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Production of raw cassava starch-degrading enzyme by Penicillium and its use in conversion of raw cassava flour to ethanol

Production of raw cassava starch-degrading enzyme by Penicillium and its use in conversion of raw... Downloaded from https://academic.oup.com/jimb/article/38/6/733/5994333 by DeepDyve user on 08 October 2022 J Ind Microbiol Biotechnol (2011) 38:733–742 DOI 10.1007/s10295-010-0910-7 OR IGINAL PAPER Production of raw cassava starch-degrading enzyme by Penicillium and its use in conversion of raw cassava flour to ethanol • • • Hai-Juan Lin Liang Xian Qiu-Jiang Zhang • • • • Xue-Mei Luo Qiang-Sheng Xu Qi Yang Cheng-Jie Duan • • Jun-Liang Liu Ji-Liang Tang Jia-Xun Feng Received: 8 April 2010 / Accepted: 10 November 2010 / Published online: 1 December 2010 Society for Industrial Microbiology 2010 Abstract A newly isolated strain Penicillium sp. GXU20 Keywords Cassava  Raw cassava starch-degrading produced a raw starch-degrading enzyme which showed enzyme  Penicillium sp.  Raw starch hydrolysis optimum activity towards raw cassava starch at pH 4.5 and Simultaneous saccharification and fermentation 50C. Maximum raw cassava starch-degrading enzyme (RCSDE) activity of 20 U/ml was achieved when GXU20 was cultivated under optimized conditions using wheat Introduction bran (3.0% w/v) and soybean meal (2.5% w/v) as carbon and nitrogen sources at pH 5.0 and 28C. This represented Cassava (Manihot esculenta Cranz) is the world’s fourth about a sixfold increment as compared with the activity most important crop and is grown in many countries in obtained under basal conditions. Starch hydrolysis degree Africa, Asia, and Latin America. Cassava is a starch- of 95% of raw cassava flour (150 g/l) was achieved after containing root crop and is one of the most important 72 h of digestion by crude RCSDE (30 U/g flour). Ethanol sources of calories in the tropics. Cassava is also widely yield reached 53.3 g/l with fermentation efficiency of 92% employed as a raw material for many industrial applica- after 48 h of simultaneous saccharification and fermenta- tions in the animal feed industry and starch industry, and tion of raw cassava flour at 150 g/l using the RCSDE more recently for production of fuel ethanol. Cassava can (30 U/g flour), carried out at pH 4.0 and 40C. This strain be cultivated on arid and semiarid land where other crops, and its RCSDE have potential applications in processing of such as corn, do not thrive [5]. raw cassava starch to ethanol. In Guangxi, China, approximately 200,000 hectares of cassava is under cultivation with production of about 6,000,000 tons of fresh cassava annually, accounting for around 60% of cassava production in China [3, 5]. The starch content in fresh cassava is higher than 30%, and it is H.-J. Lin  L. Xian  Q.-J. Zhang  X.-M. Luo  Q.-S. Xu  estimated that 3.0 tons of dry cassava or 7.2 tons of fresh Q. Yang  C.-J. Duan  J.-L. Liu  J.-L. Tang  J.-X. Feng (&) cassava can produce 1 ton of fuel ethanol [10]. Guangxi Guangxi Key Laboratory of Subtropical Bioresource COFCO Bio-energy Co. Ltd. of Beihai, China, is now Conservation and Utilization, The Key Laboratory of Ministry producing 200,000 tons of fuel ethanol from cassava starch of Education for Microbial and Plant Genetic Engineering, and College of Life Science and Technology, annually [17]. Currently in Guangxi, China, 10% fuel Guangxi University, 100 Daxue Road, 530004 Nanning, ethanol is used in cars. Guangxi, People’s Republic of China Both amylose and amylopectin in cassava starch can be e-mail: jiaxunfeng@sohu.com hydrolyzed by amylolytic enzymes to release fermentable H.-J. Lin  L. Xian  X.-M. Luo  Q.-S. Xu  Q. Yang  sugars which can be converted into ethanol by Saccharo- C.-J. Duan  J.-L. Liu  J.-L. Tang  J.-X. Feng myces cerevisiae [24]. In conventional cassava starch- National Engineering Research Center for Non-food Biorefinery, to-ethanol processing, the starch is first cooked at 90Cor and Guangxi Key Laboratory of Bioindustry Technology, higher temperature with a-amylase (EC 3.2.1.1) [4]. The Guangxi ASCR, 98 Daling Road, 530003 Nanning, Guangxi, liquefied starch is then separately or simultaneously People’s Republic of China 123 Downloaded from https://academic.oup.com/jimb/article/38/6/733/5994333 by DeepDyve user on 08 October 2022 734 J Ind Microbiol Biotechnol (2011) 38:733–742 saccharified by glucoamylase (EC 3.2.1.3) to glucose, production was a thermally resistant dried yeast from which is then fermented by S. cerevisiae. This traditional Angel Yeast Co., Ltd., China. All chemicals used were of process requires a large amount of energy for cooking the analytical grade. starch, thus resulting in high cost of ethanol production [25]. At the Guangxi COFCO Bio-energy Co. Ltd. at Isolation and identification of microorganism Beihai, pulp of manioc flour at concentration of 30–40% (w/v) is first liquefied at about 105C by spraying enzyme. Medium containing 10 g/l raw cassava flour, 3 g/l NaNO , The liquors without saccharification are converted to eth- 1 g/l KH PO , 0.5 g/l KCl, 0.5 g/l MgSO  7H O, 2 4 4 2 anol by simultaneous action of glucoamylase and S. cere- 0.01 g/l FeSO 7H O, and 15 g/l agar was used for iso- 4 2 visiae in fermentors. To conserve energy, identification of lating raw cassava starch-degrading microorganisms. Raw enzymes capable of hydrolyzing raw cassava starch gran- cassava flour was separately sterilized by Co radiation at ules into glucose in a single step without cooking for uti- dose of 20 kGy and was added to the isolation medium lization in simultaneous saccharification and fermentation when it had cooled down to about 45C. Three isolation (SSF) would be an important advance towards increasing agar plates without inoculation were found to be negative the efficiency and profitability of fuel ethanol production for microbial growth after incubation at 28C for 5 days from cassava starch. along with plates spread with 50 ll of different dilutions of It is more difficult for amylolytic enzymes to digest raw soil suspensions, indicating complete sterilization of the starch granules than gelatinized starch granules because of raw cassava flour. Starch hydrolysis was assessed as particle size and the densely compacted polycrystalline clearing zones around the colonies in the isolation agar structure present in raw starch granules [18, 20, 27]. Pre- plates by staining with KI/I solution. Colonies showing vious research has reported that many fungi such as high starch hydrolysis activities on the plates were further Aspergillus sp. [22, 23] and Rhizopus sp. [15, 26] possess evaluated by measuring their enzyme production levels in the ability to produce raw starch-degrading enzymes which liquid fermentation in basal fermentation medium of 50 ml can act on raw corn starch [11, 21–23] and raw potato contained in 250-ml shake flasks and by checking the starch [8] effectively. Production of a raw sago starch- product after enzyme hydrolysis of raw cassava flour by degrading enzyme by the fungus Acremonium sp. has also high-performance liquid chromatography (HPLC). The been reported [13]. However, to our knowledge, no basal fermentation medium contained 10 g/l raw cassava enzymes capable of digesting raw cassava starch have been flour, 2 g/l tryptone, 2.5 g/l (NH ) SO , 3 g/l KH PO , 4 2 4 2 4 reported. 0.025 g/l MgSO 7H O, 0.2 g/l FeSO 7H O, and 0.13 g/l 4 2 4 2 In this study, a fungal strain capable of producing high CaCl at pH 5.5. Flask fermentation was carried out at 28C yield of raw cassava starch-degrading enzyme (RCSDE) at 180 rpm for 5 days. One strain, named GXU20, which was isolated and identified as Penicillium sp. The culture was isolated from forest soil of Shiwandashan Mountain, conditions for producing RCSDE were optimized, and then Guangxi, China, was found to produce the highest amount the crude enzyme preparation was tested for hydrolysis of of RCSDE and was chosen for further study. raw cassava flour at concentration of 150 g/l, which is the Strain GXU20 was further identified based on the amount typically used in the starch industry. SSF of raw method described by Dritsa et al. [6]. The internal tran- cassava flour (150 and 250 g/l) to ethanol at low enzyme scribed spacer (ITS) region of strain GXU20 was amplified load was also investigated. by polymerase chain reaction (PCR), and the PCR product was sequenced and analyzed by searching GenBank. Strain GXU20 was identified as Penicillium sp. The primers used Materials and methods in the PCR reaction were ITS1 (5 -TCCGTAGGTGAA 0 0 CCTGCGG-3 ) and ITS4 (5 -TCCTCCGCTTATTGATA Materials TG-3 ). The PCR reaction conditions were as follows: 95C for 4.5 min, then 30 cycles at 95C for 30 s, 40C for 30 s, Soil samples for microbial isolation were collected from and 72C for 1 min, with a final extension of 10 min at forest in Shiwandashan Mountain of Guangxi Zhuang 72C. Autonomous Region, China and fields close to a starch processing factory in Nanning, China. Raw starch materials Enzyme assay (cassava, corn, potato, sweet potato, glutinous rice, rice, and buckwheat) and wheat bran were obtained from a local RCSDE activity was determined by measuring the reducing market in Nanning, China. The starch content of the raw sugars released on hydrolysis of raw cassava starch. Raw cassava flour used was determined to be 76%. The yeast cassava starch flour (2% w/v) in 450 ll, pH 4.0 citrate/ (Saccharomyces cerevisiae) used in SSF for ethanol phosphate buffer (a mixture of 100 mM citric acid and 123 Downloaded from https://academic.oup.com/jimb/article/38/6/733/5994333 by DeepDyve user on 08 October 2022 J Ind Microbiol Biotechnol (2011) 38:733–742 735 200 mM Na HPO ) was kept at 40C for 5 min, and then an flour solution in the pH range of 3.0–7.0 (pH 3.0, 3.5, 4.0, 2 4 appropriate dilution of 50 ll of crude enzyme was added. 