Efficient cell surface display of organophosphorous hydrolase using N-terminal domain of ice nucleation protein in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Recombinant Escherichia coli systems expressing organophosphorous hydrolase (OPH) have been used for detoxifying toxic organophosphate compounds. However, a whole cell biocatalyst system has an intrinsic problem due to substrate diffusion limitation by its cell membrane. As a strategy for reducing this diffusion barrier limitation to enhance whole cell biocatalytic activity, we engineered E. coli cells to target OPH on cell surface using ice nucleation protein (InaK) as a surface targeting motif, especially N-terminal domain of InaK (InaK-N). The whole cell OPH activities of the cells expressing InaK/OPH fusion constructs were higher (∼2.5-fold for InaK-N and ∼5.7-fold for combined N-and C-terminal domain of InaK (InaK-NC)) than that of the cells expressing cytosolic OPH. Interestingly, the membrane targeting efficiency of the cells expressing InaK-N/OPH fusion proteins was ∼2.2-fold higher compared to the cells expressing InaK-NC/OPH even though both whole cell and total cell lysate OPH activities were lower. Therefore, we found that the small size N-terminal domain of InaK is more efficient for targeting OPH on the cell surface, and the surface display of OPH using N-terminal InaK domain can reduce the mass-transfer problem in whole cell bioconversion system. This work was presented at 13 ^th YABEC symposium held at Seoul, Korea, October 20–22, 2007     

Lactic acid production using free cells of bacteria and filamentous fungi and cells immobilized in polyvinyl alcohol cryogel: A comparative analysis of the characteristics of biocatalysts and processes

The results from studies and a comparative analysis of process characteristics are presented for lactic acid (LA) production from glucose, performed under batch conditions of the long-duration use of different biocatalyst samples comprising cells of Lactobacillus сasei bacteria and Rhizopus oryzae filamentous fungi immobilized in polyvinyl alcohol (PVA) cryogel or applied as concentrated suspensions. It is established that for LA production, the bacteria and fungi must be used in the form of PVA-cryogel-immobilized cells because their half-life in this form is considerably longer than that of concentrated cell suspensions. After 200 h of the batch use of the same immobilized cell samples, the amounts of accumulated LA were similar for both fungal (920 ± 5 g) and bacterial (895 ± 5 g) biocatalysts. The fungal biocatalyst, however, was characterized by a twice higher rate of substrate conversion to product (0.92 g LA per 1 g glucose) than the bacterial biocatalyst. The half-life of the immobilized fungal biocatalyst was 80 days (96 working cycles), ten times longer than that of the bacterial biocatalyst. A comparison of our data and the literature data demonstrated the promise of using fungal cells immobilized in PVA cryogel to produce LA: the process based on their use is superior to all known processes in its main indicators, i.e., the rate of LA conversion to glucose and the maximum accumulated concentration of the product.     

Functional display of active bovine adrenodoxin on the surface of E. coli by chemical incorporation of the [2Fe-2S] cluster

The display of heterologous proteins on the surface of living cells bears promising options for a wide variety of biotechnological applications. Up to now, however, cellular surface display was merely restricted to simple polypeptide chains. Here we present for the first time the efficient display of a protein (bovine adrenodoxin) that contains an inorganic, prosthetic group in its active form on the surface of Escherichia coli. For this purpose apo-adrenodoxin was transported to the cell surface and anchored within the outer membrane by the autotransporter pathway. Incorporation of the iron-sulfur cluster was achieved by a single-vial, one-step titration under anaerobic conditions. The biological function of surface-displayed holo-adrenodoxin could be established through adrenodoxin-dependent steroid conversion by two different cytochrome P450 enzymes and the number of functional molecules on the cell surface could be determined to be more than 10(5) per cell. Neither the expression of adrenodoxin nor the incorporation of the chemical iron-sulfur cluster reduced the viability of the bacterial cells.     

2-KGA混菌发酵大小菌耦合关系研究及建模

研究了维生素C前体2-酮基-L-古龙酸(2-KGA)混菌发酵巨大芽孢杆菌(俗称大菌)与普通生酮古龙酸菌(俗称小菌)的耦合关系。经研究发现:(1)大菌通过群体感应调整自身生长行为;(2)大菌活菌代谢物及其自溶物质弥补小菌代谢缺陷促进小菌生长;(3)小菌可能通过释放溶菌酶促使大菌自溶;(4)大菌自溶释放一些蛋白酶类物质提高小菌山梨糖脱氢酶(SDH)活性从而加速产物形成。基于以上研究建立了2-KGA混菌发酵的动力学模型,并用实验数据证明了模型的有效性。     

