best potential for degradation of phenols.Considering lipases, although they were not identified for three strains of Arthrobacter by Pathma and Sakthivel (2013) but they were produced by Actinomadura sediminis UTMC 2870 (in a complex medium) reaching 300 -1700 U/mL (IMANPARAST et al., 2018).Furthermore, five strains of Streptomyces have also been reported as lipase producers (HAMEDI et al., 2019).All these exemples, also the results presented in this work (Tab.1), can confirm that actinobacteria can be succesfully indicated as alternative sources (in relation to fungi) for industrial enzymes.To ilustrate more possibilities with both strains, CDPI-30 and CDPA-32, it has been recently reported CMEs containing, aproximatedly, 840 U/g and 15 U/g of lipases and pectinases, respectively, obtained from SSF with 67% (w/w) of humidity, 35 % (w/w) of licuri pressed cake, 35 % (w/w) of wheat bran and 30 % (w/w) of licuri shells under the same conditions of incubation as in this present work (RODRIGUES, et al., 2022).

Amlyase
In this present work (Tab. 2 and Fig. 1), amylases presented singular characteristics in comparison to other amylases from different actinobacteria, as observed, the optimum was restricted to pH 7.0 and not pH 8.0 as observed for other amylases.As an example, the α-amylase from Streptomyces sp.Al-Dhabi-46 presented optimal activities at pH 7.0 -8.0 and 40 °C and, under these conditions, this enzyme was able to maintain up to 80 % of its activity for a shorter period (30 -20 min) with lower t1/2 values (50 -55 min) (AL-DHABI et al., 2020).Another α-amylase from Streptomyces sp.D1 had the best activities in the pH range of 8.4 -10.0 and demonstrated low resistance to incubation at temperatures above 80 °C, but with 6 h of incubation at 65 and 70 °C, it was possible to maintain 65 -70 % of its initial activity (CHAKRABORTY et al., 2009).Longer periods of stability (to pH and temperature) were observe with amylases CDPA-32 and CDPI-30 (Tab.2), for example, at 75 °C it was possible to estimate t1/2 around 7.0 -8.0 h and, at pH 7.0, t1/2 was estimated around 5 -6 h; pH 8.0 resulted in a higher value of t1/2 (7 h) only for amylase CDPA-32.
Considering now the substrate for amylases in a CME from Arthrobacter sp.226, the maximum activity was obtained with 10 mg/mL of starch but, with 15 mg/mL, 80 % of residual activity was still observed (OTTONI et al., 2020); in this present work (Fig. 1c), it was not possible to observe a saturation point until 15 mg/mL.For a recombinant α-amylase from Pseudoalteromonas sp.2-3, the kinetic constants were estimated as: Vmax = 0.27 mg/(mL.min)and Km = 6.94 mg/mL (SANCHEZ et al., 2019) which are lower values in comparison to what was calculated in this work (Tab.3).It is also worth mentioning that in the two examples provided above, the conditions for determining amylase activity, especially temperature, were different from those applied for the amylases CDPA-32 and CDPI-30 since both actinobacteria exemplified are cold adapted.For that matter, it is necessary to investigate more amylases from actinobacteria so there can be more data available and, consequently, more possibilities for biotecnological application.Additionally, for the three selected enzymes in this presente work (amylase, lipases and xylanases), it is necessary to perform more detailed kinetic studies (with higher substrate concentration) so it is possible to know more about their biocatalitic performances.
The proposal of using crude multienzymatic extracts over purified enzymes has the advantage of reduced costs, but it is important to remember that the enzyme can present different properties in both forms.For example, the presence of isoforms with different behaviors can be observed with amylases (GEBREMARIAM et al., 2013), or maybe the presence of compounds capable of interfering in thermostability (SILVA et al., 2018).Amylases, for example, can bee applied to textile, paper or chemical industries without the necessity of purification as it is requested for food and drugs.

