Tamarind extract pretreatment: Valorization of sugarcane bagasse for cellulase production by Aspergillus flavus

Effective pretreatment is crucial for cellulase production from sugarcane bagasse. Pretreatment with tamarind extract could reduce the hazardous effect associated with chemical pretreatment. The present work investigated tamarind (Tamarindus indica) extract in combination with H2SO4 and thermal pretreatment of sugarcane bagasse for cellulase production by Aspergillus flavus. The sugarcane bagasse was pretreated with tamarind extract pH 2 and pH 4, followed by 1% H2SO4 and thermal treatment at 121°C for 15 min. The pretreatment slurry was analysed for reducing sugar while solid bagasse was analysed for weight loss. Aspergillus flavus grew on sugarcane bagasse under solid state fermentation and the Carboxy Methyl Cellulase (CMCase) and Filter Paper Assay (FPA) activities were compared on the various pretreatments. The pretreatments changed the visible morphology of the sugarcane bagasse observed by the swelling, fibrous appearance and colour change. Pretreatment slurry yielded highest soluble reducing sugar at 60.01 mg/ml in tamarind extract (pH 4/1% H2SO4 ) and highest weight loss of solids at 73.70% in tamarind extract (pH 2/1% H2SO4 /thermal 121°C). Aspergillus flavus performed better on tamarind extract (pH 2/1% H2SO4 ) by producing optimal CMCase and FPA activities at 0.100 U/ml and 0.409 U/ml respectively after 3 days of fermentation. Cellulase was maximally active at temperature of 50 °C. The tamarind extract pretreatment successfully proved to be an alternative organo-chemical pretreatment of sugarcane bagasse as evidenced by the physical properties, soluble reducing sugars and cellulase activities. Keywords: Aspergillus flavus, Cellulase, Pretreatment, Sugarcane bagasse, Tamarind extract

A Simplified Procedure for Cellulase Filter Paper Assay

A new procedures to minimize labor intensiveness and complexity that has long been recognized in cellulase filter paper activity measurement (FPAase) described by the international union of pure and applied chemistry (IUPAC) was developed. It follows the main idea of IUPAC finding at least two cellulase dilutions have optical densities slightly more and less than a reference optical density of an arbitrary fixed 2 mg absolute glucose amount after a red-ox color reaction and due to cellulase-filter paper hydrolysis. The yielding glucose amount difference as compared to this reference is expressed in terms of absorbance difference percent determined by 3,5-dinitrosalicylic acid (DNS), in case of cellulase is cellubiase rich. If not, an external supplemental portion should be added. The intersection of the line of these two cellulase dilutions with abscissa intersect a vertical at a hypothetical 0% absorbance difference percent corresponding to a critical cellulase dilution exactly release this fixed arbitrary 2 mg glucose amount value. The factor 0.37 of this critical cellulase dilution equals to its filter paper units expressed in FPU per ml. A cellulase Cellic Ctech2 from novozymes has been tested with this methodology giving a satisfactory results with IUPAC procedures.

A Simplified Procedures for Cellulase Filter Paper Assay

A new procedures to minimize labor intensiveness and complexity that has long been recognized in cellulase filter paper activity measurement (FPAase) described by the international union of pure and applied chemistry (IUPAC) was developed. It follows the main idea of IUPAC with only exception finding at least two cellulase dilutions have optical densities slightly more and less than a reference optical density of an arbitrary fixed 2 mg absolute glucose amount after a red-ox color reaction and due to cellulase-filter paper hydrolysis. The yielding glucose amount difference as compared to this reference is expressed in terms of absorbance difference percent determined by 3,5-dinitrosalicylic acid (DNS), in case of cellulase is cellubiase rich. If not, an external supplemental portion should be added. The intersection of the line of these two cellulase dilutions with abscissa intersect a vertical at a hypothetical 0% absorbance difference percent corresponding to a critical cellulase dilution exactly release this fixed arbitrary 2 mg glucose amount value. The factor 0.37 of this critical cellulase dilution equals to its filter paper units expressed in FPU per ml.

A Simplified Procedures for Cellulase Filter Paper Assay

<p>A new procedures to minimize labor intensiveness and complexity that has long been recognized in cellulase filter paper activity measurement (FPAase) described by the international union of pure and applied chemistry (IUPAC)was developed. It follows the main idea of IUPAC with only exception finding at least two cellulase dilutions have optical densities slightly more and less than a reference optical density of an arbitrary fixed 2 mg absolute glucose amount after a red-ox color reaction and due to cellulase-filter paper hydrolysis. The yielding glucose amount difference as compared to this reference is expressed in terms of absorbance difference percent determined by 3,5-dinitrosalicylic acid (DNS), in case of cellulase is cellubiase rich. If not, an external supplemental portion should be added. The intersection of the line of these two cellulase dilutions with abscissa intersect a vertical at a hypothetical 0% absorbance difference percent corresponding to a critical cellulase dilution exactly release this fixed arbitrary 2 mg glucose amount value. The factor 0.37 of this critical cellulase dilution equals to its filter paper units expressed in FPU per ml.</p><br>

