Arbakariya Ariff

Pullulanase: Role in Starch Hydrolysis and Potential Industrial Applications

The use of pullulanase (EC 3.2.1.41) has recently been the subject of increased applications in starch-based industries especially those aimed for glucose production. Pullulanase, an important debranching enzyme, has been widely utilised to hydrolyse the α-1,6 glucosidic linkages in starch, amylopectin, pullulan, and related oligosaccharides, which enables a complete and efficient conversion of the branched polysaccharides into small fermentable sugars during saccharification process. The industrial manufacturing of glucose involves two successive enzymatic steps: liquefaction, carried out after gelatinisation by the action of α-amylase; saccharification, which results in further transformation of maltodextrins into glucose. During saccharification process, pullulanase has been used to increase the final glucose concentration with reduced amount of glucoamylase. Therefore, the reversion reaction that involves resynthesis of saccharides from glucose molecules is prevented. To date, five groups of pullulanase enzymes have been reported, that is, (i) pullulanase type I, (ii) amylopullulanase, (iii) neopullulanase, (iv) isopullulanase, and (v) pullulan hydrolase type III. The current paper extensively reviews each category of pullulanase, properties of pullulanase, merits of applying pullulanase during starch bioprocessing, current genetic engineering works related to pullulanase genes, and possible industrial applications of pullulanase.

The treatment of oil palm empty fruit bunch fibre for subsequent use as substrate for cellulase production by Chaetomium globosum Kunze

The feasibility of using treated oil palm empty fruit bunch (OPEFB) fibre as a substrate for cellulase production by Chaetomium globosum Kunze was studied using a shaking flask fermentation system. The use of 2-mm chemically untreated OPEFB fibre increased cellulase production by about two times compared to 10-mm fibre. The effect of the different chemicals (NaOH, HCl, HNO3, EDA and EDTA) on the 2-mm fibre was also investigated. Treatment with these chemicals significantly (P < 0·05) increased the cellulose and reduced the lignin contents. Fermentation using OPEFB fibre treated with HNO3(0·5% v/v) gave the highest cellulase production and this was related to its high cellulose content. Cellulase production increased further when autoclaved (121°C, 15 psi for 5 min), chemically treated OPEFB fibre was used. When autoclaved 2-mm OPEFB fibre treated with HNO3 was used as a substrate, the maximum FPase activity and yield obtained were 0·95 U ml−1 and 120·7 U g−1 cellulose, respectively. The cellulase produced by C. globosum contained a high proportion of β-glucosidase. The ratio of specific activity of β-glucosidase to FPase was about 8. The production of all three major components of cellulase (endoglucanase, cellobiohydrolase and β-glucosidase) using pretreated OPEFB fibre were about three times higher than those obtained in fermentations using pure cellulose (Avicel and carboxymethylcellulose).

Direct fermentation of gelatinized sago starch to acetone-butanol-ethanol by Clostridium acetobutylicum

Direct fermentation of gelatinized sago starch into solvent (acetone–butanol–ethanol) by Clostridium acetobutylicum P262 was studied using a 250 ml Schott bottle anaerobic fermentation system. Total solvent production from fermentation using 30 g sago starch/l (11.03g/l) was comparable to fermentation using corn starch and about 2-fold higher than fermentation using potato or tapioca starch. At the range of sago starch concentration investigated (10–80 g/l), the highest total solvent production (18.82 g/l) was obtained at 50 g/l. The use of a mixture of organic and inorganic nitrogen source (yeast extract + NH4NO3) enhanced growth of C. acetobutylicum, starch hydrolysis and solvent production (24.47 g/l) compared to the use of yeast extract alone. This gave the yield based on sugar consumed of 0.45 g/g. Result from this study also showed that the individual concentrations of nitrogen and carbon influenced solvent production to a greater extent than did carbon to nitrogen (C/N) ratio.

