Intergeneric fusant development using chitinase preparation of Rhizopus stolonifer NCIM 880
© The Author(s) 2016
Received: 2 August 2016
Accepted: 8 November 2016
Published: 14 November 2016
Fungal chitinase have tremendous applications in biotech industries, with this approach we focused on extracellular chitinase from Rhizopus stolonifer NCIM 880 for the formation of fungal protoplasts. The maximum chitinase production reached after 24 h at 2.5% colloidal chitin concentration in presence of starch as an inducer. Chitinase was extracted efficiently at 65% cold acetone concentration and then purified by using DEAE-Cellulose column chromatography. Purified chitinase having molecular weight 22 kDa with single polypeptide chain was optimally active at pH 5.0 and temperature 30 °C. The purified chitinase revealed kinetic properties like Km 1.66 mg/ml and Vmax 769 mM/min. Crude chitinase extract efficiently formed protoplasts from A. niger, A. oryzae, T. viride and F. moniliforme. The formed protoplasts of A. niger and T. viride showed 70 and 66% regeneration frequency respectively. Further, intergeneric fusants were developed successfully and identified at molecular level using RNA profiling. Thus, this study could be useful for strain improvement of various fungi for biotechnological applications.
Chitinase (E.C. 184.108.40.206) is a glycosyl hydrolase which catalyzes degradation of chitin polymer (Henrissat and Bairoch 1993). Chitinases have been detected in vast array of organisms including bacteria, fungi, insects, viruses, plants, and animals and may have various functions in different organisms (Mathivanan et al. 1998; Park et al. 2002; Tanabe et al. 2000; Tikhonov et al. 2002; Waghmare and Ghosh 2010a, b). Bacterial chitinases are produced to meet nutritional needs, so that chitin can be used as carbon and nitrogen sources. Plant chitinases function in self defense against pathogens having chitinous cell wall, whereas yeast and fungal chitinases are required for development and growth of the respective organisms (Gohel et al. 2006). Chitinase from Autographa californica nucleopolyhedrovirus was successfully expressed in transgenic tobacco plant to enhance resistance against pest and fungal pathogens (Maro et al. 2010). Rhizopus is a filamentous fungus and known for production of commercially important compounds such as fumaric acid and cortisone (Manosroi et al. 2006; Engel et al. 2008). Several fungi producing chitinase having diversified properties have been reviewed recently (Narayanan et al. 2014).
Protoplast fusion is an important tool to perform strain improvement as well as to manipulate and develop hybrid strains in filamentous fungi (Lalithakumari 2000). Fungal protoplasts have been used as an effective experimental biochemical tool to study cell wall synthesis, enzyme synthesis and their secretion, as well as in strain improvement for biotechnological applications. Dahiya et al. (2005) reported the effectiveness of Enterobacter sp. NRG4 chitinase in the generation of protoplasts from Trichoderma reesei, Pleurotus florida, Agaricus bisporus, and A. niger (Kitamato et al. 1988; Dahiya et al. 2006). Mizuno et al. (1997) isolated protoplast from Schizophyllum commune using the culture filtrate of B. circulans KA-304. An enzyme complex from B. circulans WL-12 with high chitinase activity was effective in generating protoplasts from Phaffia rhodozyma (Dahiya et al. 2005).
The present study deals with the application of crude extract obtained from the R. stolonifer NCIM 880 for the cost effective production of protoplasts from different fungi and to develop intergeneric fusant of A. niger and T. viride.
Materials and methods
Cultures of Rhizopus stolonifer NCIM 880, Aspergillus niger NCIM 545 Aspergillus oryzae NCIM 1212 and Fusarium moniliforme NCIM 1099 used in this study, were obtained from National Collection for Industrial Microorganisms (NCIM), Pune, India and maintained on PDA medium.
Screening of chitinase production
The selected R. stolonifer was tested for the chitinase production. The chitinase activity was screened on the colloidal chitin agar (NaNO3—0.3%, K2HPO4—0.1%, KCl—0.05%, MgSO4·7H2O—0.05%, FeSO4—0.001%, agar—2.3%, colloidal chitin—3.0%, pH 7.0) at 30 °C incubation temperature. After 48 h incubation period the plate was observed for the zone of hydrolysis around the growth.
