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Ultrasound mediated enzymatic hydrolysis of cellulose and carboxymethyl cellulose

Sulaiman AZ, Ajit A, Chisti Y

Biotechnol Prog, 2013 Nov-Dec;29(6):1448-57.
PMID: 23926080 DOI: 10.1002/btpr.1786

A recombinant Trichoderma reesei cellulase was used for the ultrasound-mediated hydrolysis of soluble carboxymethyl cellulose (CMC) and insoluble cellulose of various particle sizes. The hydrolysis was carried out at low intensity sonication (2.4-11.8 W cm(-2) sonication power at the tip of the sonotrode) using 10, 20, and 40% duty cycles. [A duty cycle of 10%, for example, was obtained by sonicating for 1 s followed by a rest period (no sonication) of 9 s.] The reaction pH and temperature were always 4.8 and 50°C, respectively. In all cases, sonication enhanced the rate of hydrolysis relative to nonsonicated controls. The hydrolysis of CMC was characterized by Michaelis-Menten kinetics. The Michaelis-Menten parameter of the maximum reaction rate Vmax was enhanced by sonication relative to controls, but the value of the saturation constant Km was reduced. The optimal sonication conditions were found to be a 10% duty cycle and a power intensity of 11.8 W cm(-2) . Under these conditions, the maximum rate of hydrolysis of soluble CMC was nearly double relative to control. In the hydrolysis of cellulose, an increasing particle size reduced the rate of hydrolysis. At any fixed particle size, sonication at a 10% duty cycle and 11.8 W cm(-2) power intensity improved the rate of hydrolysis relative to control. Under the above mentioned optimal sonication conditions, the enzyme lost about 20% of its initial activity in 20 min. Sonication was useful in accelerating the enzyme catalyzed saccharification of cellulose.

Matched MeSH terms: Trichoderma/enzymology*
Sago pith residue as an alternative cheap substrate for fermentable sugars production

Linggang S, Phang LY, Wasoh MH, Abd-Aziz S

Appl Biochem Biotechnol, 2012 May;167(1):122-31.
PMID: 22528646 DOI: 10.1007/s12010-012-9592-0

Sago pith residue is one of the most abundant lignocellulosic biomass which can serve as an alternative cheap substrate for fermentable sugars production. This residue is the fibrous waste left behind after the starch extraction process and contains significant amounts of starch (58%), cellulose (23%), hemicellulose (9.2%) and lignin (3.9%). The conversion of sago pith residue into fermentable sugars is commonly performed using cellulolytic enzymes or known as cellulases. In this study, crude cellulases were produced by two local isolates, Trichoderma asperellum UPM1 and Aspergillus fumigatus, UPM2 using sago pith residue as substrate. A. fumigatus UPM2 gave the highest FPase, CMCase and β-glucosidase activities of 0.39, 23.99 and 0.78 U/ml, respectively, on day 5. The highest activity of FPase, CMCase and β-glucosidase by T. asperellum UPM1 was 0.27, 12.03 and 0.42 U/ml, respectively, on day 7. The crude enzyme obtained from A. fumigatus UPM2 using β-glucosidase as the rate-limiting enzyme (3.9, 11.7 and 23.4 IU) was used for the saccharification process to convert 5% (w/v) sago pith residue into reducing sugars. Hydrolysis of sago pith residue using crude enzyme containing β-glucosidase with 23.4 IU, produced by A. fumigatus UPM2 gave higher reducing sugars production of 20.77 g/l with overall hydrolysis percentage of 73%.

Matched MeSH terms: Trichoderma/enzymology*
Isozyme analysis and relationships among three species in Malaysian Trichoderma isolates

Siddiquee S, Tan SG, Yusof UK

J Microbiol Biotechnol, 2010 Sep;20(9):1266-75.
PMID: 20890090

Isozyme and protein electrophoresis data from mycelial extracts of 27 isolates of Trichoderma harzianum, 10 isolates of T. aureoviride and 10 isolates of T. longibrachiatum from Southern Peninsular Malaysia were investigated. The eight enzyme and a single protein pattern systems were analyzed. Three isozyme and total protein patterns were shown to be useful for the detection of three Trichoderma species. The isozyme and protein data were analyzed using the Nei and Li Dice similarity coefficient for pairwise comparison between individual isolates, species isolate group, and for generating a distance matrix. The UPGMA cluster analysis showed a higher degree of relationship between T. harzianum and T. aureoviride than to T. longibrachiatum. These results suggested that the T. harzianum isolates had high levels of genetic variation compared to the other isolates of Trichoderma species.

