Growth associated biosynthesis of medium chain length poly-3-hydroxyalkanoates (mcl-PHA) in Pseudomonas putida Bet001 isolated from palm oil mill effluent was studied. Models with substrate inhibition terms described well the kinetics of its growth. Selected fatty acids (C8:0 to C18:1) and ammonium were used as carbon and nitrogen sources during growth and PHA biosynthesis, resulting in PHA accumulation of about 50 to 69% (w/w) and PHA yields ranging from 10.12 g L(-1) to 15.45 g L(-1), respectively. The monomer composition of the PHA ranges from C4 to C14, and was strongly influenced by the type of carbon substrate fed. Interestingly, an odd carbon chain length (C7) monomer was also detected when C18:1 was fed. Polymer showed melting temperature (T m) of 42.0 (± 0.2) °C, glass transition temperature (T g) of -1.0 (± 0.2) °C and endothermic melting enthalpy of fusion (ΔHf) of 110.3 (± 0.1) J g(-1). The molecular weight (M w) range of the polymer was relatively narrow between 55 to 77 kDa.
Free laccase and fungal biomass from white-rot fungi were compared in the thermokinetics study of the laccase-catalyzed decolorization of an azo dye, i.e., Trypan Blue. The decolorization in both systems followed a first-order kinetics. The apparent first-order rate constant, k1', value increases with temperature. Apparent activation energy of decolorization was similar for both systems at ∼ 22 kJ mol(-1), while energy for laccase inactivation was 18 kJ mol(-1). Although both systems were endothermic, fungal biomass showed higher enthalpy, entropy, and Gibbs free energy changes for the decolorization compared to free laccase. On the other hand, free laccase showed reaction spontaneity over a wider range of temperature (ΔT = 40 K) as opposed to fungal biomass (ΔT = 15 K). Comparison of entropy change (ΔS) values indicated metabolism of the dye by the biomass.
This review is focused on the production of microbial lipases by high cell density fermentation. Lipases are among the most widely used of the enzyme catalysts. Although lipases are produced by animals and plants, industrial lipases are sourced almost exclusively from microorganisms. Many of the commercial lipases are produced using recombinant species. Microbial lipases are mostly produced by batch and fed-batch fermentation. Lipases are generally secreted by the cell into the extracellular environment. Thus, a crude preparation of lipases can be obtained by removing the microbial cells from the fermentation broth. This crude cell-free broth may be further concentrated and used as is, or lipases may be purified from it to various levels. For many large volume applications, lipases must be produced at extremely low cost. High cell density fermentation is a promising method for low-cost production: it allows a high concentration of the biomass and the enzyme to be attained rapidly and this eases the downstream recovery of the enzyme. High density fermentation enhances enzyme productivity compared with the traditional submerged culture batch fermentation. In production of enzymes, a high cell density is generally achieved through fed-batch operation, not through perfusion culture which is cumbersome. The feeding strategies used in fed-batch fermentations for producing lipases and the implications of these strategies are discussed. Most lipase-producing microbial fermentations require oxygen. Oxygen transfer in such fermentations is discussed.
The biosynthesis and characterization of medium chain length poly-3-hydroxyalkanoates (mcl-PHA) produced by Pseudomonas putida Bet001 isolated from palm oil mill effluent was studied. The biosynthesis of mcl-PHA in this newly isolated microorganism follows a growth-associated trend. Mcl-PHA accumulation ranging from 49.7 to 68.9% on cell dry weight (CDW) basis were observed when fatty acids ranging from octanoic acid (C(8:0)) to oleic acid (C(18:1)) were used as sole carbon and energy source. Molecular weight of the polymer was found to be ranging from 55.7 to 77.7 kDa. Depending on the type of fatty acid used, the (1)H NMR and GCMSMS analyses of the chiral polymer showed a composition of even and odd carbon atom chain with monomer length of C4 to C14 with C8 and C10 as the principal monomers. No unsaturated monomer was detected. Thermo-chemical analyses showed the accumulated PHA to be semi-crystalline polymer with good thermal stability, having a thermal degradation temperature (T(d)) of 264.6 to 318.8 (± 0.2) (o)C, melting temperature (T(m)) of 43. (± 0.2) (o)C, glass transition temperature (T(g)) of -1.0 (± 0.2) (o)C and apparent melting enthalpy of fusion (ΔH(f)) of 100.9 (± 0.1) J g(-1).