Production of chitinase enzyme
The fungal growth was inoculated into the flask containing 100 ml colloidal chitin medium as discussed above. The incubation was carried out at 30 °C temperature on rotary shaker for 96 h. The chitinase production was monitored by measuring chitinase activity in the cell free broth by using colloidal chitin as substrate. The methodology of the assay is described in enzyme assay section. The protein content was estimated by the Lowry method using bovine serum albumin as standard protein (Lowry et al. 1951). The effect of substrate concentration on chitinase production was studied in presence of various concentrations of colloidal chitin in the medium mentioned above, viz. 2.0, 2.5, 3.0 and 3.5%. The effect on production of chitinase was checked by addition of starch at concentration of 1% (w/v).
Purification of chitinase
The culture of R. stolonifer was inoculated into the colloidal chitin containing medium and incubated for 48 h. After incubation, the supernatant was collected by centrifugation at 8000 rpm for 20 min. The obtained supernatant subjected to 30–75% ammonium sulphate precipitation and 45 to 75% cold acetone precipitation. Precipitate was collected by centrifugation at 8000 rpm for 20 min at 4 °C and dissolved in 50 mM Na-phosphate buffer having pH 7.4 and dialysed against the same buffer for overnight. Column was packed with activated DEAE-cellulose equilibrated with 50 mM sodium phosphate buffer as per earlier study (Waghmare et al. 2015). The height of column was 20 cm with the 2.5 cm diameter and protein was eluted with the 0.1–0.5 M NaCl gradient. The 50 fractions were collected having 5 ml volume of each fraction with the flow rate of 1 ml/min. All the steps were carried out at 4 °C. The collected fractions checked for the protein content by Lowry method and chitinase activity.
Effect of pH and temperature on chitinase activity
The effect of pH on enzyme activity was studied for pH range within 3.0–10.0, using citrate-phosphate buffer (pH 3.0–5.0), sodium-phosphate buffer (pH 6.0–8.0) and glycine-NaOH buffer (pH 9.0–10.0), whereas effect of temperature on enzyme activity was studied between temperatures 10–90 °C, and chitinase activity determined as % relative activity. In the stability study of pH and temperature, enzyme was kept for 24 h at specific pH and temperature and residual activity was determined.
Enzyme kinetics and substrate specificity
To study the Km and Vmax, enzyme was incubated with 0.2–2% colloidal chitin as substrate and reducing sugar was determined by DNSA method. The values of Km and Vmax were determined graphically using software SigmPlot version11. Substrate specificity study of chitinase was carried out using different substrate such as colloidal chitin, glycol chitin, CM-Cellulose and p-nitrophenyl-N-acetyl-β-d-glucosaminide (pNP-GlcNAc).
Purity of the fractions, showing chitinase activity, was checked by SDS-PAGE as per method discussed in Laemmli et al. (1970). The proteins were separated on 12% resolving gel and 4% stacking gel. The bands were visualized by silver staining technique (Merril 1987). The molecular weight of chitinase was determined by comparison with standard molecular marker proteins (Phosphorylase b 98 kDa, Bovine Serum Albumin 66 kDa, Ovalbumin 43 kDa, Carbonic Anhydrase 29 kDa, Soyabean Trypsin Inhibitor 20 kDa).
Chitinase activity was quantitated by using colloidal chitin as substrate, the assay mixture includes 1 ml colloidal chitin (10 mg/ml), 0.5 ml 50 mM acetate buffer (pH 5.0) and 1 ml enzyme. The assay mixture incubated at 30 °C temperature for 1 h and activity was terminated by addition of 0.5 ml NaOH (0.5 M). The reducing sugar was determined relative to the N-acetyl-β-d-glucosamine standard (100–500 µg/ml concentration), by using dinitro salicylic acid (DNSA) method (Miller 1959). One unit of enzyme activity was defined as the amount of enzyme required to release 1 µmol of reducing sugar from colloidal chitin, per minute.
The spore suspension of A. niger, A. oryzae, F. moniliforme and T. viride was inoculated into 100 ml medium containing 20% potato infusion and 2% dextrose having pH 6.0. The flask was incubated on rotary shaker at 120 rpm for 48 h at 30 °C temperature. After incubation mycelia were separated by filtration and washed with sterile distilled water followed by 50 mM sodium phosphate buffer of pH 7.0. The washed mycelia (50 mg) were incubated with 5 ml dialysed chitinase. The protoplasts formed were examined with light microscope at 400× magnification.