4.5, 5.0, 5.5, 6.0, 6.5, 7.0) using citrate/phosphate buffer at After incubating for 30 min, the amount of reducing sugars 40C. The optimum temperature for enzyme activity was was determined by the dinitrosalicylic acid (DNS) method analyzed from 20Cto60C (20C, 25C, 30C, 35C, as described by Miller [14]. One unit of RCSDE activity 40C, 45C, 50C, 55C, and 60C) in citrate/phosphate was defined as the amount of enzyme releasing 1 lmol buffer (pH 4.5). glucose equivalent per min under the assay conditions. The compounds produced by the enzyme from strain Substrate specificity GXU20 upon hydrolysis of raw cassava starch were ana- lyzed by HPLC. A reaction mixture containing 2 ml crude The substrate specificity of RCSDE from Penicillium sp. preparation of RCSDE enzyme solution and 6 ml 2% (w/v) GXU20 was determined by using different raw starches raw cassava flour (suspended in pH 4.0 citrate/phosphate (soluble starch, cassava, glutinous rice, sweet potato, rice, buffer) was kept at 40C. A 500 ll aliquot was removed corn, buckwheat, and potato) as substrate. Enzyme activity and heated in a boiling water bath for 5 min at different towards various substrates was determined using the time intervals of 5 min, 10 min, 30 min, and 4 h. The enzyme assay. Results are shown as percentages, with the supernatants were analyzed by HPLC using a Hypersil NH RCSDE activity set at 100% for comparison with other raw column. The column was run at room temperature with starch-degrading enzyme activities. 70% (v/v) acetonitrile in deionized water as the mobile phase at flow rate of 1.0 ml/min. The sugar standards used Determination of raw cassava starch adsorbability were glucose, maltose, and maltotriose. Enzyme affinity towards raw cassava starch granules was Optimization of RCSDE production by Penicillium sp. studied by mixing 1 ml crude enzyme with raw cassava GXU20 in liquid fermentation flour (2%, 4%, 8%, 16%, or 20% w/v) in 1 ml citrate/ phosphate buffer (pH 4.0) and incubating at 40C. A RCSDE production was optimized by altering various control, to exclude the possibility of enzyme precipitation environmental (pH and temperature) and culture (carbon or adsorption of the enzyme to the wall of the tube during and nitrogen sources) conditions in the basal fermentation the reaction, was included by incubating the enzyme medium. The effect of initial pH on RCSDE production without raw cassava flour in the reaction buffer. Samples was determined by growing Penicillium sp. GXU20 in were drawn out at different times of 15, 30, and 60 min. basal fermentation medium at varying pH (4.0, 4.5, 5.0, After centrifugation at 12,000 rpm for 5 min, the RCSDE 5.5, 6.0) at 28C. To study the effect of temperature on activity of the supernatants was determined, and then the production of the enzyme by strain GXU20, the culture was adsorption percentage was calculated as incubated in basal fermentation medium at 26C, 28C, Adsorptionð%Þ¼ðA  BÞ 100=A; 30C, 32Cor 34C at optimal pH. where A is the RCSDE activity of the control and B is the The effect of carbon sources was studied by replacing residual RCSDE activity in the supernatant after adsorption raw cassava flour in the basal fermentation medium with on raw cassava starch granules. other gelatinized and raw natural crude starch sources at final concentration of 1% (w/v). Similarly, the effect of nitrogen sources was studied by replacing tryptone and Scanning electron microscopy (NH ) SO with various nitrogen sources (1% w/v) in the 4 2 4 basal fermentation medium. The optimal concentration of Scanning electron micrographs were obtained for native the best carbon source (0.5%, 1.0%, 1.5%, 2.0%, 2.5%, cassava starch granules and raw cassava starch granules 3.0%, 3.5%, 4.0% w/v) and nitrogen source (1.0%, 1.5%, treated with RCSDE prepared from Penicillium sp. 2.0%, 2.5%, 3.0%, 3.5% w/v) for RCSDE production were GXU20. Raw cassava starch flour (2% w/v) was suspended also investigated. All optimization experiments were carried in 25 ml citrate/phosphate buffer (pH 4.0), and then 5 ml out on an orbital shaker at 200 rpm. The crude enzyme crude RCSDE (30 U) was added. The reaction was incu- preparations were analyzed for RCSDE activity as described bated for 6 h at 40C, stopped by the addition of 10 ml in the ‘‘Enzyme assay’’ section. absolute ethanol [9], and then the supernatant was removed by centrifugation for 5 min at 4,200 rpm. The sample was Effect of pH and temperature on crude RCSDE activity washed with absolute ethanol twice. The treated starch granules were dried at 35C to constant weight. The The effect of pH on RCSDE activity was studied by micrographs were obtained by using a S-3400N scanning assaying the crude enzyme using 2% (w/v) raw cassava electron microscope (Hitachi, Ltd.). 123 Downloaded from https://academic.oup.com/jimb/article/38/6/733/5994333 by DeepDyve user on 08 October 2022 736 J Ind Microbiol Biotechnol (2011) 38:733–742 Hydrolysis of raw cassava flour by crude enzyme Nucleotide sequence accession number preparation from Penicillium sp. GXU20 The ITS sequence of Penicillium sp. GXU20 was deposited in Raw cassava flour slurry (50, 100, 150, 200, or 250 g/l) the GenBank database with accession number GU726770. was prepared in citrate/phosphate buffer (pH 4.0), and RCSDE was added at 30 U per g of cassava flour. Statistical analysis The reaction mixture had a final volume of 20 ml. The extent of raw cassava starch hydrolysis was determined Each experiment was repeated three times. Average values at 12 h intervals after the mixtures were incubated at with standard deviation from two or three replications in 40C with shaking at 150 rpm. The effect of enzyme one experiment are presented. dose on hydrolysis of raw cassava flour at concentration of 200 g/l was studied by varying its dose from 15 to 60 U/g raw cassava flour. To determine the degree of Results hydrolysis of starch, the starch residues were washed with sterile water twice and then dried at 40C to con- Isolation and identification of microorganism producing stant weight [20]. RCSDE The degree of hydrolysis of raw cassava starch was calculated by the following formula: During the screening process, clearing zones were observed around the growing colonies of the isolated Degree of hydrolysis(% ) ¼ðW  W Þ 100=W ; 1 2 3 strains after the media were flooded with iodine solution. where W is the amount of dry cassava flour before the This suggests that the isolated strains secrete RCSDE into hydrolysis reaction, W is the amount of flour after the 2 the isolation medium in the plates. Based on the results hydrolysis reaction, and W is the amount of starch in from the clearing zones and enzyme production in liquid cassava flour before the hydrolysis reaction. fermentation, one isolated strain, designated GXU20, was found to produce the highest yield of RCSDE at 3 U/ml Simultaneous saccharification and fermentation of raw and was selected for further study. cassava flour to ethanol The ITS sequence of strain GXU20 (accession number GU726770) shared 100% identity with the ITS sequences of SSF of raw cassava flour to ethanol was investigated. Penicillium sp. (GenBank accession numbers EU301633, The reaction system consisted of crude RCSDE (30 U/g FJ977097, and DQ123663). The GXU20 colony on potato cassava flour) from Penicillium sp. GXU20, cassava flour dextrose agar (PDA) was light green in color and around 150 g/l (or 250 g/l), urea 3 g/l, and yeast 1 g/l. The pH 5.0 cm in diameter on the 7th day of incubation at 28C. of the mixture was adjusted to 4.0 using 2 M HCl, and Microscopic examination showed that the penicilli were studies were carried out in 250-ml conical flasks with