金黄节杆菌CYC705腈水解酶催化亚氨基二乙酸合成的研究

将金黄节杆菌CYC705(Arthrobacter aurescens CYC705) 腈水解酶用于生物催化合成亚氨基二乙酸(IDA),从生物催化剂的形式、生物催化反应过程优化和反应体系放大三个方面进行了考察。在氨基载体固定化酶、环氧基载体固定化酶、海藻酸钠固定化细胞、壳聚糖固定化细胞和游离全细胞几种生物催化剂形式中,壳聚糖固定化细胞催化效率最高、稳定性最好。通过反应体系、反应温度、金属离子、底物浓度、固定化细胞投量等因素的优化,确定了最佳的生物催化反应条件：以50 mmol/L pH=6.6的磷酸氢二钠-柠檬酸缓冲液作为反应体系,底物亚氨基二乙腈(IDAN)的浓度为200 mmol/L,添加CoCl2至终浓度为1 mmol/L,反应温度37 ˚C,固定化细胞投量为0.25 g每5 mL反应体积。在此条件下,反应2h可将IDAN完全转化为IDA。进一步将反应体系放大10倍,催化200 mmol/L的IDAN完全转化为IDA仅需1h。     

Optimization of lipase-catalyzed sorbitol monoester synthesis in organic medium

The influence of acid/polyol molar ratio and reaction time on lipase-catalyzed esterification of oleic acid (OA) and sorbitol was studied to determine optimal conditions for monoester synthesis. A simple mathematical model was developed to determine relationships between various parameters of technical and/or economical importance. A direct relationship. independent of OA initial concentration and reaction time, was shown between the percentage of monoester in total esters, monoester concentration (the maximum was 25–30 mM for 70–80% monoester), and sorbitol conversion rate. A high sorbitol conversion was always associated with a low percentage of monoester in total ester. No absolute optimum could be found, so that compromises should be chosen, with the help of the results presented herein, depending on the constraints on the process. Two possible optima are proposed. In both examples, monoester (25–30 mM) is 80% pure. In the first case, productivity is maximized (15 mmol·L⁻¹·h⁻¹), but OA and sorbitol conversions are only 40 and 60%, respectively. In the second case, a high OA conversion (>99%) is favored at the expense of monoester productivity (1.8 mmol·L⁻¹·h⁻¹), 60% of the sorbitol being converted. It was shown that, although sorbitol monoester has better surface properties than diester, the addition of 20% diester did not modify the interfacial activity of monoester and slightly increased its surface activity.     

甘油诱导氧化葡萄糖酸杆菌山梨醇脱氢酶

在山梨醇培养基中,梯度增大甘油质量浓度诱导提高氧化葡萄糖酸杆菌静息细胞山梨醇脱氢酶活力。当甘油质量浓度为20 g/L时,山梨醇脱氢酶的比酶活达到114 U/mg,比未用甘油诱导的提高了67%。甘油诱导后的山梨醇脱氢酶对山梨醇的亲和力增加至1.34倍(Ks从51 mmol/L变为38 mmol/L);同时其储存稳定性提高,用甘油诱导的菌体静息细胞储存30 d后,其山梨醇脱氢酶比酶活是新制备菌体静息细胞的75%,未诱导的只有30%。该工作的新颖性已为陕西省科学技术信息研究所科技查新中心2008年9月18日出具的第2008729号《科技查新报告》所证实。     

Colorimetric monitoring of the activity of recombinant Escherichia coli expressing styrene monooxygenase

Biocatalysis using recombinant Escherichia coli expressing styrene monooxygenase (SMO) activity is recognized as a promising process for the production of enantiopure aryl epoxides such as (S)-styrene oxide. Generally the activity of the whole cell biocatalyst is determined by measuring the conversion of styrene into (S)-styrene oxide by gas chromatography, which is however improper for the high-throughput analysis of many samples. Here we present that styrene monooxygenase catalyzed conversion of indole into indigo can be employed to monitor the activity of recombinant E. coli expressing styrene monooxygenase. We first confirmed that the colorimetric method was effective to monitor the whole cell activity of SMO by comparing the E. coli expressing styAB with that expressing only styB in a solid culture system. Next we used the monitoring method to investigate the effect of chaperone coexpression on the SMO whole cell activity in a liquid culture system.     

Aureobasidium subglaciale F134 is a bifunctional whole-cell biocatalyst for Baeyer-Villiger oxidation or selective carbonyl reduction controllable by temperature

The microbial production of either ester/lactones or enantio-enriched alcohols through Baeyer-Villiger oxidation or stereoselective reduction of ketones, respectively, is possible by using whole cells of A. subglaciale F134 as a bifunctional biocatalyst. The chemoselective pattern of acetophenone biotransformation catalyzed by these cells can be regulated through reaction temperature, directing the reaction either towards oxidation or reduction products. The Baeyer-Villiger oxidation activity of A. subglaciale F134 whole cells is particularly dependent on reaction temperature. Acetophenone was transformed efficiently to phenol via the primary Baeyer-Villiger product phenyl acetate at 20℃ after 48 h with 100% conversion. In contrast, at 35℃, enantio-enriched (S)-1-phenylethanol was obtained as the sole product with 64% conversion and 89% ee. In addition, A. subglaciale F134 cells also catalyze the selective reduction of various structurally different aldehydes and ketones to alcohols with 40% to 100% yield, indicating broad substrate spectrum and good enantioselectivity in relevant cases. Our study provides a bifunctional biocatalyst system that can be used in Baeyer-Villiger oxidation as well as in asymmetric carbonyl reduction, setting the stage for future work concerning the identification and isolation of the respective enzymes.     