Lipase-catalyzed synthesis of 6-O-glucosyldecanoate from d-glucose and decanoic acid was performed in dimethyl sulfoxide (DMSO), a mixture of DMSO and tert-butanol and tert-butanol alone with a decreasing order of polarity. The highest conversion yield (> 65%) of decanoic acid was obtained in the blended solvent of intermediate polarity mainly because it could dissolve relatively large amounts of both the reactants. The reaction obeyed Michaelis-Menten type of kinetics. The affinity of the enzyme towards the limiting substrate (decanoic acid) was not affected by the polarity of the solvent, but increased significantly with temperature. The esterification reaction was endothermic with activation energy in the range of 60-67 kJ mol⁻¹. Based on the Gibbs energy values, in the solvent blend of DMSO and tert-butanol the position of the equilibrium was shifted more towards the products compared to the position in pure solvents. Monoester of glucose was the main product of the reaction.
Ultrasonic irradiation greatly improved the Candida antarctica lipase B mediated ring opening polymerization of ε-caprolactone to poly-6-hydroxyhexanoate in the ionic liquid 1-ethyl-3-methylimidazolium tetraflouroborate. Compared to the conventional nonsonicated reaction, sonication improved the monomer conversion by 63% and afforded a polymer product of a narrower molecular weight distribution and a higher degree of crystallinity. Under sonication, the polydispersity index of the product was ~1.44 compared to a value of ~2.55 for the product of the conventional reaction. With sonication, nearly 75% of the monomer was converted to product, but the conversion was only ~16% for the reaction carried out conventionally. Compared to conventional operation, sonication enhanced the rate of polymer propagation by >2-fold and the turnover number of the lipase by >3-fold.
Four different lipases were compared for ultrasound-mediated synthesis of the biodegradable copolymer poly-4-hydroxybutyrate-co-6-hydroxyhexanoate. The copolymerization was carried out in chloroform. Of the enzymes tested, Novozym 435 exhibited the highest copolymerization rate, in fact the reaction rate was observed to increase with about 26-fold from 30 to 50°C (7.9×10(-3)Ms(-1)), sonic power intensity of 2.6×10(3)Wm(-2) and dissipated energy of 130.4Jml(-1). Copolymerization rates with the Candida antarctica lipase A, Candida rugosa lipase, and Lecitase Ultra™ were lower at 2.4×10(-4), 1.3×10(-4) and 3.5×10(-4)Ms(-1), respectively. The catalytic efficiency depended on the enzyme. The efficiency ranged from 4.15×10(-3)s(-1)M(-1) for Novozym 435-1.48×10(-3)s(-1)M(-1) for C. rugosa lipase. Depending on the enzyme and sonication intensity, the monomer conversion ranged from 8.2% to 48.5%. The sonication power, time and temperature were found to affect the rate of copolymerization. Increasing sonication power intensity from 1.9×10(3) to 4.5×10(3)Wm(-2) resulted in an increased in acoustic pressure (P(a)) from 3.7×10(8) to 5.7×10(8)Nm(-2) almost 2.4-3.7 times greater than the acoustic pressure (1.5×10(8)Nm(-2)) that is required to cause cavitation in water. A corresponding acoustic particle acceleration (a) of 9.6×10(3)-1.5×10(4)ms(-2) was calculated i.e. approximately 984-1500 times greater than under the action of gravity.
The effects of organic solvents and their binary mixture in the glucose functionalization of bacterial poly-3-hydroxyalkanoates catalyzed by Lecitase™ Ultra were studied. Equal volume binary mixture of DMSO and chloroform with moderate polarity was more effective for the enzyme catalyzed synthesis of the carbohydrate polymer at ≈38.2 (±0.8)% reactant conversion as compared to the mono-phasic and other binary solvents studied. The apparent reaction rate constant as a function of medium water activity (aw) was observed to increase with increasing solvent polarity, with optimum aw of 0.2, 0.4 and 0.7 (±0.1) observed in hydrophilic DMSO, binary mixture DMSO:isooctane and hydrophobic isooctane, respectively. Molecular sieve loading between 13 to 15gL(-1) (±0.2) and reaction temperature between 40 to 50°C were found optimal. Functionalized PHA polymer showed potential characteristics and biodegradability.