biverticillate and the conidia were smooth with the shape of a 100 ml working volume at 40C. Ethanol content, sphere or ellipse, which are the characteristics of Penicillium reducing sugars, and residual starch were assayed every sp. Based on the ITS sequence and microscopic features, the 12 h. strain GXU20 was identified as a Penicillium sp. Fermentation efficiency was calculated as the percent- HPLC analysis revealed that the product of hydrolysis of age of experimental to maximum theoretical yield. raw cassava flour by crude enzyme produced by Penicil- lium sp. GXU20 was solely glucose (Fig. 1), even early on Measurement of starch in the first 5 min of the reaction. This result indicated that the extracellular RCSDE produced by strain GXU20 was The starch content of raw cassava flour was measured by mainly glucoamylase. treating raw cassava flour or samples in 2 M HCl in a boiling water bath for 15 min. The pH of the mixture was Optimization of RCSDE production by Penicillium sp. neutralized with 2 M NaOH after it cooled down to room GXU20 during liquid fermentation temperature [7]. Reducing sugars were determined by the DNS method [14]. Under the operative conditions tested, the optimum pH and temperature for maximum production of RCSDE by Protein determination Penicillium sp. GXU20 in cultivation were pH 5.0 (Fig. 2a) and 28C (Fig. 2b). The RCSDE yield remained Protein concentration was determined by using the Micro constant when GXU20 was cultivated in the pH range BCA protein assay reagent kit (Pierce, Rockford, IL). from 4.5 to 5.5. 123 Downloaded from https://academic.oup.com/jimb/article/38/6/733/5994333 by DeepDyve user on 08 October 2022 J Ind Microbiol Biotechnol (2011) 38:733–742 737 Fig. 1 HPLC chromatograms of hydrolysate of raw cassava starch Fig. 2 Effects of different culture factors on RCSDE production by hydrolyzed by crude RCSDE preparation from Penicillium sp. GXU20: Penicillium sp. GXU20 in liquid fermentation. The basal medium (a) sugar standards glucose (G1), maltose (G2), and maltotriose (G3), at pH 5.5 and cultivation at 28C was used as a control, producing and products of raw cassava starch hydrolyzation by crude RCSDE 3 U/ml RCSDE. The role of pH and temperature was studied using from strain GXU20 after: (b) 5 min of hydrolysis, (c) 10 min of the basal medium. Data are means ± standard deviation from three hydrolysis, (d) 30 min of hydrolysis, and (e) 4 h of hydrolysis replications. The results presented are from a representative exper- iment; similar results were obtained in the other two independent Among the different starch materials tested, gelatinized experiments. (a) Effect of initial pH on RCSDE production. (b) Effect starch sources rather than raw starch sources supported of temperature on RCSDE production higher enzyme production by strain GXU20 (Fig. 3a). In particular 3.5% (w/v) wheat bran yielded a maximum pH of 4.0 used for S. cerevisiae fermentation in the ethanol RCSDE production of 17 U/ml (Fig. 3b), while soybean process. The enzyme was optimally active at 50C meal (Fig. 3c) at 2.5% (w/v) was found to be the best (Fig. 4b). It showed about 87% relative activity at 40C. nitrogen source for RCSDE production, achieving pro- In determining the substrate specificity, it was found that ductivity of 9.6 U/ml (Fig. 3d). crude enzyme preparation from GXU20 had the ability to When wheat bran and soybean meal were synergistically hydrolyze all the raw starch flours to varying extents employed together as the carbon and nitrogen sources at the (Table 1). Results indicated that crude enzyme hydrolyzed optimum, pH 5.0 and 28C, the RCSDE yield was 20 U/ml, raw starch materials in the following order: corn, rice, representing approximately a sixfold increase as compared cassava, potato, sweet potato, buckwheat, glutinous rice, with the yield under the basal cultivation conditions. soluble starch. Raw corn and raw rice showed relatively higher enzyme susceptibility than other starches to the Properties of the crude RCSDE crude RCSDE. Studies on the adsorbability of the crude RCSDE prep- The optimum pH of the crude RCSDE produced by Peni- aration from Penicillium sp. GXU20 towards raw cassava cillium GXU20 was pH 4.5 (Fig. 4a), which is close to the starch granules showed that the adsorption increased with 123 Downloaded from https://academic.oup.com/jimb/article/38/6/733/5994333 by DeepDyve user on 08 October 2022 738 J Ind Microbiol Biotechnol (2011) 38:733–742 Fig. 3 Effect of carbon and nitrogen sources on RCSDE production experiment; similar results were obtained in the other two indepen- by strain GXU20. The basal medium at pH 5.5 and cultivation at 28C dent experiments. (a) Effect of various carbon sources on RCSDE was used as a control, producing 3 U/ml RCSDE. The carbon and production. (b) Raw cassava flour in the basal medium was replaced nitrogen components in the basal medium were replaced with with varying concentrations of wheat bran. (c) Effect of various different carbon and nitrogen sources while keeping the other nitrogen sources on RCSDE production. (d) Tryptone and (NH ) SO 4 2 4 ingredients the same. Data are means ± standard deviation from in the basal medium were replaced with varying concentrations of three replications. The results presented are from a representative soybean meal increasing raw cassava starch concentration from 2% to granules may allow the RCSDE to penetrate into the inner 20%, but not with increased incubation time at pH 4.0 and granule more extensively. 40C (Table 2). Hydrolysis of raw cassava flour by crude RCSDE Scanning electron microscopy (SEM) from Penicillium sp. GXU20 The scanning electron micrographs revealed that treated As shown in Fig. 6, the RCSDE preparation could hydrolyze cassava starch granules were severely damaged, suggesting raw cassava starch in a short incubation time of 36 h. that the granules were significantly hydrolyzed by RCSDE Approximately 100% of the cassava starch (at 50 g/l) was from Penicillium sp. GXU20. As shown in Fig. 5, the hydrolyzed after 36 h of incubation. With a constant RCSDE surface of untreated cassava granules was smooth (Fig. 5a). dose of 30 U/g cassava flour, the degree of starch hydrolysis However, a rough surface and eroded granules were decreased as the cassava flour concentration increased. At observed (Fig. 5b) in those hydrolyzed by RCSDE from cassava flour concentrations of 100, 150, 200, and 250 g/l, Penicillium sp. GXU20. The surface of the treated cassava the degree of starch hydrolysis after 36 h of incubation was starch granules appeared to be composed of a number of 90%, 80%, 74%, and 68%, respectively. When the incuba- small pores. The random pinholes on the surface of the tion time was increased from 36 to 72 h, the degree of starch 123 Downloaded from https://academic.oup.com/jimb/article/38/6/733/5994333 by DeepDyve user on 08 October 2022 J Ind Microbiol Biotechnol (2011) 38:733–742 739 hydrolysis increased to 100%, 95%, 87%, and 80%, respectively (Fig. 6a). These results indicate that the majority of the hydrolysis occurred during the first 36 h. When 200 g/l raw cassava flour was incubated with an RCSDE dose from 15 to 60 U/g cassava flour, a similar degree of starch hydrolysis was observed, except for the enzyme dose of 15 U/g cassava flour (Fig. 6b). Simultaneous saccharification and fermentation of raw cassava flour to ethanol At flour concentration of 150 g/l, an amount typically used in the starch industry, ethanol yield of 53.3 g/l and fer- mentation efficiency of 92% were achieved after 48 h of SSF (Fig. 7a). No significant increase in ethanol yield was observed after 60 and 72 h of fermentation. At flour con- centration of 250 g/l, ethanol yield of 75.6 g/l (Fig. 7b) and fermentation efficiency of 76% were obtained after 48 h of SSF. Prolonging the duration of SSF to 72 h resulted in little increase in ethanol content (80.5 g/l) with fermenta- tion efficiency of 84%. Discussion RCSDE secreted by strain GXU20 showed high activity towards raw cassava starch. Production of this enzyme was significantly enhanced using wheat bran and soybean meal as the carbon and nitrogen sources for strain GXU20 cul- tivated at pH 5.0 and 28C. An earlier study also indicated Fig. 4 Effect of pH and temperature on activity of crude RCSDE produced by Penicillium sp. GXU20. Data are means ± standard that wheat bran and soybean meal were effective for raw deviation from three replications. The results presented are from a starch-degrading glucoamylase production by Aspergillus representative experiment; similar results were obtained in the other niger [19]. Wheat bran and soybean meal as cheap energy two independent experiments. (a) Influence of pH on crude RCSDE sources are generally preferred for industrial processes activity. (b) Influence