Regeneration of protoplasts
To study the regeneration ability of protoplasts, the formed individual protoplasts were spread on medium containing NaNO3—0.3%, K2HPO4—0.1%, MgSO4·7H2O—0.05%, FeSO4—0.001%, agar—2.0%, pH 7.0, with an osmotic stabilizer KCl—0.05% and sucrose 2%. Then, the plates were incubated at room temperature for 2 days. After 24 h the plates were observed for formation of mycelia under microscope. The regeneration frequency of protoplasts was measured as per the method described by Patil et al. (2015). Formation of fungal hyphae on soil media was observed under microscope at 400× magnification.
Fusion of protoplasts
The protoplasts fusion of A. niger and T. viride was carried out by two methods i.e. method 1—self fusion and method 2—using Polyethylene glycol (PEG). In method-1, 1 ml protoplast suspension of A. niger and T. viride fungi were mixed in tube and 2 ml protoplast suspension of each kept separately in respective tubes. In method-2, same additions as per method one with additional 1 ml 30% PEG was done. Then these tubes were incubated at room temperature for 24 h. After incubation 0.1 ml aliquots from each tube was spread on Potato Dextrose Agar plates and plates were incubated at room temperature for 3–4 days. The plates were observed for fused colonies.
Molecular characterization of fusant
The fusant obtained on the basis of colony morphology subjected to molecular characterization by using RNA profiling study. The total RNA from fusant (F1, F2, F3) and parent fungi (A. niger, T. viride) were extracted as per the method described by Sanchez-Rodriguez et al. (2008).
Results obtained were the mean of three determinants and ANOVA was carried on all data at p < 0.05 using GraphPad Software.
Screening of chitinase production of R. stolonifer
The mycelia of R. stolonifer were spread on colloidal chitin agar medium containing colloidal chitin as a sole source of carbon for the production of chitinase. After 24 h incubation period the mycelial growth was observed on the plate, but the zone of hydrolysis was observed after 48 h which confirms the ability of R. stolonifer to produce extracellular chitinase.
Production of chitinase
Purification of chitinase
Characterization of purified chitinase
Substrate specificity of chitinase
Relative activity (%)
Fungal protoplast formation
RNA profiling of fusant
The extracellular chitinase was extracted from fungi R. stolonifer NCIM 880 in presence of chitin as a substrate similar to earlier studies using R. oligosporus (Yanai et al. 1992) and R. oryzae (Chen et al. 2013). The fungi R. stolonifer NCIM 880 produces maximum chitinase at 2.5% colloidal chitin concentration in the medium comparatively higher than the reported 1% of Penicillium sp. LYG 0704 (Lee et al. 2009), 1% of A. carneus (Abde-Naby et al. 1992), 2% of P. ochrochloron MTCC 517 (Patil et al. 2013). Increase in chitinase production along with fungal biomass was observed in presence of starch, which suggests that R. stolonifer utilizes starch more rapidly than the colloidal chitin.
The extracellular chitinase produced by R. stolonifer was extracted by Ammonium sulphate and cold acetone precipitation method. Among these two methods cold acetone precipitation method was found more suitable for precipitation which gives 12 fold more yield than the ammonium sulphate precipitation. Similarly Lee et al. (2009) reported precipitation of chitinase produced from Penicillium sp. LYG 0704 by using isopropanol (Lee et al. 2009). The chitinase was purified by one step purification technique using DEAE-Cellulose ion exchange column chromatography. The eluted single peak showing chitinase activity indicates that R. stolonifer produces single enzyme which hydrolyzes chitin efficiently, where as R. oligosporus produces two chitinases (Yanai et al. 1992). The purified enzyme was optimally active at pH 5.0 and temperature 30 °C which was similar to that of chitinase produced by various fungi such as Fusarium chlamydosporum (Mathivanan et al. 1998), Metarhizium anisopliae (Kang et al. 1999), Penicillium sp. LYG0704 (Lee et al. 2009). Chitinase purified by ion exchange chromatography of R. stolonifer shows single polypeptide chain with low molecular weight as confirmed by SDS-PAGE. The approximate molecular weight of chitinase produced by R. stolonifer was 22 kDa, which is comparatively lower than the 50 kDa of R. oryzae (Chen et al. 2013), 68 kDa of P. ochrochloron MTCC 517 (Patil et al. 2013). Km of R. stolonifer chitinase was 1.66 mg/ml which was lower as compared to other chitinases like 4.02 mg/ml of Rhizomucor miehei (Yang et al. 2016), but higher than the 0.82 mg/ml chitinase of Lecanicillium lecanii (Nguyen et al. 2015).