One-pot synthesis of sugar-functionalized oligomeric caprolactone was carried out by lipase-catalyzed esterification of ε-caprolactone (ECL) with methyl-d-glucopyranoside (MGP) followed by the elongation of functionalized oligomer chain. Functionalization was performed in a custom-fabricated glass reactor equipped with Rushton turbine impeller and controlled temperature at 60 °C using tert-butanol as reaction medium. The overall reaction steps include MGP esterification of ECL monomer and its subsequent elongation by free 6-hydroxyhexanoate monomer units. A ping-pong bi-bi mechanism without ternary complex was proposed for esterification of ECL and MGP with apparent values of kinetic constant, namely maximal velocity (Vmax ), Michaelis constant for MGP (KmMGP ), and Michaelis constant for ECL (KmECL ) at 3.848 × 10-3 M H-1 , 8.189 × 10-2 M, and 6.050 M, respectively. Chain propagation step of MGP-functionalized ECL oligomer exhibits the properties of living polymerization mechanism. Linear relationship between conversion (%) and number average molecular weight, Mn (g mol-1 ), of functionalized oligomer was observed. Synthesized functionalized oligomer showed narrow range of molecular weight from 1,400 to 1,600 g mol-1 with more than 90% conversion achieved. Structural analysis confirmed the presence of covalent bond between the hydroxyl group in MGP with carboxyl end group of ECL oligomer.
Lactobacillus plantarum BET003 isolated from Momordica charantia fruit was used to ferment its juice. Momordica charantia fresh juice was able to support good growth of the lactic acid bacterium. High growth rate and cell viability were obtained without further nutrient supplementation. In stirred tank reactor batch fermentation, agitation rate showed significant effect on specific growth rate of the bacterium in the fruit juice. After the fermentation, initially abundant momordicoside 23-O-β-Allopyranosyle-cucurbita-5,24-dien-7α,3β,22(R),23(S)-tetraol-3-O-β-allopyranoside was transformed into its corresponding aglycone in addition to the emergence of new metabolites. The fermented M. charantia juice consistently reduced glucose production by 27.2%, 14.5%, 17.1% and 19.2% at 15-minute intervals respectively, when compared against the negative control. This putative anti-diabetic activity can be attributed to the increase in availability and concentration of aglycones as well as other phenolic compounds resulting from degradation of glycosidic momordicoside. Biotransformation of M. charantia fruit juice via lactic acid bacterium fermentation reduced its bitterness, reduced its sugar content, produced aglycones and other metabolites as well as improved its inhibition of α-glucosidase activity compared with the fresh, non-fermented juice.
The biosynthesis of medium-chain-length poly-3-hydroxyalkanoates by Pseudomonas putida Bet001 cultivated on mixed carbon sources was investigated. The mixed carbon sources consisted of heptanoic acid (HA) and oleic acid (OA). A relatively low PHA content at 1.2% (w/w) and 11.4% (w/w) was obtained when HA or OA was used as the sole carbon source. When these fatty acids were supplied as a mixture, PHA content increased threefold. Interestingly, the mixture-derived PHA composed of both odd and even monomer units, namely. 3-hydroxyheptanoate, 3-hydroxyoctanoate, 3-hydroxydecanoate, and 3-hydroxydodecanoate and no unsaturated monomer was detected. It is hypothesized that the even-numbered monomers were derived primarily from OA, whereas the odd-numbered monomer was derived from HA. This also points out to an efficient and yet distinct fatty acids metabolism that fed the PHA biosynthesis machinery of this particular microorganism. PHA obtained was elastomeric because melting temperature (Tm ) and crystallinity were absent. It showed good thermal stability with degradation temperature (Td ) ranging from 275.96 to 283.05 °C.