of temperature on crude RCSDE activity compared with more expensive, chemically refined ingre- dients such as tryptone and yeast extract. Compared with traditional cassava starch-to-ethanol Table 1 Substrate specificity of crude RCSDE from strain GXU20 processing, enzyme that is capable of degrading raw cas- towards various raw starches at pH 4.0 and 40C sava starch is economically attractive, since it can save Substrate Specific activity (U/mg protein) Relative activity (%) energy costs. The crude RCSDE produced by strain GXU20 is effectively active towards raw cassava flour Cassava 6.65 ± 0.095 100 ± 1.4 at pH 4.0 and 40C, similar to the conditions used Corn 14.05 ± 0.64 211 ± 9.7 for fermentation of the thermally resistant dried yeast Rice 13.73 ± 0.34 206 ± 5.2 (S. cerevisiae) from Angel Yeast Co., Ltd., China. This is Potato 6.37 ± 0.026 96 ± 0.4 an important advantage for the potential application of Sweet potato 4.22 ± 0.07 63 ± 1.0 RCSDE for use in digesting raw cassava flour in SSF. In Buckwheat 3.62 ± 0.069 54 ± 1.0 addition, the SEM observation strongly supports the Glutinous 2.26 ± 0.018 34 ± 0.3 effective action of the crude RCSDE towards raw cassava rice starch granules. Aggarwal [2] reported a similar SEM Soluble 0.26 ± 0.006 4 ± 0.09 starch observation pattern after hydrolysis of raw corn starch by glucoamylase from Rhizopus. Data are means ± standard deviation from three replications. The In the RCSDE adsorbability study, it was not observed experiment was repeated three times, and similar results were obtained each time that the amount of reducing sugars formed would be 123 Downloaded from https://academic.oup.com/jimb/article/38/6/733/5994333 by DeepDyve user on 08 October 2022 740 J Ind Microbiol Biotechnol (2011) 38:733–742 Table 2 Adsorbability of Concentration of raw Adsorption to raw cassava starch granules (%) RCSDE towards raw cassava cassava flour (% w/v) starch granules 15 min 30 min 60 min 2 3.1 ± 1.9 14.4 ± 0.9 10.0 ± 1.1 Data are means ± standard 4 9.2 ± 0.7 16.4 ± 1.7 13.1 ± 0.4 deviation from three 8 13.2 ± 0.4 18.0 ± 1.3 14.9 ± 0.7 replications. The experiment was repeated three times, and 16 18.8 ± 0.6 27.3 ± 1.6 23.7 ± 0.7 similar results were obtained 20 21.1 ± 1.2 29.8 ± 0.6 27.2 ± 1.6 each time Fig. 5 Scanning electron micrographs of raw cassava starch gran- ules: (a) untreated, and (b) treated with crude RCSDE preparation Fig. 6 Effect of substrate concentration and enzyme dose on from Penicillium sp. GXU20 hydrolysis of raw cassava flour by crude RCSDE produced by Penicillium sp. GXU20. All hydrolyses were carried out at pH 4.0 and proportional to the amount of RCSDE adsorption. A sim- 40C. Data are means ± standard deviation from two replications. The results presented are from a representative experiment; similar ilar result was also observed by Kimura and Robyt [9], who results were obtained in the other two independent experiments. noted that the enzyme could adsorb onto raw potato starch. (a) Hydrolysis of raw cassava flour at different concentrations with Binding of raw starch-degrading enzyme to starch granules constant RCSDE dose (30 U/g cassava flour). (b) Hydrolysis of raw has been shown to be necessary for degrading starch cassava flour (200 g/l) with varying doses of crude RCSDE granules by fungi glucoamylase [16]. Mamo and Gessesse preparation [12] found that over 70% of the glucoamylase from Aspergillus sp. GP-21 could strongly absorb onto raw corn starch-degrading glucoamylase from Penicillium sp. X-1 and raw potato starch after incubation for 15 min at room showed no adsorbability to raw corn starch, despite varying temperature. However, Sun et al. [21] found that the raw the incubation conditions considerably. 123 Downloaded from https://academic.oup.com/jimb/article/38/6/733/5994333 by DeepDyve user on 08 October 2022 J Ind Microbiol Biotechnol (2011) 38:733–742 741 corn starch concentration of 20% (w/v) with enzyme dose of 250 U per gram substrate after 12 h of incubation. For synergistic action of commercial a-amylase with purified glucoamylase, Sun [21] reported that hydrolysis content of 92.4% was obtained in hydrolysis of 15% (w/v) raw corn starch slurry after 2 h of incubation (500 U a-amylase plus 1,000 U raw starch-degrading glucoamylase for 15 g raw corn starch). The Penicillium sp. GXU20 RCSDE used in this study may have potential applications in direct deg- radation of raw cassava starch in the food and fermentation industries. To further explore the potential use of the crude RCSDE preparation from strain GXU20, crude RCSDE was tested in SSF of raw cassava flour to ethanol. SSF of raw cassava flour (150 g/l) to ethanol was carried out effectively in a short time of 48 h using the crude RCSDE. The residual starch of 0.63% (w/v) and fermentation efficiency of 92% indicated that the SSF process was completed successfully. During the SSF process, raw cassava starch was easily digested by the RCSDE, since the level of reducing sugars was kept low (\0.5% w/v) after 24 h, when end-product inhibition would be likely. The cassava-to-ethanol process presented herein has a distinct advantage over the conventional process using cooked cassava starch, as the new process makes it unnec- essary to consume energy for starch cooking. Since this research was carried out in a 250-ml Erlen- Fig. 7 Ethanol production from raw cassava flour by SSF using crude meyer flask, it is necessary to scale up production and carry RCSDE from Penicillium sp. GXU20. Data are means ± standard deviation from two replications. The results presented are from a out application trials of the RCSDE before any conclusions representative experiment; similar results were obtained in the other can be drawn regarding industrial utilization of this crude two independent experiments. (a) SSF of raw cassava flour to ethanol enzyme. Further work on purification and application at starch concentration of 150 g/l. (b) SSF of raw cassava flour to scale-up is in progress. ethanol at higher starch concentration of 250 g/l. (filled diamonds ethanol yield, filled squares reducing sugar, filled triangles residual starch, filled circles fermentation efficiency) Acknowledgments This work was supported by grants from the National High Technology Research and Development Program of China (863 Program) (No. 2007AA021307), and the National Science The potential application of the crude RCSDE prepa- and Technology Pillar Program in the Eleventh Five-Year Plan Period ration from Penicillium sp. GXU20 was evaluated by of China (No. 2007BAD75B05). studying the degree of hydrolysis of raw cassava flour at pH 4.0 and 40C. The hydrolysis degree of 95% attained after 72 h of incubation strongly suggests that low tem- References perature (40C) and concentration of enzyme (30 U/g cassava flour) are sufficient for RCSDE to hydrolyze raw 1. Abe J-I, Bergmann FW, Obata K, Hizukuri S (1988) Production of the raw-starch digesting amylase of Aspergillus sp. K-27. Appl cassava flour at substrate concentration of 150 g/l. Using Microbiol Biotechnol 27:447–450 fungi crude enzyme, Abe et al. [1] reported that 86% of 2. Aggarwal P, Dollimore D (1998) A thermal analysis investigation corn was hydrolyzed by raw starch-digesting amylase from of partially hydrolyzed starch. Thermochim Acta 319:17–25 Aspergillus sp. K27 at 30C after 24 h with 250 U of 3. Cardona CA, Sanchez OJ (2007) Fuel ethanol production: process design trends and integration opportunities. 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Dritsa V, Rigas F, Natsis K, Marchant R (2007) Characterization and Xu [11] reported a purified a-amylase from Bacillus sp. of a fungal strain isolated from a polyphenol polluted site. for which hydrolysis degree of 50% was obtained at raw Bioresour Technol 98:1741–1747 123 Downloaded from https://academic.oup.com/jimb/article/38/6/733/5994333 by DeepDyve user on 08 October 2022 742 J Ind Microbiol Biotechnol (2011) 38:733–742 7. Gonzalez CF, Farina JI, de Defigueroa LIC (2008) Optimized 18. Quigley TA, Kelly CT, Doyle EM, Fogart WM (1998) Patterns of amylolytic enzymes production in Saccharomycopsis fibuligera raw starch digestion by the glucoamylase of Cladosporium gos- DSM-70554: an approach to efficient cassava starch utilization. sypiicola ATCC 38026. Process Biochem 33:677–681 Enzyme Microbiol Technol 42:272–277 19. Rajoka MI, Yasmeen A (2005) Induction, and production studies 8. 