Matched MeSH terms: Trichoderma/enzymology
Effect of agitation and aeration rates on chitinase production using Trichoderma virens UKM1 in 2-l stirred tank reactor

Abd-Aziz S, Fernandez CC, Salleh MM, Illias RM, Hassan MA

Appl Biochem Biotechnol, 2008 Aug;150(2):193-204.
PMID: 18633736 DOI: 10.1007/s12010-008-8140-4

Shrimps have been a popular raw material for the burgeoning marine and food industry contributing to increasing marine waste. Shrimp waste, which is rich in organic compounds is an abundant source of chitin, a natural polymer of N-acetyl-D-glucosamine (GluNac), a reducing sugar. For this respect, chitinase-producing fungi have been extensively studied as biocontrol agents. Locally isolated Trichoderma virens UKM1 was used in this study. The effect of agitation and aeration rates using colloidal chitin as control substrate in a 2-l stirred tank reactor gave the best agitation and aeration rates at 200 rpm and 0.33 vvm with 4.1 U/l per hour and 5.97 U/l per hour of maximum volumetric chitinase activity obtained, respectively. Microscopic observations showed shear sensitivity at higher agitation rate of the above system. The oxygen uptake rate during the highest chitinase productivity obtained using sun-dried ground shrimp waste of 1.74 mg of dissolved oxygen per gram of fungal biomass per hour at the kappaL a of 8.34 per hour.

Matched MeSH terms: Trichoderma/enzymology*
Statistical optimization of process conditions for cellulase production by liquid state bioconversion of domestic wastewater sludge

Alam MZ, Muyibi SA, Wahid R

Bioresour Technol, 2008 Jul;99(11):4709-16.
PMID: 17981027

A two-level fractional factorial design (FFD) was used to determine the effects of six factors, i.e. substrate (domestic wastewater sludge - DWS) and co-substrate concentration (wheat flour - WF), temperature, initial pH, inoculum size and agitation rate on the production of cellulase enzyme by Trichoderma harzianum in liquid state bioconversion. On statistical analysis of the results from the experimental studies, optimum process conditions were found to be temperature 32.5 degrees C, substrate concentration (DWS) 0.75% (w/w), co-substrate (WF) concentration 2% (w/w), initial pH 5, inoculum size 2% (v/w) and agitation 175 rpm. Analysis of variance (ANOVA) showed a high coefficient of determination (R2) of 0.975. Cellulase activity reached 10.2 FPU/ml at day 3 during the fermentation process which indicated about 1.5-fold increase in production compared to the cellulase activity obtained from the results of design of experiment (6.9 FPU/ml). Biodegradation of DWS was also evaluated to verify the efficiency of the bioconversion process as a waste management method.

Matched MeSH terms: Trichoderma/enzymology*
Use of fungi for the bioconversion of rice straw into cellulase enzyme

Khan MH, Ali S, Fakhru'l-Razi A, Alam Z

J Environ Sci Health B, 2007 May;42(4):381-6.
PMID: 17474017

Cellulase production was carried out by solid state bioconversion (SSB) method using rice straw, a lignocellulosic material and agricultural waste, as the substrate of three Trichoderma spp. and Phanerochaete chrysosporium in lab-scale experiments. The results were compared to select the best fungi among them for the production of cellulase. Phanerochaete chrysosporium was found to be the best among these species of fungi, which produced the highest cellulase enzyme of 1.43 IU/mL of filter paper activity (FPase) and 2.40 IU/mL of carboxymethylcellulose activity (CMCase). The "glucosamine" and "reducing sugar" parameters were observed to evaluate the growth and substrate utilization in the experiments. In the case of Phanerochaete Chrysosporium, the highest glucosamine concentration was 1.60 g/L and a high concentration of the release of reducing sugar was measured as 2.58 g/L obtained on the 4th day of fermentation. The pH values were also recorded. The range of the pH was about 5.15 to 5.56 in the case of Phanerochaete Chrysosporium.