Biosynthesis and in vivo depolymerization of intracellular medium-chain-length poly-3-hydroxyalkanoates (mcl-PHA) in Pseudomonas putida Bet001 grown on lauric acid were studied. Highest mcl-PHA fraction (>50 % of total biomass) and cell concentration (8 g L-1 ) were obtained at carbon-to-nitrogen (C/N) ratio 20, starting cell concentration 1 g L-1 , and 48 H fermentation. The mcl-PHA comprised of 3-hydroxyhexanoate (C6 ), 3-hydroxyoctanote (C8 ), 3-hydroxydecanoate (C10 ), and 3-hydroxydodecanoate (C12 ) monomers. In vivo action was studied in a mineral liquid medium without carbon source, and in different buffer solutions with varied pH, molarity, ionic strength, and temperature. The monomer liberation rate reflected the mol percentage distribution of the initial polymer subunit composition. Rate and percentage of in vivo depolymerization were highest in 0.2 M Tris-HCl buffer (pH 9, strength = 0.2 M, 30 °C) at 0.21 g L-1 H-1 and 98.6 ± 1.3 wt%, respectively. There is a congruity vis-à-vis to specific buffer type, molarity, pH, ionic strength, and temperature values for superior in vivo depolymerization activities. Direct products from in vivo depolymerization matched the individual monomeric composition of native mcl-PHA. It points to exo-type reaction for the in vivo process, and potential biological route to chiral molecules.
In vivo and in vitro depolymerizations of intracellular medium-chain-length poly-3-hydroxyalkanoates (mcl-PHA) in Pseudomonas putida Bet001 grown on lauric acid was studied. Both processes were studied under optimum conditions for mcl-PHA depolymerization viz. 0.2 M Tris-HCl buffer, pH 9, ionic strength (I) = 0.2 M at 30°C. For in vitro depolymerization studies, cell-free system was obtained from lysing bacterial cells suspension by ultrasonication at optimum conditions (frequency 37 kHz, 30% of power output, <25°C for 120 min). The comparison between in vivo and in vitro depolymerizations of intracellular mcl-PHA was made. In vitro depolymerization showed lower depolymerization rate but higher yield compared to in vivo depolymerization. The monomer liberation rate reflected the mol% distribution of the initial polymer subunit composition, and the resulting direct individual products of depolymerization were identical for both in vivo and in vitro processes. It points to exo-type reaction for both processes, and potential biological route to chiral molecules.
The present study reported for the first time covalent immobilization of protocatechuate 3,4-dioxygenase (3,4-POD) onto functionalized multi-walled carbon nanotubes (F-MWCNT) for degrading the toxic 3,4-dihydroxybenzoic acid (3,4-DHBA) pollutant in water. The F-MWCNTs had a maximum 3,4-POD loading of 1060 μg/mg. Immobilized 3,4 POD had 44% of relative structural changes to its free configurations. Nevertheless, >90% of relative activity and about 50% of catalytic efficiency were retained to the free enzyme. Immobilized 3,4-POD demonstrated higher alkaline stability and thermostability than the free 3,4-POD. The free and immobilized 3,4-POD lost 82% and 66% of relative activities, respectively after 180 min of incubations at 90 °C. Excellent shelf-life was observed for the immobilized 3,4-POD with residual activity of 56% compared with 41% and 39% of the free 3,4-POD at 4 °C and 25 °C over 30 days storage. Immobilized 3,4-POD showed >60% of catalytic activity retention even after ten-cycle uses, defraying the expenses of free 3,4-POD productions for long term uses. Finally, the immobilized 3,4-POD removed 71% of 3,4-DHBA from water in <4 h, paving its future application for water purification with reduced costs and time.
Zingiber zerumbet Smith is an important herb that contains bioactive phytomedicinal compound, zerumbone. To enhance cell growth and production of this useful compound, we investigated the growth conditions of cell suspension culture. Embryogenic callus generated from shoot bud was used to initiate cell suspension culture. The highest specific growth rate of cells was recorded when it was cultured in liquid Murashige and Skoog basal medium containing 3% sucrose with pH 5.7 and incubated under continuous shaking condition of 70 rpm for 16 h light and 8 h dark cycle at 24°C. Our results also revealed that the type of carbohydrate substrate, light regime, agitation speed, and incubation temperature could affect the production of zerumbone. Although the zerumbone produced in this study was not abundant compared to rhizome of Z. zerumbet, the possibility of producing zerumbone during early stage could serve as a model for subsequent improvement.
