Glucoamylase immobilized on montmorillonite: Synthesis, characterization and starch hydrolysis activity in a fixed bed reactor

Glucoamylase from Aspergillus Niger was immobilized on montmorillonite clay (K-10) by two procedures, adsorption and covalent binding. The immobilized enzymes were characterized using XRD, surface area measurements and 27Al MAS NMR and the activity of the immobilized enzymes for starch hydrolysis was tested in a fixed bed reactor (FBR). XRD shows that enzyme intercalates into the inter-lamellar space of the clay matrix with a layer expansion up to 2.25 nm. Covalently bound glucoamylase demonstrates a sharp decrease in surface area and pore volume that suggests binding of the enzyme at the pore entrance. NMR studies reveal the involvement of octahedral and tetrahedral Al during immobilization. The performance characteristics in FBR were evaluated. Effectiveness factor (η) for FBR is greater than unity demonstrating that activity of enzyme is more than that of the free enzyme. The Michaelis constant (Km) for covalently bound glucoamylase was lower than that for free enzyme, i.e., the affinity for substrate improves upon immobilization. This shows that diffusional effects are completely eliminated in the FBR. Both immobilized systems showed almost 100% initial activity after 96 h of continuous operation. Covalent binding demonstrated better operational stability.

Ionomer Compatibilized Low Density Polyethylene - Tapioca Starch Blends Biodegradability and Photodegradability

Biodegradation is the chemical degradation of materials brought about by the action of naturally occurring microorganisms. Biodegradation is a relatively rapid process under suitable conditions of moisture, temperature and oxygen availability. The logic behind blending biopolymers such as starch with inert polymers like polyethylene is that if the biopolymer component is present in sufficient amount, and if it is removed by microorganisms in the waste disposal environment, then the base inert plastic should slowly degrade and disappear. The present work focuses on the preparation of biodegradable and photodegradable blends based on low density polyethylene incorporating small quantities of ionomers as compatibilizers. The thesis consists of eight chapters. The first chapter presents an introduction to the present research work and literature survey. The details of the materials used and the experimental procedures undertaken for the study are described in the second chapter. Preparation and characterization of low density polyethylene (LDPE)-biopolymer (starch/dextrin) blends are described in the third chapter. The result of investigations on the effect of polyethylene-co-methacrylic acid ionomers on the compatibility of LDPE and starch are reported in chapter 4. Chapter 5 has been divided into two parts. The first part deals with the effect of metal oxides on the photodegradation of LDPE. The second part describes the function of metal stearates on the photodegradation of LDPE. The results of the investigations on the role of various metal oxides as pro-oxidants on the degradation of ionomer compatibilized LDPE-starch blends are reported in chapter 6. Chapter 7 deals with the results of investigations on the role of various metal stearates as pro-oxidants on the degradation of ionomer compatibilized LDPE-starch blends. The conclusion of the investigations is presented in the last chapter of the thesis.

Biodegradability Studies on LDPE-Starch Blends using Amylase-Producing Vibrios

Low-density polyethylene, (LDPE) was mixed with two grades of tapioca starch–lowgrade and high-grade. Various compositions were prepared and mechanical and thermal studies performed. The biodegradability of these samples was checked using a culture medium containing Vibrios (an amylase-producing bacteria), which was isolated from a marine benthic environment. The soil burial test and reprocessability of these samples were checked. The studies on biodegradability show that these blends are partially biodegradable. These low-density polyethylene-starch blends are reprocessable without sacrificing much of their mechanical properties

Investigations on the preparation of composite polysaccharide films and micro / nanoparticles for novel applications

chemical Sciences and Technology Division, National Institute for Interdisciplinary Science and Technology, CSIR

Glucoamylase immobilized on montmorillonite: influence of nature of binding on surface properties of clay-support and activity of enzyme

Glucoamylase was immobilized on acid activated montmorillonite clay via two different procedures namely adsorption and covalent binding. The immobilized enzymes were characterized by XRD, NMR and N2 adsorption measurements and the activity of immobilized glucoamylase for starch hydrolysis was determined in a batch reactor. XRD shows intercalation of enzyme into the clay matrix during both immobilization procedures. Intercalation occurs via the side chains of the amino acid residues, the entire polypeptide backbone being situated at the periphery of the clay matrix. 27Al NMR studies revealed the different nature of interaction of enzyme with the support for both immobilization techniques. N2 adsorption measurements indicated a sharp drop in surface area and pore volume for the covalently bound glucoamylase that suggested severe pore blockage. Activity studies were performed in a batch reactor. The adsorbed and covalently bound glucoamylase retained 49% and 66% activity of the free enzyme respectively. They showed enhanced pH and thermal stabilities. The immobilized enzymes also followed Michaelis–Menten kinetics. Km was greater than the free enzyme that was attributed to an effect of immobilization. The immobilized preparations demonstrated increased reusability as well as storage stability.