Effect of different flocculants on the flocculation performance of microalgae, Chaetoceros calcitrans, cells

The possibility of using flocculation technique for the separation of microalgae, Chaetoceros calcitrans, biomass from the culture broth was investigated. The flocculation experiments were conducted in 500 mL beaker using culture broth obtained from 10 L photobioreactor. The harvesting efficiency of 90 and 60% was obtained in flocculation without flocculants conducted for 10 days at 27oC (in light and dark) and 4oC (dark), respectively. Harvesting efficiency higher than 90% with short settling time was achieved by adjusting the culture pH to 10.2 using either sodium hydroxide (NaOH) or potassium hydroxide (KOH). Improved cell viability (> 80%) and settling time with a slight improvement of flocculation efficiency was achieved by the addition of polyelectrolytes flocculant (Magnafloc® LT 27 and LT 25). However, the flocculants were only functioned when the pH of the microalgae culture was pre-adjusted to a certain value that promotes cells entrapment and surface charge neutralization prior to flocculation process. The flocculation efficiency and cell viability obtained in flocculation with Magnafloc® (LT 25 and LT 27) was comparable to that obtained in flocculation with chitosan. When chitosan and Magnafloc® (LT 25 and LT 27) were used as flocculants, the highest flocculation efficiency of C. calcitrans cells was observed at pH 8 and 10.2, respectively. Substantial increased in sedimentation rate was observed with increasing flocculants dosage though the flocculation efficiency and cell viability were not significantly varied.

The kinetics and mechanism of lead (II) biosorption by powderized Rhizopus oligosporus

The kinetics and mechanism of lead biosorption by powderized Rhizopus oligosporus were studied using shake flask experiment. The optimum biomass concentration and initial solution pH for lead sorption at initial lead concentrations ranging from 50–200 mg/l was obtained at 0.5 g/l and pH5, respectively. In term of the ratio of initial lead concentration to biomass concentration ratio, the highest lead adsorption was obtained at 750 mg/g which gave the maximum lead uptake capacity of 126 mg/g. The experimental data of lead sorption by R.oligosporus fitted well to the Langmuir sorption isotherm model, indicating that the sorption was similar to that for an ion-exchange resin. This means that the sorption is a single layer metal adsorption that occurred as a molecular surface coverage. This assumption was confirmed by the examination of lead sorption using transmission electron microscope and energy dispersive X-ray analysis, which showed that during sorption most of the lead was adsorbed on the surface of cell.

The mechanism of cadmium removal from aqueous solution by nonmetabolizing free and immobilized live biomass of Rhizopus oligosporus

A preliminary study on the removal of cadmium by nonmetabolizing live biomass of Rhizopus oligosporus from aqueous solution is presented. The equilibrium of the process was in all cases well described by the Langmuir sorption isotherm, suggesting that the process was a chemical, equilibrated and saturable mechanism which reflected the predominantly site-specific mechanism on the cell surface. A curve of Scatchard transformation plots reflected the covalent nature of Cd2+ adsorption by the cells. The maximum cadmium uptake capacities were 34.25 mg/g for immobilized cells and 17.09 mg/g for free cells. Some factorial experiments in shake flasks were performed in order to investigate the effect of different initial cadmium concentrations and biomass concentrations on the equilibrium. Experimental results showed a reverse trend of the influence of the immobilized and free biomass concentration on the cadmium specific uptake capacity. The immobilized cells had a higher specific cadmium uptake capacity with increasing biomass concentrations compared to free cells. In a bioreactor, the cadmium uptake capacity of immobilized cells (qmax = 30.1–37.5 mg/g) was similar to that observed in shake flask experiments (qmax = 34.25 mg/g) whereas with free cells the bioreactor qmax of 4.8–13.0 mg/g; was much lower than in shake flasks (qmax = 17.09 mg/g), suggesting that cadmium biosorption by immobilized cells of R. oligosporus might be further improved in bigger reactors. EDAX and transmission electron microscopic experiments on the fungal biomass indicated that the presence of Cd2+ sequestrated to the cell wall was due to bioadsorption.

