Teh-Liang Chen

Production of an alkaline lipase by Acinetobacter radioresistens

Abstract Alkaline lipase production by Acinetobacter radioresistens was investigated and the role of culture conditions was examined. The enzyme had an optimum pH of 10 and was stable over a pH range of 6–10; it could have great potential for application in the detergent industry. The optimal temperature and pH for lipase fermentation were 30°C and 7, which were related to cell growth, protease formation, and stability and reactivity of lipase. A high lipase yield could be obtained during prolonged cultivation in the presence of n -hexadecane. With additional olive oil supplementation, the volumetric productivity of lipase could be improved; however, the lipase yield decreased with increasing concentrations of olive oil. The increase in the rate of lipase formation by olive oil in the presence of n -hexadecane was attributed to its enhancement of the uptake of n -hexadecane; the hydrolytic products of olive oil promoted the emulsification of n -hexadecane. Because olive oil repressed the synthesis of lipase, optimization in formulating the medium composition is considered necessary.

Effect of oxygen transfer on lipase production by Acinetobacter radioresistens.

The influence of oxygen on alkaline lipase production by Acinetobacter radioresistens was studied under two operating modes: controlled dissolved oxygen (DO) concentration and controlled aeration rate. Compared with cell growth, the lipase production depended more extensively on oxygen. The intrinsic factor determining cell growth and lipase production was oxygen transfer rate (OTR) rather than DO concentration. Improvements in OTR, either by aeration or agitation, resulted in an increase in lipase yield and/or a reduction in fermentation time. The formation of A. radioresistens lipase could be described by a mixed-growth-associated model, and the enzyme was mainly a growth-associated product. The overall productivity for the lipase, which depended more strongly on agitation than aeration, could be related with kLa. DO concentration could not be employed in this correlation, though it has been useful as a criterion for ensuring no oxygen limitation in an aerobic fermentation.

Production of Acinetobacter radioresistens lipase using Tween 80 as the carbon source

Lipase of Acinetobacter radioresistens was produced using Tween 80 as the carbon source. Compared with olive oil, the hydrolysis rate of Tween 80 in the fermentation broth was much slower. Taking advantage of the slow hydrolysis rate, Tween 80 could provide oleic acid for cell growth and lipase production through a mode of controlled release, and the known repression of lipase synthesis by oleic acid could be avoided. The optimal lipase yield obtained with batch culture in a 2.5-L tank fermentor was 25 U/mL with a fermentation time of 6 h, when using 0.3% (v/v) Tween 80. The existence of an optimal yield with respect to concentration of Tween 80 was a result of the compromise between cell growth and the repression of lipase synthesis. To further the lipase productivity, a fed-batch culture with the pH-stat feeding method was employed, and the lipase productivity achieved was 120 U/mL in 16 h. Various strategies for production of A. radioresistens lipase were compared.

Improvements of enzyme activity and enantioselectivity via combined substrate engineering and covalent immobilization

Esterases, lipases, and serine proteases have been applied as versatile biocatalysts for preparing a variety of chiral compounds in industry via the kinetic resolution of their racemates. In order to meet this requirement, three approaches of enzyme engineering, medium engineering, and substrate engineering are exploited to improve the enzyme activity and enantioselectivity. With the hydrolysis of (R,S)‐mandelates in biphasic media consisting of isooctane and pH 6 buffer at 55°C as the model system, the strategy of combined substrate engineering and covalent immobilization leads to an increase of enzyme activity and enantioselectivity from VS/(Et) = 1.62 mmol/h g and VS/VR = 43.6 of (R,S)‐ethyl mandelate (1) for a Klebsiella oxytoca esterase (named as SNSM‐87 from the producer) to 16.7 mmol/h g and 867 of (R,S)‐2‐methoxyethyl mandelate (4) for the enzyme immobilized on Eupergit C 250L. The analysis is then extended to other (R,S)‐2‐hydroxycarboxylic acid esters, giving improvements of the enzyme performance from VS/(Et) = 1.56 mmol/h g and VS/VR = 41.9 of (R,S)‐ethyl 3‐chloromandelate (9) for the free esterase to 39.4 mmol/h g and 401 of (R,S)‐2‐methoxyethyl 3‐chloromandelate (16) for the immobilized enzyme, VS/(Et) = 5.46 mmol/h g and VS/VR = 8.27 of (R,S)‐ethyl 4‐chloromandelate (10) for free SNSM‐87 to 33.5 mmol/h g and 123 of (R,S)‐methyl 4‐chloromandelate (14) for the immobilized enzyme, as well as VS/(Et) = 3.0 mmol/h g and VS/VR = 7.94 of (R,S)‐ethyl 3‐phenyllactate (11) for the free esterase to 40.7 mmol/h g and 158 of (R,S)‐2‐methoxyethyl 3‐phenyllactate (18) for the immobilized enzyme. The great enantioselectivty enhancement is rationalized from the alteration of ionization constants of imidazolium moiety of catalytic histidine for both enantiomers and conformation distortion of active site after the covalent immobilization, as well as the selection of leaving alcohol moiety via substrate engineering approach. Biotechnol. Bioeng. 2008;101: 460–469.