Protoplast have been formed from the various genera of fungi by using mixtures of different enzymatic preparations including β,1-3 glucanase, chitinase and protease (Robinson and Deacon 2001; Solis et al. 1996; Stasz et al. 1988; Tilburn et al. 1983; Waghmare et al. 2011). In this study we have used crude extract of chitinase produced from R. stolonifer to form protoplasts from various fungi such as A. niger, A. oryzae, T. viride, F. moniliforme. The protoplasts were released efficiently from all these tested fungi alone by using chitinase of R. stolonifer, compared with protoplasts generated by using enzymatic preparations of T. harzianum contains β,1-3 glucanase and chitinase (Yanai et al. 1992). So these results indicate that chitinase of R. stolonifer has a broad activity for protoplasts formation among various genera of fungi.
The regeneration of protoplasts into mycelium is an important aspect to study the stability and gene expression of fungi. Protoplasts of A. niger formed by using chitinase preparation of R. stolonifer shown 70% regeneration frequency in medium containing osmotic stabilizer KCl- 0.05% and sucrose 2% at pH 7.0, which was higher than the earlier report (Bekker et al. 2009). The intergeneric fusion between A. niger and T. viride was achieved using 30% PEG and fusants were observed on the basis of phenetic and molecular characterization. In-vivo RNA profiling study has been found convenient for the identification of fusants. Earlier, Santos et al. (1994) reported identification of pathogenic Candida species on the basis of transfer RNA profiling. Similarly, Zhou et al. (2012) discussed genome wide identification on profiling of microRNA-like RNAs from Metarhizium anisopliae during development. Likewise, Patil et al. (2015) reported intergeneric fusion between A. oryzae and T. harzianum. Strom and Bushley (2016) described role of heterokaryotic fungi with distinct traits for antibiotic and enzyme production, fermentation, biocontrol, and bioremediation.
Here, we first time report the production of extracellular chitinase from R. stolonifer NCIM 880. Crude chitinase preparation has ability to form protoplasts of various genera of fungi with high regeneration frequency rate. The successfully formed intergeneric fusant of A. niger and T. viride have been identified using RNA profiling. Thus, the formed heterokaryotic fungi would be useful in various sectors of industries for biotechnological applications.
SRW and KDS directed and designed experiments. NRD, AGN, PTG, SVA performed experiments. SRW, KDS, DBJ, NHN: analysis of results and preparation of manuscript. All authors read and approved the manuscript.
All authors are thankful to Department of Microbiology, Shivaji University, Kolhapur, Maharashtra, India for providing laboratory facilities.
All authors have declared that they have no competing interests.
This article does not contain any studies with human or animal participants.