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World Energy 86:162–169 J Microbiol Biotechnol 23:603–613 11. Liu XD, Xu Y (2008) A novel raw starch digesting a-amylase 22. Sun H, Zhao P, Peng M (2008) Application of maltitol to improve from a newly isolated Bacillus sp. YX-1: purification and char- production of raw starch digesting glucoamylase by Aspergillus acterization. Bioresour Technol 99:4315–4320 niger F-08. World J Microbiol Biotechnol 24:2613–2618 12. Mamo G, Gessesse A (1999) Production of raw-starch digesting 23. Sun H-Y, Wang L, Liu J-W, Peng M (2008) Improved production amyloglucosidase by Aspergillus sp. GP-21 in solid state fer- of raw starch degrading enzyme by Aspergillus oryzae F-30 using mentation. J Ind Microbiol Biotechnol 22:622–626 methyl glucoside sesqui-stearate. Appl Biochem Biotechol 13. Marlida Y, Saarib N, Hassanb Z, Radu S (2000) Improvement in 159:78–84 raw sago starch degrading enzyme production from Acremonium 24. Turner P, Mamo G, Karlsson EN (2007) Potential and utilization sp. endophytic fungus using carbon and nitrogen sources. of thermophiles and thermostable enzymes in biorefining. Enzyme Microbiol Technol 27:511–515 Microbiol Cell Fact 6:9 14. Miller GL (1959) Use of dinitrosaIicyIic acid reagent for deter- 25. Verma G, Nigam P, Singh D, Chaudhary K (2000) Bioconversion mination of reducing sugar. Anal Chem 31:426–428 of starch to ethanol in a single-step process by coculture of 15. Morita H, Matsunaga M, Mizuno K, Fujio Y (1998) A compar- amylolytic yeasts and Saccharomyces cerevisiae 21. Bioresour ison of raw starch-digesting glucoamylase production in liquid Technol 72:261–266 and solid cultures of Rhizopus strains. J Gen Appl Microbiol 26. Wang L-S, Ge X-Y, Zhang W-G (2007) Improvement of ethanol 44:211–216 yield from raw corn flour by Rhizopus sp. World J Microbiol 16. Norouzian D, Akbarzadeh A, Scharer JM, Young MM (2006) Biotechnol 23:461–465 Fungal glucoamylases. Biotechnol Adv 24:80–85 27. Zhang T, Oates CG (1999) Relationship between a-amylase 17. Qiu H, Huang J, Yang J, Rozelle S, Yuhua Zhang, Yahui Zhang, degradation and physico-chemical properties of sweet potato Yanli Zhang (2010) Bioethanol development in China and the starches. Food Chem 65:157–163 potential impacts on its agricultural economy. Appl Energy 87:76–83 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Industrial Microbiology and Biotechnology Oxford University Press

Production of raw cassava starch-degrading enzyme by Penicillium and its use in conversion of raw cassava flour to ethanol

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Copyright © 2022 Society for Industrial Microbiology and Biotechnology
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Abstract

Downloaded from https://academic.oup.com/jimb/article/38/6/733/5994333 by DeepDyve user on 08 October 2022 J Ind Microbiol Biotechnol (2011) 38:733–742 DOI 10.1007/s10295-010-0910-7 OR IGINAL PAPER Production of raw cassava starch-degrading enzyme by Penicillium and its use in conversion of raw cassava flour to ethanol • • • Hai-Juan Lin Liang Xian Qiu-Jiang Zhang • • • • Xue-Mei Luo Qiang-Sheng Xu Qi Yang Cheng-Jie Duan • • Jun-Liang Liu Ji-Liang Tang Jia-Xun Feng Received: 8 April 2010 / Accepted: 10 November 2010 / Published online: 1 December 2010 Society for Industrial Microbiology 2010 Abstract A newly isolated strain Penicillium sp. GXU20 Keywords Cassava  Raw cassava starch-degrading produced a raw starch-degrading enzyme which showed enzyme  Penicillium sp.  Raw starch hydrolysis optimum activity towards raw cassava starch at pH 4.5 and Simultaneous saccharification and fermentation 50C. Maximum raw cassava starch-degrading enzyme (RCSDE) activity of 20 U/ml was achieved when GXU20 was cultivated under optimized conditions using wheat Introduction bran (3.0% w/v) and soybean meal (2.5% w/v) as carbon and nitrogen sources at pH 5.0 and 28C. This represented Cassava (Manihot esculenta Cranz) is the world’s fourth about a sixfold increment as compared with the activity most important crop and is grown in many countries in obtained under basal conditions. Starch hydrolysis degree Africa, Asia, and Latin America. Cassava is a starch- of 95% of raw cassava flour (150 g/l) was achieved after containing root crop and is one of the most important 72 h of digestion by crude RCSDE (30 U/g flour). Ethanol sources of calories in the tropics. Cassava is also widely yield reached 53.3 g/l with fermentation efficiency of 92% employed as a raw material for many industrial applica- after 48 h of simultaneous saccharification and fermenta- tions in the animal feed industry and starch industry, and tion of raw cassava flour at 150 g/l using the RCSDE more recently for production of fuel ethanol. Cassava can (30 U/g flour), carried out at pH 4.0 and 40C. This strain be cultivated on arid and semiarid land where other crops, and its RCSDE have potential applications in processing of such as corn, do not thrive [5]. raw cassava starch to ethanol. In Guangxi, China, approximately 200,000 hectares of cassava is under cultivation with production of about 6,000,000 tons of fresh cassava annually, accounting for around 60% of cassava production in China [3, 5]. The starch content in fresh cassava is higher than 30%, and it is H.-J. Lin  L. Xian  Q.-J. Zhang  X.-M. Luo  Q.-S. Xu  estimated that 3.0 tons of dry cassava or 7.2 tons of fresh Q. Yang  C.-J. Duan  J.-L. Liu  J.-L. Tang  J.-X. Feng (&) cassava can produce 1 ton of fuel ethanol [10]. Guangxi Guangxi Key Laboratory of Subtropical Bioresource COFCO Bio-energy Co. Ltd. of Beihai, China, is now Conservation and Utilization, The Key Laboratory of Ministry producing 200,000 tons of fuel ethanol from cassava starch of Education for Microbial and Plant Genetic Engineering, and College of Life Science and Technology, annually [17]. Currently in Guangxi, China, 10% fuel Guangxi University, 100 Daxue Road, 530004 Nanning, ethanol is used in cars. Guangxi, People’s Republic of China Both amylose and amylopectin in cassava starch can be e-mail: jiaxunfeng@sohu.com hydrolyzed by amylolytic enzymes to release fermentable H.-J. Lin  L. Xian  X.-M. Luo  Q.-S. Xu  Q. Yang  sugars which can be converted into ethanol by Saccharo- C.-J. Duan  J.-L. Liu  J.-L. Tang  J.-X. Feng myces cerevisiae [24]. In conventional cassava starch- National Engineering Research Center for Non-food Biorefinery, to-ethanol processing, the starch is first cooked at 90Cor and Guangxi Key Laboratory of Bioindustry Technology, higher temperature with a-amylase (EC 3.2.1.1) [4]. The Guangxi ASCR, 98 Daling Road, 530003 Nanning, Guangxi, liquefied starch is then separately or simultaneously People’s Republic of China 123 Downloaded from https://academic.oup.com/jimb/article/38/6/733/5994333 by DeepDyve user on 08 October 2022 734 J Ind Microbiol Biotechnol (2011) 38:733–742 saccharified by glucoamylase (EC 3.2.1.3) to glucose, production was a thermally resistant dried yeast from which is then fermented by S. cerevisiae. This traditional Angel Yeast Co., Ltd., China. All chemicals used were of process requires a large amount of energy for cooking the analytical grade. starch, thus resulting in high cost of ethanol production [25]. At the Guangxi COFCO Bio-energy Co. Ltd. at Isolation and identification of microorganism Beihai, pulp of manioc flour at concentration of 30–40% (w/v) is first liquefied at about 105C by spraying enzyme. Medium containing 10 g/l raw cassava flour, 3 g/l NaNO , The liquors without saccharification are converted to eth- 1 g/l KH PO , 0.5 g/l KCl, 0.5 g/l MgSO  7H O, 2 4 4 2 anol by simultaneous action of glucoamylase and S. cere- 0.01 g/l FeSO 7H O, and 15 g/l agar was used for iso- 4 2 visiae in fermentors. To conserve energy, identification of lating raw cassava starch-degrading microorganisms. Raw enzymes capable of hydrolyzing raw cassava starch gran- cassava flour was separately sterilized by Co radiation at ules into glucose in a single step without cooking for uti- dose of 20 kGy and was added to the isolation medium lization in simultaneous saccharification and fermentation when it had cooled down to about 45C. Three isolation (SSF) would be an important advance towards increasing agar plates without inoculation were found to be negative the efficiency and profitability of fuel ethanol production for microbial growth after incubation at 28C for 5 days from cassava starch. along with plates spread with 50 ll of different dilutions of It is more difficult for amylolytic enzymes to digest raw soil suspensions, indicating complete sterilization of the starch granules than gelatinized starch granules because of raw cassava flour. Starch hydrolysis was assessed as particle size and the densely compacted polycrystalline clearing zones around the colonies in the isolation agar structure present in raw starch granules [18, 20, 27]. Pre- plates by staining with KI/I solution. Colonies showing vious research has reported that many fungi such as high starch hydrolysis activities on the plates were further Aspergillus sp. [22, 23] and Rhizopus sp. [15, 26] possess evaluated by measuring their enzyme production levels in the ability to produce raw starch-degrading enzymes which liquid fermentation in basal fermentation medium of 50 ml can act on raw corn starch [11, 21–23] and raw potato contained in 250-ml shake flasks and by checking the starch [8] effectively. Production of a raw sago starch- product after enzyme hydrolysis of raw cassava flour by degrading enzyme by the fungus Acremonium sp. has also high-performance liquid chromatography (HPLC). The been reported [13]. However, to our knowledge, no basal fermentation medium contained 10 g/l raw cassava enzymes capable of digesting raw cassava starch have been flour, 2 g/l tryptone, 2.5 g/l (NH ) SO , 3 g/l KH PO , 4 2 4 2 4 reported. 