Extractive fermentation for improved production and recovery of lipase derived from Burkholderia cepacia using a thermoseparating polymer in aqueous two-phase systems

An extractive fermentation technique was developed using a thermoseparating reagent to form a two-phase system for simultaneous cell cultivation and downstream processing of extracellular Burkholderia cepacia lipase. A 10% (w/w) solution of ethylene oxide-propylene oxide (EOPO) with a molecular mass of 3900 g/mol and pH 8.5, a 200 rpm speed, and 30 °C were selected as the optimal conditions for lipase production (55 U/ml). Repetitive batch fermentation was performed by continuous replacement of the top phase every 24h, which resulted in an average cell growth mass of 4.7 g/L for 10 extractive batches over 240 h. In scaling-up the process, a bench-scale bioreactor was tested under the conditions that had been optimized in flasks. The production rate and recovery yield were higher in the bioreactor compared to fermentation performed in flasks.

Effects of temperature on the physical properties of pink guava juice at two different concentrations

Abstract The effect of temperature on the physical properties of pink guava juice (Psidium guajava L.) variety Beaumont: B-30 with two different total soluble solids (9°Brix and 11°Brix) was investigated. While the flow behaviour index (n) and density (ρ) increased, consistency coefficient (K), specific heat capacity (CP) and thermal conductivity (k) decreased as temperature increased. The linear regression equations or models for consistency coefficient, flow behaviour index, density, specific heat capacity and thermal conductivity were determined with correlation coefficients ranged from 0.8078 to 0.9597.

Mycelium-bound lipase from a locally isolated strain of Aspergillus flavus link: Pattern and factors involved in its production

Aspergillus flavus produces a lipase (EC 3.1.1.3) which is partly bound to the mycelium during growth. The production of the mycelium-bound lipase is concomitant with growth, and declines when growth ceases. Maximum productivity of the enzyme is obtained when the culture is incubated at 30°C, an initial culture pH of 6·5 and with 2% (w/v) each of corn oil and yeast extract as carbon and organic nitrogen source. Yeast extract affects not only the production of lipase but also the secretion of proteases into the culture medium. Production of the latter enzymes, which inactivate the free lipase, is enhanced by adding yeast extract (1–2%, w/v) to the culture medium. However, at 5% (w/v) yeast extract concentration, proteolytic activity is not detected and consequently, the activity of free lipase may easily be measured. Free lipase activity can easily be detected when 0·001 mol dm−3 EDTA is added to the culture medium. The presence of the chelating agent enhances the production and maintains the stability of the extracted mycelium-bound lipase.

Isolation of Pediococcus acidilactici Kp10 with ability to secrete bacteriocin-like inhibitory substance from milk products for applications in food industry

BackgroundLactic acid bacteria (LAB) can be isolated from traditional milk products. LAB that secrete substances that inhibit pathogenic bacteria and are resistant to acid, bile, and pepsin but not vancomycin may have potential in food applications.ResultsLAB isolated from a range of traditional fermented products were screened for the production of bacteriocin-like inhibitory substances. A total of 222 LAB strains were isolated from fermented milk products in the form of fresh curds, dried curds, and ghara (a traditional flavor enhancer prepared from whey), and fermented cocoa bean. Eleven LAB isolates that produced antimicrobial substances were identified as Lactococcus lactis, Lactobacillus plantarum, and Pediococcus acidilactici strains by biochemical methods and 16S rDNA gene sequencing. Of these, the cell-free supernatant of Kp10 (P. acidilactici) most strongly inhibited Listeria monocytogenes. Further analysis identified the antimicrobial substance produced by Kp10 as proteinaceous in nature and active over a wide pH range. Kp10 (P. acidilactici) was found to be catalase-negative, able to produce β-galactosidase, resistant to bile salts (0.3%) and acidic conditions (pH 3), and susceptible to most antibiotics.ConclusionTraditionally prepared fermented milk products are good sources of LAB with characteristics suitable for industrial applications. The isolate Kp10 (P. acidilactici) shows potential for the production of probiotic and functional foods.