This work was supported by Department of Science and Technology, Government of India, New Delhi under the DST-PURSE Scheme sanctioned to Shivaji University, Kolhapur, Maharashtra, India.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
- Abde-Naby MA, El-Shayeb NMA, Sherief AA (1992) Purification and some properties of Aspergillus carneus. Appl Biochem Biotech 37:141–154View ArticleGoogle Scholar
- Bekker C, Wiebenga A, Aguilar G, Wösten HAB (2009) An enzyme cocktail for efficient protoplast formation in Aspergillus niger. J Microbiol Methods 76:305–306View ArticlePubMedGoogle Scholar
- Chen WM, Chen CS, Jiang ST (2013) Purification and characterization of an extracellular chitinase from Rhizopus oryzae. J Mar Sci Tech 21:361–366Google Scholar
- Dahiya N, Tewari R, Tiwari RP, Hoondal GS (2005) Production of an antifungal chitinase from Enterobacter sp. NRG4 and its application in protoplast production. World J Microb Biot 21:1611–1616View ArticleGoogle Scholar
- Dahiya N, Tewari R, Hoondal GS (2006) Biotechnological aspects of chitinolytic enzymes: a review. Appl Microbiol Biot 71:773–782View ArticleGoogle Scholar
- Engel CAR, Straathof AJJ, Zijlmans TW, van Gulik WM, van der Wielen LAM (2008) Fumaric acid production by fermentation. Appl Microbiol Biot 78:379–389View ArticleGoogle Scholar
- Gohel V, Singh A, Vimal M, Ashwini P, Chatpar HS (2006) Bioprospecting and antifungal potential of chitinolytic microorganisms. Afr J Biotech 5:54–72Google Scholar
- Henrissat B, Bairoch M (1993) New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J 293:781–788View ArticlePubMedPubMed CentralGoogle Scholar
- Kang SC, Park S, Lee DG (1999) Purification and characterization of a novel chitinase from the entomopathogenic fungus Metarhizium anisopliae. J Invertebr Pathol 73:276–281View ArticlePubMedGoogle Scholar
- Kitamato Y, Mori N, Yamamoto M, Ohiwa T, Ichikava Y (1988) A simple method for protoplasts formation and improvement of protoplast regeneration from various fungi using an enzyme from Trichoderma harzianum. Appl Microbiol Biot 28:445–450View ArticleGoogle Scholar
- Laemmli UK (1970) Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature 227:680–685View ArticlePubMedGoogle Scholar
- Lalithakumari D (2000) Fungal protoplasts—a biotechnological tool. Oxford and IBH Publishing Company Private Limited, New DelhiGoogle Scholar
- Lee YG, Chung KC, Wic SG, Lee JC, Bae HJ (2009) Purification and properties of a chitinase from Penicillium sp. LYG 0704. Protein Expres Purif 65:244–250View ArticleGoogle Scholar
- Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275PubMedGoogle Scholar
- Manosroi J, Chisti Y, Manosroi A (2006) Biotransformation of cortexolone to hydrocortisone by molds using a rapid color-development assay. Appl Biochem Microbiol 42:479–483View ArticleGoogle Scholar
- Maro A, Terracciano I, Sticco L, Fiandra L, Ruocco M, Corrado G, Parente A, Rao R (2010) Purification and characterization of a viral chitinase active against plant pathogens and herbivores from transgenic tobacco. J Biotechnol 147:1–6View ArticlePubMedGoogle Scholar
- Mathivanan N, Kabilan V, Murugesan K (1998) Purification, characterization and antifungal activity of chitinase from Fusarium chlamydosporum, a mycoparasite to groudnut rust, Puccinia arachidis. Can J Microbiol 44:646–651View ArticlePubMedGoogle Scholar
- Merril CR (1987) Detection of proteins separated by electrophoresis. Adv Electrophor 1:111–139Google Scholar
- Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428View ArticleGoogle Scholar
- Mizuno K, Kimura O, Tachiki T (1997) Protoplast formation from Schizophyllum commune by a culture filtrate of Bacillus circulans KA-304 grown on a cell-wall preparation of S. commune as a carbon source. Biosci Biotech Bioch 61:852–857View ArticleGoogle Scholar
- Narayanan K, Karthik A, Parameswaran B, Pandey A (2014) Production, purification and properties of fungal chitinases—a review. Indian J Exp Biol 52:1025–1035Google Scholar
- Nguyen HQ, Quyen DT, Nguyen SLT, Vu VH (2015) An extracellular antifungal chitinase from Lecanicillium lecanii: purification, properties, and application in biocontrol against plant pathogenic fungi. Turk J Biol 39:6–14View ArticleGoogle Scholar