Production of extracellular laccase by the white-rot fungus Pycnoporus sanguineus was examined in batch submerged cultures in shake flasks, baffled shake flasks and a stirred tank bioreactor. The biomass growth in the various culture systems closely followed a logistic growth model. The production of laccase followed a Luedeking-Piret model. A modified Luedeking-Piret model incorporating logistic growth effectively described the consumption of glucose. Biomass productivity, enzyme productivity and substrate consumption were enhanced in baffled shake flasks relative to the cases for the conventional shake flasks. This was associated with improved oxygen transfer in the presence of the baffles. The best results were obtained in the stirred tank bioreactor. At 28 °C, pH 4.5, an agitation speed of 600 rpm and a dissolved oxygen concentration of ~25 % of air saturation, the laccase productivity in the bioreactor exceeded 19 U L(-1 )days(-1), or 1.5-fold better than the best case for the baffled shake flask. The final concentration of the enzyme was about 325 U L(-1).
Wound management and healing in several physiological or pathological conditions, particularly when comorbidities are involved, usually proves to be difficult. This presents complications leading to socio-economic and public health burdens. The accelerative wound healing potential of biocompatible poly(3-hydroxyalkanoates)-co-(6-hydroxyhexanoate) (PHA-PCL) composite hydrogel is reported herein. The biosynthesized PHA-PCL macromer was cross-linked with PEGMA to give a hydrogel. Twenty-four rats weighing 200-250 g each were randomly assigned to four groups of six rats. Rats in group I (negative control) were dressed with sterilized gum acacia paste in 10% normal saline while PEGMA-alone hydrogel (PH) was used to dress group II (secondary control) rats. Group III rats were dressed with PHAs-PCL cross-linked PEGMA hydrogel (PPH). For the positive control (group IV), the rats were dressed with Intrasite(®) gel. Biochemical, histomorphometric and immunohistomorphometric analyses revealed a significant difference in area closure and re-epithelialization on days 7 and 14 in PPH or Intrasite(®) gel groups compared to gum acacia or PEGMA-alone groups. Furthermore, wounds dressed with PPH or Intrasite(®) gel showed evident collagen deposition, enhanced fibrosis and extensively organized angiogenesis on day 14 compared to the negative control group. While improvement in wound healing of the PH dressed group could be observed, there was no significant difference between the negative control group and the PH dressed group in any of the tests. The findings suggested that topical application of PPH accelerated the rats' wound healing process by improving angiogenesis attributed to the increased microvessel density (MVD) and expressions of VEGF-A in tissue samples. Thus, PPH has been demonstrated to be effective in the treatment of cutaneous wounds in rats, and could be a potential novel agent in the management and acceleration of wound healing in humans and animals.
Purification of cyclodextrin glycosyl transferase (CGTase) from Bacillus cereus using polyethylene glycol (PEG)-potassium phosphates aqueous two-phase flotation (ATPF) system was studied in this paper. The effects of varying PEG molecular weight, tie-line length (TLL) value, volume ratio (VR), pH value, crude concentration and gas nitrogen flotation time were investigated. The optimal condition for purification of CGTase was attained at 18.0% (w/w) PEG 8000, 7.0% (w/w) potassium phosphates, VR of 3.0, 20% (w/w) crude load at pH 7, and 80 min nitrogen flotation time at a flow rate of 5 L/min. With this optimal condition, purification factor (PFT) of 21.8 and a yield (YT) of 97.1% were attained. CGTase was successfully purified in a single downstream processing step using the ATPF.
Aqueous two-phase system (ATPS) extractive bioconversion provides a technique which integrates bioconversion and purification into a single step process. Extractive bioconversion of gamma-cyclodextrin (γ-CD) from soluble starch with cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) enzyme derived from Bacillus cereus was evaluated using polyethylene glycol (PEG)/potassium phosphate based on ATPS. The optimum condition was attained in the ATPS constituted of 30.0% (w/w) PEG 3000 g/mol and 7.0% (w/w) potassium phosphate. A γ-CD concentration of 1.60 mg/mL with a 19% concentration ratio was recovered after 1 h bioconversion process. The γ-CD was mainly partitioned to the top phase (YT=81.88%), with CGTase partitioning in the salt-rich bottom phase (KCGTase=0.51). Repetitive batch processes of extractive bioconversion were successfully recycled three times, indicating that this is an environmental friendly and a cost saving technique for γ-CD production and purification.