0.025 g/l MgSO 7H O, 0.2 g/l FeSO 7H O, and 0.13 g/l 4 2 4 2 In this study, a fungal strain capable of producing high CaCl at pH 5.5. Flask fermentation was carried out at 28C yield of raw cassava starch-degrading enzyme (RCSDE) at 180 rpm for 5 days. One strain, named GXU20, which was isolated and identified as Penicillium sp. The culture was isolated from forest soil of Shiwandashan Mountain, conditions for producing RCSDE were optimized, and then Guangxi, China, was found to produce the highest amount the crude enzyme preparation was tested for hydrolysis of of RCSDE and was chosen for further study. raw cassava flour at concentration of 150 g/l, which is the Strain GXU20 was further identified based on the amount typically used in the starch industry. SSF of raw method described by Dritsa et al. [6]. The internal tran- cassava flour (150 and 250 g/l) to ethanol at low enzyme scribed spacer (ITS) region of strain GXU20 was amplified load was also investigated. by polymerase chain reaction (PCR), and the PCR product was sequenced and analyzed by searching GenBank. Strain GXU20 was identified as Penicillium sp. The primers used Materials and methods in the PCR reaction were ITS1 (5 -TCCGTAGGTGAA 0 0 CCTGCGG-3 ) and ITS4 (5 -TCCTCCGCTTATTGATA Materials TG-3 ). The PCR reaction conditions were as follows: 95C for 4.5 min, then 30 cycles at 95C for 30 s, 40C for 30 s, Soil samples for microbial isolation were collected from and 72C for 1 min, with a final extension of 10 min at forest in Shiwandashan Mountain of Guangxi Zhuang 72C. Autonomous Region, China and fields close to a starch processing factory in Nanning, China. Raw starch materials Enzyme assay (cassava, corn, potato, sweet potato, glutinous rice, rice, and buckwheat) and wheat bran were obtained from a local RCSDE activity was determined by measuring the reducing market in Nanning, China. The starch content of the raw sugars released on hydrolysis of raw cassava starch. Raw cassava flour used was determined to be 76%. The yeast cassava starch flour (2% w/v) in 450 ll, pH 4.0 citrate/ (Saccharomyces cerevisiae) used in SSF for ethanol phosphate buffer (a mixture of 100 mM citric acid and 123 Downloaded from https://academic.oup.com/jimb/article/38/6/733/5994333 by DeepDyve user on 08 October 2022 J Ind Microbiol Biotechnol (2011) 38:733–742 735 200 mM Na HPO ) was kept at 40C for 5 min, and then an flour solution in the pH range of 3.0–7.0 (pH 3.0, 3.5, 4.0, 2 4 appropriate dilution of 50 ll of crude enzyme was added. 4.5, 5.0, 5.5, 6.0, 6.5, 7.0) using citrate/phosphate buffer at After incubating for 30 min, the amount of reducing sugars 40C. The optimum temperature for enzyme activity was was determined by the dinitrosalicylic acid (DNS) method analyzed from 20Cto60C (20C, 25C, 30C, 35C, as described by Miller [14]. One unit of RCSDE activity 40C, 45C, 50C, 55C, and 60C) in citrate/phosphate was defined as the amount of enzyme releasing 1 lmol buffer (pH 4.5). glucose equivalent per min under the assay conditions. The compounds produced by the enzyme from strain Substrate specificity GXU20 upon hydrolysis of raw cassava starch were ana- lyzed by HPLC. A reaction mixture containing 2 ml crude The substrate specificity of RCSDE from Penicillium sp. preparation of RCSDE enzyme solution and 6 ml 2% (w/v) GXU20 was determined by using different raw starches raw cassava flour (suspended in pH 4.0 citrate/phosphate (soluble starch, cassava, glutinous rice, sweet potato, rice, buffer) was kept at 40C. A 500 ll aliquot was removed corn, buckwheat, and potato) as substrate. Enzyme activity and heated in a boiling water bath for 5 min at different towards various substrates was determined using the time intervals of 5 min, 10 min, 30 min, and 4 h. The enzyme assay. Results are shown as percentages, with the supernatants were analyzed by HPLC using a Hypersil NH RCSDE activity set at 100% for comparison with other raw column. The column was run at room temperature with starch-degrading enzyme activities. 70% (v/v) acetonitrile in deionized water as the mobile phase at flow rate of 1.0 ml/min. The sugar standards used Determination of raw cassava starch adsorbability were glucose, maltose, and maltotriose. Enzyme affinity towards raw cassava starch granules was Optimization of RCSDE production by Penicillium sp. studied by mixing 1 ml crude enzyme with raw cassava GXU20 in liquid fermentation flour (2%, 4%, 8%, 16%, or 20% w/v) in 1 ml citrate/ phosphate buffer (pH 4.0) and incubating at 40C. A RCSDE production was optimized by altering various control, to exclude the possibility of enzyme precipitation environmental (pH and temperature) and culture (carbon or adsorption of the enzyme to the wall of the tube during and nitrogen sources) conditions in the basal fermentation the reaction, was included by incubating the enzyme medium. The effect of initial pH on RCSDE production without raw cassava flour in the reaction buffer. Samples was determined by growing Penicillium sp. GXU20 in were drawn out at different times of 15, 30, and 60 min. basal fermentation medium at varying pH (4.0, 4.5, 5.0, After centrifugation at 12,000 rpm for 5 min, the RCSDE 5.5, 6.0) at 28C. To study the effect of temperature on activity of the supernatants was determined, and then the production of the enzyme by strain GXU20, the culture was adsorption percentage was calculated as incubated in basal fermentation medium at 26C, 28C, Adsorptionð%Þ¼ðA  BÞ 100=A; 30C, 32Cor 34C at optimal pH. where A is the RCSDE activity of the control and B is the The effect of carbon sources was studied by replacing residual RCSDE activity in the supernatant after adsorption raw cassava flour in the basal fermentation medium with on raw cassava starch granules. other gelatinized and raw natural crude starch sources at final concentration of 1% (w/v). Similarly, the effect of nitrogen sources was studied by replacing tryptone and Scanning electron microscopy (NH ) SO with various nitrogen sources (1% w/v) in the 4 2 4 basal fermentation medium. The optimal concentration of Scanning electron micrographs were obtained for native the best carbon source (0.5%, 1.0%, 1.5%, 2.0%, 2.5%, cassava starch granules and raw cassava starch granules 3.0%, 3.5%, 4.0% w/v) and nitrogen source (1.0%, 1.5%, treated with RCSDE prepared from Penicillium sp. 2.0%, 2.5%, 3.0%, 3.5% w/v) for RCSDE production were GXU20. Raw cassava starch flour (2% w/v) was suspended also investigated. All optimization experiments were carried in 25 ml citrate/phosphate buffer (pH 4.0), and then 5 ml out on an orbital shaker at 200 rpm. The crude enzyme crude RCSDE (30 U) was added. The reaction was incu- preparations were analyzed for RCSDE activity as described bated for 6 h at 40C, stopped by the addition of 10 ml in the ‘‘Enzyme assay’’ section. absolute ethanol [9], and then the supernatant was removed by centrifugation for 5 min at 4,200 rpm. The sample was Effect of pH and temperature on crude RCSDE activity washed with absolute ethanol twice. The treated starch granules were dried at 35C to constant weight. The The effect of pH on RCSDE activity was studied by micrographs were obtained by using a S-3400N scanning assaying the crude enzyme using 2% (w/v) raw cassava electron microscope (Hitachi, Ltd.). 