Matched MeSH terms: Trichoderma/enzymology*
Isolation and characterisation of a partial peptide synthetase gene from Trichoderma asperellum

Chutrakul C, Peberdy JF

FEMS Microbiol Lett, 2005 Nov 15;252(2):257-65.
PMID: 16214297

Many species of Trichoderma have attracted interest as agents for the biological control of soil borne fungal pathogens of a range of crop plants. Research on the biochemical mechanisms associated with this application has focused on the ability of these fungi to produce enzymes which lyse fungal cell walls, and antifungal antibiotics. An important group of the latter are the non-ribosomal peptides called peptaibols. In this study Trichoderma asperellum, a strain used in biological control in Malaysia, was found to produce the peptaibol, trichotoxin. This type of peptide molecule is synthesised by a peptide synthetase (PES) enzyme template encoded by a peptide synthetase (pes) gene. Using nucleotide sequences amplified from adenylation (A-) domains as probes, to hybridise against a lambda FIXII genomic library from T. asperellum, 25 clones were recovered. These were subsequently identified as representative of four groups based on their encoding properties for specific amino acid incorporation modules in a PES. This was based on analysis of their amino acid sequences which showed up to 86% identity to other PESs including TEX 1.

Matched MeSH terms: Trichoderma/enzymology*
Identification patterns of Trichoderma strains using morphological characteristics, phylogenetic analyses and lignocellulolytic activities

Asis A, Shahriar SA, Naher L, Saallah S, Fatihah HNN, Kumar V, et al.

Mol Biol Rep, 2021 Apr;48(4):3285-3301.
PMID: 33880673 DOI: 10.1007/s11033-021-06321-0

Trichoderma is a genus of soil-borne fungus with an abundance of reports of its economic importance in the agriculture industry. Thus, the correct identification of Trichoderma species is necessary for its commercial purposes. Globally, Trichoderma species are routinely identified from micro-morphological descriptions which can be tedious and prone to errors. Thus, we emphasize that the accurate identification of Trichoderma strains requires a three-pronged approach i.e. based on its morphological characteristics, multilocus gene sequences of the rDNA [internal transcribed spacer (ITS) 1 and 2 regions], translation elongation factor 1-α (TEF-1α), Calmodulin (CAL) and its lignocellulolytic activities. We used this approach to identify a total of 53 Trichoderma strains which were isolated from a wet paddy field located at Tuaran, Sabah, Malaysia. The 53 strains were positively identified as belonging to three Trichoderma species, namely T. asperellum (43 strains), T. harzianum (9 strains), and T. reesei (one strain) on the basis of its morphological characteristics and multilocus gene sequences. Phylogenetic trees constructed based on the UPGMA method of the ITS 1 and 2 regions of the rDNA, TEF-1α and CAL revealed three distinct groups with the T. asperellum, T. harzianum and T. reesei strains placed under the section of Trichoderma, Pachybasium and Longibrachiatum, respectively. In addition, the lignocellulolytic activities of the isolates were measured based on the diameters of the halo zones produced when degrading cellulose, lignin, and starch, respectively. This diagnostic assay can be used to identify Trichoderma as it produces polyphenol oxidase when Tannic Acid Media is used for the lignin test, endoglucanases when Jensen media is used for cellulose, and it hydrolyzes starch to glucose when the modified Melin-Nokrans media is used for the starch test. Accurate identification of Trichoderma species is needed as these strains can potentially be used as a biocontrol agent to prevent diseases and to increase yield in agriculture crops.

Matched MeSH terms: Trichoderma/enzymology
Fulltext Saccharification of rice straw by cellulase from a local Trichoderma harzianum SNRS3 for biobutanol production