A study on the Immobilizaton of Enzymes on Polymeric Supports

This work was focused to study the immobilization of enzymes on polymers. A large range of polymer matrices have been employed as supports for enzyme immobilization. Here polyaniline (PAN!) and poly(0~toluidine) (POT) were used as supports. PANI and POT provides an excellent support for enzyme immobilization by virtue of its facile synthesis, superior chemical and physical stabilities, and large retention capacity. We selected industrially important starch hydrolyzing enzymes a-amylase and glucoamylase for the study. In this work the selected enzymes were immobilized via adsorption and covalent bonding methods.To optimize the catalytic efficiency and stability of the resulting biocatalysts, the attempt was made to understand the immobilization effects on enzymatic properties. The effect of pH of the immobilization medium, time of immobilization on the immobilization efficiency was observed. The starch hydrolyzing activity of free 0:-amylase and glucoamylase were compared with immobilized forms. Immobilization on solid supports changes the microenvironment of the enzyme there by influences the pH and temperature relationship on the enzymatic activity. Hence these parameters also optimized. The reusability and storage stability of immobilized enzymes an important aspect from an application standpoint, especially in industrial applications. Taking in to consideration of this, the reusability and the long tenn storage stability of the immobilized enzyme investigated.

Linear Low Density Polyethylene-Biodegradability Using Bacteria from Marine Benthic Environment and Photodegradability Using Ultraviolet Light

Various compositions of linear low density polyethylene(LLDPE) containing bio-filler(either starch or dextrin)of various particle sizes were prepared.The mechanical,thermal,FTIR,morphological(SEM),water absorption and melt flow(MFI) studies were carried out.Biodegradability of the compositions were determined using a shake culture flask containing amylase producing bacteria(vibrios),which were isolated from marine benthic environment and by soil burial test. The effect of low quantities of metal oxides and metal stearate as pro-oxidants in LLDPE and in the LLDPE-biofiller compositions was established by exposing the samples to ultraviolet light.The combination of bio-filler and a pro-oxidant improves the degradation of linear low density polyethylene.The maleation of LLDPE improves the compatibility of the c blend components and thepro-oxidants enhance the photodegradability of the compatibilised blends.The responsibility studies on the partially biodegradable LLDPE containing bio-fillers and pro-oxidants suggest that the blends could be repeatedly reprocessed without deterioration in mechanical properties.

Studies on immobilization of Bacteria

Cell immnhilizatinn technology in a rapidly expanding arna in the endeavour of microbial fnrmentatiwn.During the lnmt 15 years anveral prnceafinn have been developed and more are in developmental atage of approaching commercial utilizatinn.In the present programme it was planned to develop an optimized process for the innobilization of alpha amylase producing Bacillus polymyxa (CBTB 25) an isolate obtained from Cochin University campus primarily for the production of alpha-amylase.Optimal concentration of support material that attributaa stability and maximal activity to the immobilized cell beads was determined using different concentrations of sodium aliginate as support and estimation of amylase production.An overeall assessment of the data obtained for the various studies conducted denotes that immobilized cells synthesize alpha-amylase at comparable rates with free cells and produce reducing sugara at a higher level than free cells.Results indicated that both phosphate and citrate buffers could be used for disrupting the immobilized beads since they enforced maximal release of cells through leaching from the beads within one hour.On comparative analysis it was observed that immobilized cells could synthesize alpha amylase at similar levels with free cells of B.polymyxa.On Co-immobilization of B.Polymyxa with S.cerevisiae,the co-immobilizate beads could effeciently convert starch directly to ethanol with a yield of 14.8% at 1 : 2 ratio.