123 Downloaded from https://academic.oup.com/jimb/article/38/6/733/5994333 by DeepDyve user on 08 October 2022 736 J Ind Microbiol Biotechnol (2011) 38:733–742 Hydrolysis of raw cassava flour by crude enzyme Nucleotide sequence accession number preparation from Penicillium sp. GXU20 The ITS sequence of Penicillium sp. GXU20 was deposited in Raw cassava flour slurry (50, 100, 150, 200, or 250 g/l) the GenBank database with accession number GU726770. was prepared in citrate/phosphate buffer (pH 4.0), and RCSDE was added at 30 U per g of cassava flour. Statistical analysis The reaction mixture had a final volume of 20 ml. The extent of raw cassava starch hydrolysis was determined Each experiment was repeated three times. Average values at 12 h intervals after the mixtures were incubated at with standard deviation from two or three replications in 40C with shaking at 150 rpm. The effect of enzyme one experiment are presented. dose on hydrolysis of raw cassava flour at concentration of 200 g/l was studied by varying its dose from 15 to 60 U/g raw cassava flour. To determine the degree of Results hydrolysis of starch, the starch residues were washed with sterile water twice and then dried at 40C to con- Isolation and identification of microorganism producing stant weight [20]. RCSDE The degree of hydrolysis of raw cassava starch was calculated by the following formula: During the screening process, clearing zones were observed around the growing colonies of the isolated Degree of hydrolysis(% ) ¼ðW  W Þ 100=W ; 1 2 3 strains after the media were flooded with iodine solution. where W is the amount of dry cassava flour before the This suggests that the isolated strains secrete RCSDE into hydrolysis reaction, W is the amount of flour after the 2 the isolation medium in the plates. Based on the results hydrolysis reaction, and W is the amount of starch in from the clearing zones and enzyme production in liquid cassava flour before the hydrolysis reaction. fermentation, one isolated strain, designated GXU20, was found to produce the highest yield of RCSDE at 3 U/ml Simultaneous saccharification and fermentation of raw and was selected for further study. cassava flour to ethanol The ITS sequence of strain GXU20 (accession number GU726770) shared 100% identity with the ITS sequences of SSF of raw cassava flour to ethanol was investigated. Penicillium sp. (GenBank accession numbers EU301633, The reaction system consisted of crude RCSDE (30 U/g FJ977097, and DQ123663). The GXU20 colony on potato cassava flour) from Penicillium sp. GXU20, cassava flour dextrose agar (PDA) was light green in color and around 150 g/l (or 250 g/l), urea 3 g/l, and yeast 1 g/l. The pH 5.0 cm in diameter on the 7th day of incubation at 28C. of the mixture was adjusted to 4.0 using 2 M HCl, and Microscopic examination showed that the penicilli were studies were carried out in 250-ml conical flasks with biverticillate and the conidia were smooth with the shape of a 100 ml working volume at 40C. Ethanol content, sphere or ellipse, which are the characteristics of Penicillium reducing sugars, and residual starch were assayed every sp. Based on the ITS sequence and microscopic features, the 12 h. strain GXU20 was identified as a Penicillium sp. Fermentation efficiency was calculated as the percent- HPLC analysis revealed that the product of hydrolysis of age of experimental to maximum theoretical yield. raw cassava flour by crude enzyme produced by Penicil- lium sp. GXU20 was solely glucose (Fig. 1), even early on Measurement of starch in the first 5 min of the reaction. This result indicated that the extracellular RCSDE produced by strain GXU20 was The starch content of raw cassava flour was measured by mainly glucoamylase. treating raw cassava flour or samples in 2 M HCl in a boiling water bath for 15 min. The pH of the mixture was Optimization of RCSDE production by Penicillium sp. neutralized with 2 M NaOH after it cooled down to room GXU20 during liquid fermentation temperature [7]. Reducing sugars were determined by the DNS method [14]. Under the operative conditions tested, the optimum pH and temperature for maximum production of RCSDE by Protein determination Penicillium sp. GXU20 in cultivation were pH 5.0 (Fig. 2a) and 28C (Fig. 2b). The RCSDE yield remained Protein concentration was determined by using the Micro constant when GXU20 was cultivated in the pH range BCA protein assay reagent kit (Pierce, Rockford, IL). from 4.5 to 5.5. 123 Downloaded from https://academic.oup.com/jimb/article/38/6/733/5994333 by DeepDyve user on 08 October 2022 J Ind Microbiol Biotechnol (2011) 38:733–742 737 Fig. 1 HPLC chromatograms of hydrolysate of raw cassava starch Fig. 2 Effects of different culture factors on RCSDE production by hydrolyzed by crude RCSDE preparation from Penicillium sp. GXU20: Penicillium sp. GXU20 in liquid fermentation. The basal medium (a) sugar standards glucose (G1), maltose (G2), and maltotriose (G3), at pH 5.5 and cultivation at 28C was used as a control, producing and products of raw cassava starch hydrolyzation by crude RCSDE 3 U/ml RCSDE. The role of pH and temperature was studied using from strain GXU20 after: (b) 5 min of hydrolysis, (c) 10 min of the basal medium. Data are means ± standard deviation from three hydrolysis, (d) 30 min of hydrolysis, and (e) 4 h of hydrolysis replications. The results presented are from a representative exper- iment; similar results were obtained in the other two independent Among the different starch materials tested, gelatinized experiments. (a) Effect of initial pH on RCSDE production. (b) Effect starch sources rather than raw starch sources supported of temperature on RCSDE production higher enzyme production by strain GXU20 (Fig. 3a). In particular 3.5% (w/v) wheat bran yielded a maximum pH of 4.0 used for S. cerevisiae fermentation in the ethanol RCSDE production of 17 U/ml (Fig. 3b), while soybean process. The enzyme was optimally active at 50C meal (Fig. 3c) at 2.5% (w/v) was found to be the best (Fig. 4b). It showed about 87% relative activity at 40C. nitrogen source for RCSDE production, achieving pro- In determining the substrate specificity, it was found that ductivity of 9.6 U/ml (Fig. 3d). crude enzyme preparation from GXU20 had the ability to When wheat bran and soybean meal were synergistically hydrolyze all the raw starch flours to varying extents employed together as the carbon and nitrogen sources at the (Table 1). Results indicated that crude enzyme hydrolyzed optimum, pH 5.0 and 28C, the RCSDE yield was 20 U/ml, raw starch materials in the following order: corn, rice, representing approximately a sixfold increase as compared cassava, potato, sweet potato, buckwheat, glutinous rice, with the yield under the basal cultivation conditions. soluble starch. Raw corn and raw rice showed relatively higher enzyme susceptibility than other starches to the Properties of the crude RCSDE crude RCSDE. Studies on the adsorbability of the crude RCSDE prep- The optimum pH of the crude RCSDE produced by Peni- aration from Penicillium sp. GXU20 towards raw cassava cillium GXU20 was pH 4.5 (Fig. 4a), which is close to the starch granules showed that the adsorption increased with 123 Downloaded from https://academic.oup.com/jimb/article/38/6/733/5994333 by DeepDyve user on 08 October 2022 738 J Ind Microbiol Biotechnol (2011) 38:733–742 Fig. 3 Effect of carbon and nitrogen sources on RCSDE production experiment; similar results were obtained in the other two indepen- by strain GXU20. The basal medium at pH 5.5 and cultivation at 28C dent experiments. (a) Effect of various carbon sources on RCSDE was used as a control, producing 3 U/ml RCSDE. The carbon and production. (b) Raw cassava flour in the basal medium was replaced nitrogen components in the basal medium were replaced with with varying concentrations of wheat bran. (c) Effect of various different carbon and nitrogen sources while keeping the other nitrogen sources on RCSDE production. (d) Tryptone and (NH ) SO 4 2 4 ingredients the same. Data are means ± standard deviation from in the basal medium were replaced with varying concentrations of three replications. The results presented are from a representative soybean meal increasing raw cassava starch concentration from 2% to granules may allow the RCSDE to penetrate into the inner 20%, but not with increased incubation time at pH 4.0 and granule more extensively. 