- Park HY, Pan CH, So MY, Ah JH, Jo DH, Kim SI (2002) Purification, characterization, and cDNA cloning of rice class III chitinase. Mol Cells 28:69–76Google Scholar
- Patil NS, Waghmare SR, Jadhav JP (2013) Purification and characterization of an extracellular antifungal chitinase from Penicillium ochrochloron MTCC 517 and its application in protoplast formation. Process Biochem 48:176–183View ArticleGoogle Scholar
- Patil NS, Patil SM, Govindwar SP, Jadhav JP (2015) Molecular characterization of intergeneric hybrid between Aspergillus oryzae and Trichoderma harzianum by protoplast fusion. J Appl Microbiol 118:390–398View ArticlePubMedGoogle Scholar
- Robinson HL, Deacon JW (2001) Protoplast preparation and transient transformation of Rhizoctonia solani. Mycol Res 105:1295–1303View ArticleGoogle Scholar
- Sanchez-Rodriguez A, Portal O, Rojas LE, Ocana B, Mendoza M, Acosta M, Jimenez E, Hofte M (2008) An efficient method for the extraction of high-quality fungal total RNA to study the Mycosphaerella fijiensis–Musa spp. interaction. Mol Biotechnol 40:299–305View ArticlePubMedGoogle Scholar
- Santos MAS, El-Adlouni C, Cox AD, Luz JM, Keith G, Tuite MF (1994) Transfer RNA profiling: a new method for the identification of pathogenic Candida species. Yeast 10:625–636View ArticlePubMedGoogle Scholar
- Solis S, Flores ME, Huitron C (1996) Protoplasts from pectinolytic fungi: isolation, regeneration and pectinolytic enzyme production. Lett Appl Microbiol 23:36–42View ArticleGoogle Scholar
- Stasz TE, Harman GE, Weeder NF (1988) Protoplast preparation and fusion in two biocontrol strains of Trichoderma harzianum. Mycologia 80:141–150View ArticleGoogle Scholar
- Strom NB, Bushley KE (2016) Two genomes are better than one: history, genetics, and biotechnological applications of fungal heterokaryons. Fungal Biol Biotechnol 3:4. doi:10.1186/s40694-016-0022-x View ArticleGoogle Scholar
- Tanabe T, Kawase T, Watanabe T, Uchida Y, Mitsutomi M (2000) Purification and characterization of a 49 kDa chitinase from Streptomyces griseus HUT 6037. J Biosci Bioengi 89:27–32View ArticleGoogle Scholar
- Tikhonov VE, Lopez-Llorca LV, Salinas J, Jansson HB (2002) Purification and characterization of chitinases from the nematophagous fungi Verticillium chamydosporium and V. suchlasporium. Fungal Genet Biol 35:67–78View ArticlePubMedGoogle Scholar
- Tilburn J, Scazzocchio C, Taylor GG, Zabicky-Zissman JH, Lockington RA, Davies RW (1983) Transformation by integration in Aspergillus nidulans. Gene 26:205–221View ArticlePubMedGoogle Scholar
- Waghmare SR, Ghosh JS (2010a) Study of thermostable chitinases from Oerskovia xanthineolytica NCIM 2839. Appl Microbiol Biot 86:1849–1856View ArticleGoogle Scholar
- Waghmare SR, Ghosh JS (2010b) Chitobiose production by using novel thermostable chitinase from Bacillus licheniformis strain JS isolated from a mushroom bed. Carbohyd Res 345:2630–2635View ArticleGoogle Scholar
- Waghmare SR, Kulkarni SS, Ghosh JS (2011) Chitinase production of Oerskovia xanthineolytica NCIM 2839 by solid state fermentation and its application in fungal protoplasts formation. Current Microbiol 63:295–299View ArticlePubMedGoogle Scholar
- Waghmare SR, Gurav AA, Mali SA, Nadaf NH, Jadhav DB, Sonawane KD (2015) Purification and characterization of novel organic solvent tolerant 98 kDa alkaline protease from isolated Stenotrophomonas maltophilia strain SK. Protein Expres Purif 107:1–7View ArticleGoogle Scholar
- Yanai K, Takaya N, Kojima N, Horiuchi H, Ohta A, Takagi M (1992) Purification of two chitinases from Rhizopus oligosporus and isolation and sequencing of the encoding genes. J Bacteriol 174:7398–7406View ArticlePubMedPubMed CentralGoogle Scholar
- Yang S, Fu X, Yan Q, Jiang Z, Wang J (2016) Biochemical characterization of a novel acidic exochitinase from Rhizomucor miehei with antifungal activity. J Agric Food Chem 64:461–469View ArticlePubMedGoogle Scholar
- Zhou Q, Wang Z, Zhang J, Meng H, Huang B (2012) Genome-wide identification and profiling of microRNA-like RNAs from Metarhizium anisopliae during development. Fungal Biol 116:1156–1162View ArticlePubMedGoogle Scholar