A pseudo-exponential feeding method for control of specific growth rate in fed-batch cultures

A simple feeding method for controlling specific growth rate in fed-batch culture was developed. This method applies a constant feed rate using a concentrate reservoir and two mixing chambers in series to simulate the exponential feeding. Fed-batch cultures with Escherichia coli showed that the present feeding method could sustain the cells growing at predetermined specific growth rates, where the time length for exponential growth was dependent on the magnitude of the growth rate. The present feeding method is convenient to operate, requires no computerized control equipments, and thus could expect an extensive application in fed-batch culture.

Lipase production by acinetobacter radioresistens in a batch fill- and-draw culture

Alkaline lipase production byAcinetobacter radioresistens was performed in a batch fill- and-draw culture, and the results were compared with a batch culture. In the batch culture, the lipase yield was 18 U/mL, which was restricted by the occurrence of protease forming and excessive foaming as cell growth ceased. Because the formation of lipase was found to be chiefly growth-associated, the fill- and-draw culture, which provided an environment for continuous growth, could surpass the limitation encountered in a batch culture and increase the lipase yield to 30 U/mL. The improvement in the lipase yield was suggested to be caused by an adaptation of the cells to the medium during the repeated culture. Although the increase in the lipase yield was accompanied by a decrease in lipase productivity, the fill- and-draw culture could be a better mode for lipase production.

Effect of specific growth rate on the production of a recombinant nuclease by Escherichia coli

The effect of specific growth rate µ on the production of Vibrio vulnificusnuclease was investigated in fed-batch cultures of a high-yield recombinant Escherichia coli. A pseudo-exponential feeding method for controlling µ was employed to obtain the desired data. It was found that the nuclease content of cells γ P varied with µ, with a maximum occurred at µ = 0.05 h −1 ; however, the specific nuclease production rate qp increased with increasing µ. The synthesis of nuclease was dependent on µ, irrespective of the feeding methods. When concerning the compromise between the nuclease yield and its production rate, the linear-gradient feeding method, being simple and adaptable, was shown to be adequate for the nuclease production.

Lipase Production by Acinetobacter radioresistens in the Presence of a Nonwoven Fabric

Lipase production by Acinetobacter radioresistens was performed in a 2‐L tank fermentor equipped with a nonwoven fabric, which was made of nylon 6 fiber and coated with a hydrophobic acrylic resin. The fermentation medium contained 2% (v/v) n‐hexadecane as the carbon source. The use of the nonwoven fabric was intended for the dispersion of hydrocarbons; thus, the contact surface for the cells to assimilate n‐hexadecane can be increased without using emulsifiers. The formation of lipase was found to be growth‐associated when the cells grew on n‐hexadecane. The use of the nonwoven fabric increased the lipase yield by 130%. Further supplementation of 0.1% (v/v) olive oil to the medium could markedly shorten the fermentation time, and thus increase the volumetric productivity. The use of the nonwoven fabric offered two additional advantages in the lipase fermentation: ease of foam control and favorable partition of lipase in the aqueous phase.

Initial protein concentration effects on precipitation by salt

The influence of initial protein concentration on the performance of salting-out precipitation is examined. In the precipitation of bovine albumin by ammonium sulfate, a peak occurs in the plot of protein solubility versus initial protein concentration; that is, the solubility first increases and then falls with increasing initial protein concentration. In addition, the dependence of solubility on the initial protein concentration is less significant if using higher salt concentrations. The solubility behavior of bovine albumin may be representative, because it covers all possible alternatives; namely, the solubility is independent of, increases with, decreases with, or first increases and then decreases with the initial protein concentration. The appearance of a solubility peak can be explained based on the occurrence of a primary particle during the precipitation process. However, inclusion of the influence of initial protein concentration into the Cohn equation is not feasible with the use of a logarithmic scale, which does not sensitively reflect the change in protein solubility. Increasing initial protein concentration favors protein recovery because it reduces the resultant volume of the supernatant phase. (c) l997 John Wiley & Sons, Inc.

Recovery of Acinetobacter radioresistens Lipase by Hydrophobic Adsorption to n‐Hexadecane Coated on Nonwoven Fabric

A simple and clean adsorption/desorption process was proposed for recovering Acinetobacter radio resisten s lipase from fermentation broth. The adsorbent used was n‐hexadecane coated on a hydrophobic nonwoven fabric (NWF). n‐Hexadecane has a melting point of 16–18 °C, and its affinity for lipase decreases markedly from liquid to solid state. Accordingly, performing the adsorption and desorption above and below, respectively, the melting point would need no extraneous materials for separation. The adsorption isotherms at various temperatures were found to follow the Langmuir model. Simulation of the batch adsorption/desorption process showed that there exists an optimal amount of adsorbent for both concentration factor and enzyme recovery; the process is restrained by equilibrium. The performance of column adsorption/desorption could also be simulated using the adsorption isotherm, and it was shown that the concentration factor was proportional to the amount of adsorbent used. The benefits of this process include easy preparation of adsorbent, low operational cost, no extraneous materials needed, negligible enzyme denaturation, high efficiency, and simple process simulation.