Studies on amylolytic bacteria in Cochin backwaters

This thesis Entitled Studies on amylolytic bacteria in cochin backwaters.This thesis presents a detailed account of the disribution of amylolytic bacteria in water. sediment. fishes ( Etroplus suratensis and Liza parsia) • prawns ( Penaeus indicus and Metapenaeus dobsoni) and clams ( Sunetta scripta and Meretrix casta) from Cochin backwaters. genera-wise distribution of amylolytic bacteria, ability of selected strains to grow and produce amylase at various physico-chemical conditions. Regulation of amylase synthesis anrt characters of amylases producer by these halophilic bacteria.Amylolytic bacteria are distributed widely in water. sediment. fishes. prawns and clams of Cochin back waters. 53% of the total isolates tested were capable of producing amylase. Maximum number of arnylolytic bacteria were present in Metapenaeus dobsoni. In general, the gut region of aquatic animals harboured more amylolytic bacteria than the gill or surface. These bacteria may help in the digestion of starch present in their food.Presence of ions in the medium was found to be essential for growth and amylase production. It was found that this ionic requirement is not highly specific. Sorlium chloride could be replaced by potassium chloride. or magnesium chloride to some extent I without affecting growth and amylase production. The important function of these ions may be to maintain the osmotic balance between the cells and their environment.All the isolates showed the ability to grow and produce amylase using raw-starches from cassava. plantain and potato .This property suggests their role in the rdegradation of native starches in the environment

Polypropylene/metal oxide nanocomposites: fiber spinning and evaluation”

PP has been getting much attention over the years because it is a very durable polymer commonly used in aggressive environments including automotive battery casings, fuel containers etc. They are used to make bottles, fibers for clothing, components in cars etc. However, it has some shortcomings such as low dimensional and thermal stability. Materials such as metal oxides with sizes of the order 1–50 nm have received a great deal of attention because of their versatile applications in polymer/ inorganic nanocomposites, optoelectronic devices, biomedical materials, and other areas. They are stable under harsh process conditions and also regarded as safe materials to human beings and animals. In the present investigation, PP is modified by incorporating metal oxide nanoparticles such as ZnO and TiO2 by simple melt mixing method. Melt spinning method was used to prepare PP/metal oxide nanocomposite fibers. Various studies have been carried out on these composites and fibers. In the first part of the study, ZnO nanoparticles were prepared from ZnCl2 and NaOH in presence of chitosan, PVA, ethanol and starch. This is a simple and inexpensive method compared to other methods. Change in morphology and particle size of ZnO were studied. Least particle size was obtained in chitosan medium. The particles were characterized by using XRD, SEM, TEM, TGA and EDAX. Antibacterial properties of ZnO prepared in chitosan medium (NZO) and commercial zinc oxide (CZO) were evaluated using a gram positive and a gram negative bacteria

Fermentation Of Rice And Related Products By Bacillus Sp.

Recently’ recognition cnf immobilization ‘technology for the rapid conversion of several substrates into metabolites and repeated reuse of the biocatalysts have drawn the attention of the fermentation scientists and technologists to try these new technologies for the rapid production of pnxkmt and enhancement of the efficiencies of the systems Hence in the present study rice was selected,as a substrate since it is a rich source of starch, available and cultivated throughout the year almost in all part of our country. Rice although known for its use as a staple food in many forms as rice, idli, dosai etc., has not been used in industry extensively. However, it ii; a potential resource for’ the production of alcohol, high protein food anui for sugar and sugar syrups as it is evidenced by the few reports mentioned in the review of literature. Of the several microorganisms available, Bacillus sp, is a known candidate for the production of amylases. Hence in the present study Bacillus sp, was desired for its known efficiencies in starch conversion

Thermdstable alpha amylase production by bacillus coagulans

Microbial enzymes are in great demand owing to their importance in several industries such as brewing, baking, leather, laundry detergent, dairy. starch processing and textiles besides pharmaceuticals. About 80% of the enzymes produced through fermentation and sold in the industrial scale are hydrolytic enzymes. Due to recognition of new and new applications, an intensive screening of different kinds of enzymes with novel properties, from various microorganisms, is being pursued all over the world. Bacillus sp are largely known to produce a-amylase, among the different groups of microoganisms, at industrial level. They are known to produce both saccharifying and liquefying a-amylases (Fukumoto 1963; walker and Campbell, 1967a). which are distinguishable by their mechanisms of starch degradation by the fact that the saccharifying asamylases produce an increase in reducing power about twice that of the liquefying enzyme (Fukumoto, 1963; Pazur and Okada, 1966). Under this circumstances, the present study was undertaken, with a View to utilise a fast growing B.coagu1ans isolated from soil, for production of thermostable and alkaline oz-amylase under different fermentation processes