Lipase
As for to the optimum conditions observed for the lipases in this work (Fig. 4), a similar pH were observed with a lipase from Janibacter sp.R02 (around 7.0 and 8.0) (CASTILLA et al., 2017) and a partially purified lipase from Streptomyces sp.(pH = 7.0) (KUMAR et al., 2017).In regard to temperature, these two examples presented optimum at a higher temperature (80 °C) and a more similar conditions (40 °C), respectively.When considering pH 8.0, it was reported by Castilla et al. (2017) a reduction of around 20 % in thermophilic lipase activity after 1 h incubation in a wider temperature range (4 -90 °C).More than 80 % of lipase activities, from Streptomyces sp.
Activation was observed with the three inestigated enzymes (amylase, lipase and xylanase) during termal and pH stability determination.This effect has been reported for different enzymes, or maybe just ignore in a few cases since the activation can be quickly followed by the deactivation.Usually, with time, the enzyme activity tend to be lost due to unfolding of its 3D structure but, at certain temperature/pH/time, it is possible to observe an increase in activity (even for a brief period of time) and that is a complex behaviour that enzymes can present.
According to Bechtold and Panke (2012), the enzymatic deactivation kinetics can be quite complex with a few intermediate protein states.In a complementary way, the activation caused by a brief incubation under certain temperature/pH can be applied before the enzyme application in order to improve its performance (AGUIAR-OLIVEIRA & MAUGERI, 2011).In order to better understand the activation/deactivation of amylase, lipase and xylanase from CDPI-30 and CDPA-32, it is necessary to perform more stability/thermodynamic studies.Even so, the results obtained so far are a good indicative of interesting properties of the crude extracts and for the purified enzymes.
When analazing the Vmax values (Tab.3), for both lipases, it is possible to observe that lipase CDPA-32 could be more efficient (faster) than the lipase CDPI-30, considering the substrate pNPP.For the same substrate (pNPP), a purified lipase from Acinetobacter sp.AU07 (GURURAJ et al., 2015), presented a Vmax closer to the lipase from CDPA-32 (Tab.3) but with a Km up to 10 times smaller which could suggest a more effective kinetic (less substrate necessary to reach the same Vmax).There are few reports in the literature on kinetic studies for actinobacterial lipases and the estimated kinetic parameters were obtained with different substrates, such as: Km = 873 mg/mL with 4-methylumbelliferyl butyrate (CASTILLA et al., 2017) and Km = 6.4 mg/mL and Vmax = 21.0U/mg with pNP caproate (WANG et al., 2016).
In addition, besides the lipases produced by actinobacteria, interestingly, they also have the ability to grow MSU3 which presented peaks of 15 -20 % of activation in 30 and 90 min (SANJIVKUMAR et al., 2017).
With the intention to avoid expensive substrates (such as xylan), Danso and coworkers (2022) used wheat straw to cultivate Streptomyces sp.MS-S2 (isolated from the gut of termites) to obtain a crude extract containing xylanase and cellulase that was applied in the hydrolysis of the same agroindustrial residue to obtain reducing sugars for bioethanol production.This (and other similar works) is a good example to estimulate the studies and application of the strains CDPI-30 and CDPA-32 and their enzymes (crude or purified).

CONCLUSION
Actinobacteria are versatile for solid state fermentation (SSF), just as fungi can be, and their enzymes may present different characteristics than the "traditional" fungi enzymes, for this reason, it is important to promote more related studies with actinobacteria as a source for enzyme production through SSF.The results presented in this work, are good estimates for the behavior of amylases, lipases and xylanases from A. polychromogenes CDPI-30 and S. violaceoruber CDPA-32 and provided important information since there is a need for more current data on these enzymes from actinobacteria and SSF.As crude extracts, enzymes may exhibit more complex behaviors than purified forms, however, it is always worth noting that, in relation to operational costs, crude extracts with effective enzymatic activities obtained from microbial cultivation in agro-industrial residues are valuable bioproducts.Basic characterization of enzymes in crude extracts can present some important information needed as an initial evaluation of the enzymes viabilities as crude extracts.The CMEs obtained resulted with good activities of amylases, lipases and xylanases (and indicated to contain more enzymes) and, concerning the application, these CMEs could be directed towards processes and formulations that do not require pure enzymes as usually required for food and drugs.In addition, licuri residues proved to be good substrates for SSF and the use of agro-industrial residues is a modern necessity since good results can be obtained from such underused materials.Sequential studies will give more detailed information so the biocatalytic performances can be better manipulated.To sum up, this work contributed with valid information of actinobacteria cultivated in agroindustrial residues as a source of industrial enzymes.

Figure 2 .
Figure 2. Characterization of amylase activity in crude multi-enzymatic extracts from A. polychromogenes CDPI-30 (black square) and S. violaceoruber CDPA-32 (gray square) in different a) pH, b) temperature and c) substrate (soluble starch) concentrations.Values were expressed as a residual activity (U/Uo, %), being 100 % equivalent to the highest observed activity value (Uo).Bars indicate the deviation of triplicates and lines are only used to guide the eyes.The standard conditions for activity were: pH 7.0, 50 °C and 10 mg/mL of starch.