Rahnama N, Foo HL, Abdul Rahman NA, Ariff A, Md Shah UK

BMC Biotechnol, 2014;14:103.
PMID: 25496491 DOI: 10.1186/s12896-014-0103-y

BACKGROUND: Rice straw has shown to be a promising agricultural by-product in the bioconversion of biomass to value-added products. Hydrolysis of cellulose, a main constituent of lignocellulosic biomass, is a requirement for fermentable sugar production and its subsequent bioconversion to biofuels such as biobutanol. The high cost of commercial enzymes is a major impediment to the industrial application of cellulases. Therefore, the use of local microbial enzymes has been suggested. Trichoderma harzianum strains are potential CMCase and β-glucosidase producers. However, few researches have been reported on cellulase production by T. harzianum and the subsequent use of the crude cellulase for cellulose enzymatic hydrolysis. For cellulose hydrolysis to be efficiently performed, the presence of the whole set of cellulase components including exoglucanase, endoglucanase, and β-glucosidase at a considerable concentration is required. Biomass recalcitrance is also a bottleneck in the bioconversion of agricultural residues to value-added products. An effective pretreatment could be of central significance in the bioconversion of biomass to biofuels.
RESULTS: Rice straw pretreated using various concentrations of NaOH was subjected to enzymatic hydrolysis. The saccharification of rice straw pretreated with 2% (w/v) NaOH using crude cellulase from local T. harzianum SNRS3 resulted in the production of 29.87 g/L reducing sugar and a yield of 0.6 g/g substrate. The use of rice straw hydrolysate as carbon source for biobutanol fermentation by Clostridium acetobutylicum ATCC 824 resulted in an ABE yield, ABE productivity, and biobutanol yield of 0.27 g/g glucose, 0.04 g/L/h and 0.16 g/g glucose, respectively. As a potential β-glucosidase producer, T. harzianum SNRS3 used in this study was able to produce β-glucosidase at the activity of 173.71 U/g substrate. However, for cellulose hydrolysis to be efficient, Filter Paper Activity at a considerable concentration is also required to initiate the hydrolytic reaction. According to the results of our study, FPase is a major component of cellulose hydrolytic enzyme complex system and the reducing sugar rate-limiting enzyme.
CONCLUSION: Our study revealed that rice straw hydrolysate served as a potential substrate for biobutanol production and FPase is a rate-limiting enzyme in saccharification.

Matched MeSH terms: Trichoderma/enzymology*
Status of the effectiveness of contact lens disinfectants in Malaysia against keratitis-causing pathogens

Abjani F, Khan NA, Jung SY, Siddiqui R

Exp Parasitol, 2017 Dec;183:187-193.
PMID: 28919333 DOI: 10.1016/j.exppara.2017.09.007

The aim of this study was (i) to assess the antimicrobial effects of contact lens disinfecting solutions marketed in Malaysia against common bacterial eye pathogens and as well as eye parasite, Acanthamoeba castellanii, and (ii) to determine whether targeting cyst wall would improve the efficacy of contact lens disinfectants. Using ISO 14729 Stand-Alone Test for disinfecting solutions, bactericidal and amoebicidal assays of six different contact lens solutions including Oxysept®, AO SEPT PLUS, OPTI-FREE® pure moist®, Renu® fresh™, FreshKon® CLEAR and COMPLETE RevitaLens™ were performed using Manufacturers Minimum recommended disinfection time (MRDT). The efficacy of contact lens solutions was determined against keratitis-causing microbes, namely: Pseudomonas aeruginosa, Methicillin-resistant Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, and Acanthamoeba castellanii. In addition, using chlorhexidine as an antiamoebic compound and cellulase enzyme to disrupt cyst wall structure, we determined whether combination of both agents can enhance efficacy of marketed contact lens disinfectants against A. castellanii trophozoites and cysts, in vitro. The results revealed that all contact lens disinfectants tested showed potent bactericidal effects exhibiting 100% kill against all bacterial species tested. In contrast, none of the contact lens disinfectants had potent effects against Acanthamoeba cysts viability. When tested against trophozoites, two disinfectants, Oxysept Multipurpose and AO-sept Multipurpose showed partial amoebicidal effects. Using chlorhexidine as an antiamoebic compound and cellulase enzyme to disrupt cyst wall structure, the findings revealed that combination of both agents in contact lens disinfectants abolished viability of A. castellanii cysts and trophozoites. Given the inefficacy of contact lens disinfectants tested in this study, these findings present a significant concern to public health. These findings revealed that targeting cyst wall by using cyst wall degrading molecules in contact lens disinfecting solutions will enhance their efficacy against this devastating eye infection.

Matched MeSH terms: Trichoderma/enzymology

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