A brackishwater isolate of Pseudomonas PS-102, a potential antagonistic bacterium against pathogenic vibrios in penaeid and non-penaeid rearing systems

A Pseudomonas sp PS-102 recovered from Muttukkadu brackish water lagoon, situated south of Chennai, showed significant activity against a number of shrimp pathogenic vibrios. Out of the 112 isolates of bacterial pathogens comprising Vibrio harveyi, V. vulnificus, V. parahaemolyticus, V. alginolyticus, V. fluvialis, and Aeromonas spp, 73% were inhibited in vitro by the cell-free culture supernatant of Pseudomonas sp PS-102 isolate. The organism produced yellowish fluorescent pigment on King's B medium, hydrolysed starch and protein, and produced 36.4% siderophore units by CAS assay and 32 μM of catechol siderophores as estimated by Arnow's assay. The PS-102 isolate showed wide ranging environmental tolerance with, temperatures from 25 to 40 °C, pH from 6 to 8, salinity from 0 to 36 ppt, while the antagonistic activity peaked in cultures grown at 30 °C, pH 8.0 and at 5 ppt saline conditions. The antagonistic activity of the culture supernatant was evident even at 30% v / v dilution against V. harveyi. The preliminary studies on the nature of the antibacterial action indicated that the antagonistic principle as heat stable and resistant to proteolytic, lipolytic and amylolytic enzymes. Pseudomonas sp PS 102 was found to be safe to shrimp when PL-9 stage were challenged at 107 CFU ml−1 and by intramuscular injection into of ∼5 g sub-adults shrimp at 105 to 108 CFU. Further, its safety in a mammalian system, tested by its pathogenicity to mice, was also determined and its LD50 to BALB/c mice was found to be 109 CFU. The results of this study indicated that the organism Pseudomonas sp PS 102 could be employed as a potential probiont in shrimp and prawn aquaculture systems for management and control of bacterial infections

Banana waste as substrate for a-amylase production by Bacillus subtilis (CBTK 106) under solid-state fermentation

Bacillus subtilis CBTK 106, isolated from banana wastes, produced high titres of a-amylase when banana fruit stalk was used as substrate in a solid-state fermentation system. The e¤ects of initial moisture content, particle size, cooking time and temperature, pH, incubation temperature, additional nutrients, inoculum size and incubation period on the production of a- amylase were characterised. A maximum yield of 5 345 000 U mg~1 min~1 was recorded when pretreated banana fruit stalk (autoclaved at 121 ¡C for 60 min) was used as substrate with 70% initial moisture content, 400 lm particle size, an initial pH of 7.0, a temperature of 35 ¡C, and additional nutrients (ammonium sulphate/sodium nitrate at 1.0%, beef extract/peptone at 0.5%, glucose/sucrose/starch/maltose at 0.1% and potassium chloride/sodium chloride at 1.0%) in the medium, with an inoculum-to-substrate ratio of 10% (v/w) for 24 h. The enzyme yield was 2.65-fold higher with banana fruit stalk medium compared to wheat bran

Characterization of an extracellular alkaline serine protease from marine Engyodontium album BTMFS10

An alkaline protease from marine Engyodontium album was characterized for its physicochemical properties towards evaluation of its suitability for potential industrial applications. Molecular mass of the enzyme by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) analysis was calculated as 28.6 kDa. Isoelectric focusing yielded pI of 3–4. Enzyme inhibition by phenylmethylsulfonyl fluoride (PMSF) and aprotinin confirmed the serine protease nature of the enzyme.Km, Vmax, and Kcat of the enzyme were 4.727 9 10-2 mg/ml, 394.68 U, and 4.2175 9 10-2 s-1, respectively. Enzyme was noted to be active over a broad range of pH (6–12) and temperature (15–65 C), withmaximumactivity at pH 11 and 60 C. CaCl2 (1 mM), starch (1%), and sucrose (1%) imparted thermal stability at 65 C. Hg2?, Cu2?, Fe3?, Zn2?, Cd?, and Al3? inhibited enzyme activity, while 1 mMCo2? enhanced enzyme activity. Reducing agents enhanced enzyme activity at lower concentrations. The enzyme showed considerable storage stability, and retained its activity in the presence of hydrocarbons, natural oils, surfactants, and most of the organic solvents tested. Results indicate that the marine protease holds potential for use in the detergent industry and for varied applications.