40C (Table 2). Hydrolysis of raw cassava flour by crude RCSDE Scanning electron microscopy (SEM) from Penicillium sp. GXU20 The scanning electron micrographs revealed that treated As shown in Fig. 6, the RCSDE preparation could hydrolyze cassava starch granules were severely damaged, suggesting raw cassava starch in a short incubation time of 36 h. that the granules were significantly hydrolyzed by RCSDE Approximately 100% of the cassava starch (at 50 g/l) was from Penicillium sp. GXU20. As shown in Fig. 5, the hydrolyzed after 36 h of incubation. With a constant RCSDE surface of untreated cassava granules was smooth (Fig. 5a). dose of 30 U/g cassava flour, the degree of starch hydrolysis However, a rough surface and eroded granules were decreased as the cassava flour concentration increased. At observed (Fig. 5b) in those hydrolyzed by RCSDE from cassava flour concentrations of 100, 150, 200, and 250 g/l, Penicillium sp. GXU20. The surface of the treated cassava the degree of starch hydrolysis after 36 h of incubation was starch granules appeared to be composed of a number of 90%, 80%, 74%, and 68%, respectively. When the incuba- small pores. The random pinholes on the surface of the tion time was increased from 36 to 72 h, the degree of starch 123 Downloaded from https://academic.oup.com/jimb/article/38/6/733/5994333 by DeepDyve user on 08 October 2022 J Ind Microbiol Biotechnol (2011) 38:733–742 739 hydrolysis increased to 100%, 95%, 87%, and 80%, respectively (Fig. 6a). These results indicate that the majority of the hydrolysis occurred during the first 36 h. When 200 g/l raw cassava flour was incubated with an RCSDE dose from 15 to 60 U/g cassava flour, a similar degree of starch hydrolysis was observed, except for the enzyme dose of 15 U/g cassava flour (Fig. 6b). Simultaneous saccharification and fermentation of raw cassava flour to ethanol At flour concentration of 150 g/l, an amount typically used in the starch industry, ethanol yield of 53.3 g/l and fer- mentation efficiency of 92% were achieved after 48 h of SSF (Fig. 7a). No significant increase in ethanol yield was observed after 60 and 72 h of fermentation. At flour con- centration of 250 g/l, ethanol yield of 75.6 g/l (Fig. 7b) and fermentation efficiency of 76% were obtained after 48 h of SSF. Prolonging the duration of SSF to 72 h resulted in little increase in ethanol content (80.5 g/l) with fermenta- tion efficiency of 84%. Discussion RCSDE secreted by strain GXU20 showed high activity towards raw cassava starch. Production of this enzyme was significantly enhanced using wheat bran and soybean meal as the carbon and nitrogen sources for strain GXU20 cul- tivated at pH 5.0 and 28C. An earlier study also indicated Fig. 4 Effect of pH and temperature on activity of crude RCSDE produced by Penicillium sp. GXU20. Data are means ± standard that wheat bran and soybean meal were effective for raw deviation from three replications. The results presented are from a starch-degrading glucoamylase production by Aspergillus representative experiment; similar results were obtained in the other niger [19]. Wheat bran and soybean meal as cheap energy two independent experiments. (a) Influence of pH on crude RCSDE sources are generally preferred for industrial processes activity. (b) Influence of temperature on crude RCSDE activity compared with more expensive, chemically refined ingre- dients such as tryptone and yeast extract. Compared with traditional cassava starch-to-ethanol Table 1 Substrate specificity of crude RCSDE from strain GXU20 processing, enzyme that is capable of degrading raw cas- towards various raw starches at pH 4.0 and 40C sava starch is economically attractive, since it can save Substrate Specific activity (U/mg protein) Relative activity (%) energy costs. The crude RCSDE produced by strain GXU20 is effectively active towards raw cassava flour Cassava 6.65 ± 0.095 100 ± 1.4 at pH 4.0 and 40C, similar to the conditions used Corn 14.05 ± 0.64 211 ± 9.7 for fermentation of the thermally resistant dried yeast Rice 13.73 ± 0.34 206 ± 5.2 (S. cerevisiae) from Angel Yeast Co., Ltd., China. This is Potato 6.37 ± 0.026 96 ± 0.4 an important advantage for the potential application of Sweet potato 4.22 ± 0.07 63 ± 1.0 RCSDE for use in digesting raw cassava flour in SSF. In Buckwheat 3.62 ± 0.069 54 ± 1.0 addition, the SEM observation strongly supports the Glutinous 2.26 ± 0.018 34 ± 0.3 effective action of the crude RCSDE towards raw cassava rice starch granules. Aggarwal [2] reported a similar SEM Soluble 0.26 ± 0.006 4 ± 0.09 starch observation pattern after hydrolysis of raw corn starch by glucoamylase from Rhizopus. Data are means ± standard deviation from three replications. The In the RCSDE adsorbability study, it was not observed experiment was repeated three times, and similar results were obtained each time that the amount of reducing sugars formed would be 123 Downloaded from https://academic.oup.com/jimb/article/38/6/733/5994333 by DeepDyve user on 08 October 2022 740 J Ind Microbiol Biotechnol (2011) 38:733–742 Table 2 Adsorbability of Concentration of raw Adsorption to raw cassava starch granules (%) RCSDE towards raw cassava cassava flour (% w/v) starch granules 15 min 30 min 60 min 2 3.1 ± 1.9 14.4 ± 0.9 10.0 ± 1.1 Data are means ± standard 4 9.2 ± 0.7 16.4 ± 1.7 13.1 ± 0.4 deviation from three 8 13.2 ± 0.4 18.0 ± 1.3 14.9 ± 0.7 replications. The experiment was repeated three times, and 16 18.8 ± 0.6 27.3 ± 1.6 23.7 ± 0.7 similar results were obtained 20 21.1 ± 1.2 29.8 ± 0.6 27.2 ± 1.6 each time Fig. 5 Scanning electron micrographs of raw cassava starch gran- ules: (a) untreated, and (b) treated with crude RCSDE preparation Fig. 6 Effect of substrate concentration and enzyme dose on from Penicillium sp. GXU20 hydrolysis of raw cassava flour by crude RCSDE produced by Penicillium sp. GXU20. All hydrolyses were carried out at pH 4.0 and proportional to the amount of RCSDE adsorption. A sim- 40C. Data are means ± standard deviation from two replications. The results presented are from a representative experiment; similar ilar result was also observed by Kimura and Robyt [9], who results were obtained in the other two independent experiments. noted that the enzyme could adsorb onto raw potato starch. (a) Hydrolysis of raw cassava flour at different concentrations with Binding of raw starch-degrading enzyme to starch granules constant RCSDE dose (30 U/g cassava flour). (b) Hydrolysis of raw has been shown to be necessary for degrading starch cassava flour (200 g/l) with varying doses of crude RCSDE granules by fungi glucoamylase [16]. Mamo and Gessesse preparation [12] found that over 70% of the glucoamylase from Aspergillus sp. GP-21 could strongly absorb onto raw corn starch-degrading glucoamylase from Penicillium sp. X-1 and raw potato starch after incubation for 15 min at room showed no adsorbability to raw corn starch, despite varying temperature. However, Sun et al. [21] found that the raw the incubation conditions considerably. 123 Downloaded from https://academic.oup.com/jimb/article/38/6/733/5994333 by DeepDyve user on 08 October 2022 J Ind Microbiol Biotechnol (2011) 38:733–742 741 corn starch concentration of 20% (w/v) with enzyme dose of 250 U per gram substrate after 12 h of incubation. For synergistic action of commercial a-amylase with purified glucoamylase, Sun [21] reported that hydrolysis content of 92.4% was obtained in hydrolysis of 15% (w/v) raw corn starch slurry after 2 h of incubation (500 U a-amylase plus 1,000 U raw starch-degrading glucoamylase for 15 g raw corn starch). The Penicillium sp. GXU20 RCSDE used in this study may have potential applications in direct deg- radation of raw cassava starch in the food and fermentation industries. To further explore the potential use of the crude RCSDE preparation from strain GXU20, crude RCSDE was tested in SSF of raw cassava flour to ethanol. SSF of raw cassava flour (150 g/l) to ethanol was carried out effectively in a short time of 48 h using the crude RCSDE. The residual starch of 0.63% (w/v) and fermentation efficiency of 92% indicated that the SSF process was completed successfully. During the SSF process, raw cassava starch was easily digested by the RCSDE, since the level of reducing sugars was kept low (\0.5% w/v) after 24 h, when end-product inhibition would be likely. The cassava-to-ethanol process presented herein has a distinct advantage over the conventional process using cooked cassava starch, as the new process makes it unnec- essary to consume energy for starch cooking. Since this research was carried out in a 250-ml Erlen- Fig. 7 Ethanol production from raw cassava flour by SSF using crude meyer flask, it is necessary to scale up production and carry RCSDE from Penicillium sp. GXU20. Data are means ± standard deviation from two replications. The results presented are from a out application trials of the RCSDE before any conclusions representative experiment; similar results were obtained in the other can be drawn regarding industrial utilization of this crude two independent experiments. (a) SSF of raw cassava flour to ethanol enzyme. Further work on purification and application at starch concentration of 150 g/l. (b) SSF of raw cassava flour to scale-up is in progress. ethanol at higher starch concentration of 250 g/l. (filled diamonds ethanol yield, filled squares reducing sugar, filled triangles residual starch, filled circles fermentation efficiency) Acknowledgments This work was supported by grants from the National High Technology Research and Development Program of China (863 Program) (No. 2007AA021307), and the National Science The potential application of the crude RCSDE prepa- and Technology Pillar Program in the Eleventh Five-Year Plan Period ration from Penicillium sp. 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Journal of Industrial Microbiology and BiotechnologyOxford University Press

Published: Jun 1, 2011

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