A Simplified Procedures for Cellulase Filter Paper Assay

<p>A new procedures to minimize labor intensiveness and complexity that has long been recognized in cellulase filter paper activity measurement (FPAase) described by the international union of pure and applied chemistry (IUPAC)was developed. It follows the main idea of IUPAC with only exception finding at least two cellulase dilutions have optical densities slightly more and less than a reference optical density of an arbitrary fixed 2 mg absolute glucose amount after a red-ox color reaction and due to cellulase-filter paper hydrolysis. The yielding glucose amount difference as compared to this reference is expressed in terms of absorbance difference percent determined by 3,5-dinitrosalicylic acid (DNS), in case of cellulase is cellubiase rich. If not, an external supplemental portion should be added. The intersection of the line of these two cellulase dilutions with abscissa intersect a vertical at a hypothetical 0% absorbance difference percent corresponding to a critical cellulase dilution exactly release this fixed arbitrary 2 mg glucose amount value. The factor 0.37 of this critical cellulase dilution equals to its filter paper units expressed in FPU per ml.</p><br>

Biocatalyst: Cellulase Production in Solid State Fermentation (SSF) Using Rice Bran as Substrate

The study was aimed to analyze the biological transformation of cellulose in rice bran by Aspergillus flavus SB04 in SSF for 28 days. The culture conditions such as pH, temperature, moisture content were optimized for the effective production of the enzyme in SSF. Effect of carbon and nitrogen sources on cellulase production was further estimated in SMF and were quantified for 24hrs intervals for 7 days Maximum cellulase production for rice bran was observed to be high in glucose (carbon source) and yeast extract (nitrogen source) at initial moisture 75ml, pH 6, temperature 33°C and fermentation period was 14th day that was optimized using response surface methodology. The enzyme production was analyzed individually by dinitrosalicylic acid (DNS) method, Lowry protein estimation, and filter paper assay. The lignocellulosic degradation was observed and confirmed by FTIR and SEM. The degradation of cellulose periodically increases after 7 days, which influences the yield of cellulase enzyme.

Rapid radiochemical filter paper assay for determination of hexokinase activity and affinity for glucose-6-phosphate

Glucose phosphorylation by hexokinase (HK) is a rate-limiting step in glucose metabolism. Regulation of HK includes feedback inhibition by its product glucose-6-phosphate (G6P) and mitochondria binding. HK affinity for G6P is difficult to measure because its natural product (G6P) inhibits enzyme activity. HK phosphorylates several hexoses, and we have taken advantage of the fact that 2-deoxyglucose (2-DG)-6-phosphate does not inhibit HK activity. By this, we have developed a new method for rapid radiochemical analysis of HK activity with 2-DG as a substrate, which allows control of the concentrations of G6P to investigate HK affinity for inhibition by G6P. We verified that 2-DG serves as a substrate for the HK reaction with linear time and concentration dependency as well as expected maximal velocity and KM. This is the first simple assay that evaluates feedback inhibition of HK by its product G6P and provides a unique technique for future research evaluating the regulation of glucose phosphorylation under various physiological conditions. NEW & NOTEWORTHY Traditionally, hexokinase activity has been analyzed spectrophotometrically in which the product formation of glucose-6-phosphate (G6P) is analyzed by an indirect reaction coupled to NADPH formation during conversion of G6P to 6-P gluconolactone. By nature, this assay prevents measurements of hexokinase (HK) affinity for inhibition by G6P. We have developed a rapid radiochemical filter paper assay to study HK affinity for G6P by use of radiolabeled 2-deoxyglucose as substrate to study physiological regulation of HK affinity for G6P-induced inhibition.

The Evaluation of Substrates and Trichoderma sp. Isolates for Cellulase Production

As higher interest was on the lignocellulose-based or second generation bioethanol production, the research was then more focused on the production of cellulase, especially on the domestic enzyme. Trichoderma sp. is considered as one of the most efficient producer of cellulase. This study was conducted to investigate the performance of Trichoderma sp. on a variety of substrates to produce cellulase. Three types of substrate variations and three types of Trichoderma sp. were used in this experiment. The substrate used were wheat bran, rice bran and oil palm empty fruit bunches (EFBs), whereas Trichoderma sp. isolates were encoded as T004, T051 and T063. Production of cellulase was made by solid fermentation for 7 days. The analysis of cellulase activity was done by National Renewable Energy Laboratory (NREL) method for filter paper assay. The results showed that the type of substrate affected the performance of Trichoderma sp. All types of fungus produced cellulase on wheat bran substrate with activity of 0.52 FPU /ml for T004, 0.23 FPU/ml for T051 and 0.27 FPU /ml for T063. With the rice bran substrate and EFBs, only T004 could produce cellulase and the enzyme activity analyzed were 0.08 FPU /ml and 0.008 FPU/ml respectively. Optimation of the buffer addition on enzyme extraction process produces the highest activity 0.85 FPU/mL for T004 with wheat bran substrate. Keywords: cellulase, EFBs, rice bran , Trichoderma sp. , wheat bran