Figure 3 .
Figure 3. Estimative of a) and c) pH and b) and d) temperature (T) stability parameters: deactivation constant (kd, 1/min) and half-life (t1/2, min), for amylase in crude multi-enzymatic extracts from A. polychromogenes CDPI-30 and S. violaceoruber CDPA-32.Dotted lines are only used to guide the eyes.Linear Regression for kd determinationwas obtained with four to five points and 0.9242 ≤ R 2 ≤ 0.9932.The standard conditions for activity were: pH 7.0 and 50 °C.

Figure 4 .
Figure 4. Characterization of lipase activity in crude multi-enzymatic extracts from A. polychromogenes CDPI-30 and S. violaceoruber CDPA-32 in different a) pH, b) temperature and c) substrate (pNPP) concentrations.Values were expressed as a residual activity (U/Uo, %), being 100 % equivalent to the highest observed activity value (Uo).Bars indicate the deviation of triplicates and dotted lines are only used to guide the eyes.The standard conditions for activity were: pH = 7.0, 37 °C and 7,95 µmol/mL of pNPP.

Figure 5 .
Figure 5. Estimate of a) and c) pH and b) and d) temperature stability parameters: deactivation constant (kd, 1/min) and half-life (t1/2, min), for lipases in crude multi-enzymatic extracts from A. polychromogenes CDPI-30 and S. violaceoruber CDPA-32.Dotted lines are only used to guide the eyes.Linear Regression for kd determination was obtained with four to six points and 0.9223 ≤ R 2 ≤ 0.9970.The standard conditions for activity were: pH 7.0 and 37 °C.

Figure 6 .
Figure 6.Characterization of xylanase activity in crude multi-enzymatic extracts from A. polychromogenes CDPI-30 and S. violaceoruber CDPA-32 in different a) pH, b) temperature and c) substrate (xylan) concentrations.Values were expressed as a residual activity (U/Uo, %), being 100 % equivalent to the highest observed activity value (Uo).Bars indicate the deviation of triplicates and the lines are only used to guide the eyes.The standard conditions for activity were: pH 5, 50 °C and 10 mg/mL of xylan.

Figure 7 .
Figure 7. Estimate of a) and c) pH and b) and d) temperature stability parameters: deactivation constant (kd, 1/min) and half-life (t1/2, min), for xylanases in crude multi-enzymatic extracts from A. polychromogenes CDPI-30 and S. violaceoruber CDPA-32.Dotted lines are only used to guide the eyes.Linear Regression for kd determination was obtained with four to six points and 0.9286 ≤ R 2 ≤ 0.9920.The standard conditions for activity were: pH 5.0 and 50 °C.
using glycerol(COSTA-GUTIERREZ et al., 2021)  -the principal waste generated from the esterification and transesterification of lipids (biodiesel production, for example) -this can perfectly picture the biotechnological value of actinobacteria.XylanaseBy comparing the optimum conditions of pH = 5.0 (Fig.6.a)and 50 °C (Fig. 6.b) observed with the xylanases from both strains with xylanases from other actinobacteria, it is possible to observe more basic values of pH (around pH 7.0 and 8.0) and a lower optimum temperature (40 °C) for the xylanase from Streptomyces olivaceus MSU3 (SANJIVKUMAR et al., 2017).For the xylanase from Arthrobacter sp., cultivated in residues of wheat, rice and bagasse, it was possible to observe a higher temperature range, 60 -110 °C, and also a higher optimum pH, around 8 and 9(KHANDEPARKAR & BHOSLE, 2006).In regard the stability of these two examples, the xylanase from S. olivaceus MSU3 presented better residual activities (reaching 77 -79 %) when incubated at pH 8.0 / 40 °C / 90 min and the xylanase from Arthrobacter sp. was capable to retain 100 % of its activity at pHs 7.0 and 8.0 for 24 h.According to the results (Tab.4) the best t1/2 (around 7 h) were presented by the xylanase CDPA-30 in pH 4,0 and 45 °C, which are close to the optimum conditions.Morover, a few conditions evaluated (Tab.4) presented periods of activation, similar fact was reported with the xylanase from S. olivaceus
* Linearization by Lineweaver-Burk plot with four to five points and 0.923 < R 2 < 0.999.

Table 1 ,
specially for CMCase, xylanase, amylase, pectinase and lipase.Another aspect worth mention is that different actinobateria can produce the same enzyme but with different characterisitcs and it is crucial to perform characterization studies and analyze with caution when comparing results.