Two-step bioprocess employing whole cell and enzyme for economical biodiesel production

A preliminary research for feasible and economical bioprocess for biodiesel production was carried out. A microorganism producing lipase was isolated from grease-contaminated soil. After chemically mutating twice, the microorganism exhibited 2.5 times higher intracellular lipase activities than wild type one and was used as a whole cell biocatalyst for biodiesel production. 0.1% toluene treatment followed by freeze drying was the optimum preparation method for whole cell biocatalyst. 1 g whole cell biocatalyst corresponded to 0.15 g commercial enzyme under mild methanol inhibition condition, while corresponding to 0.7 g enzyme under severe methanol inhibition environment. A two-step process, biodiesel production by the whole cell and sequentially by commercial enzyme, was suggested. This novel process, combining the advantages of whole cell and enzyme, saved the usage of commercial enzyme and alleviated enzyme deactivation by methanol.     

Stereodivergent Biocatalytic Formal Reduction of α-Angelica Lactone to (R)- and (S)-γ-Valerolactone in a One-Pot Cascade

The formal asymmetric and stereodivergent enzymatic reduction of α-angelica lactone to both enantiomers of γ-valerolactone was achieved in a one-pot cascade by uniting the promiscuous stereoselective isomerization activity of Old Yellow Enzymes with their native reductase activity. In addition to running the cascade with one enzyme for each catalytic step, a bifunctional isomerase-reductase biocatalyst was designed by fusing two Old Yellow Enzymes, thereby generating an unprecedented case of an artificial enzyme catalyzing the reduction of nonactivated C=C bonds to access (R)-valerolactone in overall 41 % conversion and up to 91 % ee. The enzyme BfOYE4 could be used as single biocatalyst for both steps and delivered (S)-valerolactone in up to 84 % ee and 41 % overall conversion. The reducing equivalents were provided by a nicotinamide recycling system based on formate and formate dehydrogenase, added in a second step. This enzymatic system provides an asymmetric route to valuable chiral building blocks from an abundant bio-based chemical.     

Regioselective Synthesis of Palm-Based Sorbitol Esters as Biobased Surfactant by Lipase from Thermomyces lanuginosus in Nonaqueous Media

This paper describes the regioselective production of palm-based sorbitol monoesters via esterification catalyzed by Lipozyme^® TL IM (Thermomyces lanuginosus lipase adsorbed onto silica gel, Novozymes, Inc., Franklington, NC, USA). Effects of various reaction parameters including types of solvent, substrate molar ratio, molecular sieve and lipase concentration, temperature, reaction time, and fatty acid chain length were investigated. Approximately 76% conversion of sorbitol to sorbitol esters was achieved within 24 h under optimal conditions: sorbitol (0.4 M), fatty acid (0.8 M), 20 wt% Lipozyme^® TL IM in 100 mL tert-butanol at 55 °C for 24 h in the presence of 25 wt% 3 Å molecular sieve as water absorbent. The reactions were conducted in an orbital incubator shaker at a shaking rate of 200 rpm. Lipozyme^® TL IM was highly regioselective, esterifying exclusively at sorbitol's primary hydroxyl groups, producing 1-O- and 6-O-sorbitol monoesters. The biocatalyst also exhibited substrate selectivity toward shorter chain acyl donors, with caprylic acid exhibiting the highest conversion of sorbitol. In addition, Lipozyme^® TL IM was reused up to four successive reaction cycles without significant loss of activity. The biocatalytic process reported in this paper is a one-step process to produce biobased surfactants that does not involve the use of toxic or expensive solvents that are commonly employed for derivatization of sugars, or pre-derivatization of the substrates molecules.     

Chemicals from biomass: synthesis of lactic acid by alkaline hydrothermal conversion of sorbitol

BACKGROUND: Currently, the ‘green chemistry’ philosophy is being increasingly adopted by the chemical industry and, therefore, new production procedures of valuable chemicals from biomass‐derived raw materials are being sought. In this work, the synthesis of lactic acid from sorbitol under alkaline hydrothermal conditions is investigated by analyzing the influence on conversions and yields of temperature, NaOH/sorbitol molar ratio (MR), initial sorbitol concentration (SC) and reaction time. RESULTS: A 100% sorbitol conversion and a maximum 39.5% yield of lactic acid on a carbon basis are obtained at 280 °C, 50 min, 1.0 mol L^?1 SC and 2.0 MR. Glyceraldehyde was the only identified intermediate while formic acid, acrylic acid, acetic acid, oxalic acid and sodium carbonate were identified as over‐oxidation products, all of them in very low yields with the exception of formic acid (16% yield at a MR of 4 and 280 °C). Several plausible conversion routes of sorbitol involving dehydrations, keto‐enol tautomerisms, reverse aldol condensations, aldol condensations, Cannizzaro reactions and oxidations are proposed. CONCLUSIONS: Considering the high number of parallel conversion routes as a consequence of high functionality of sorbitol, the 39.5% lactic acid yield obtained is a good result. Total carbon mass in all identified products only justifies, at most, 50% of that in sorbitol due to the coexistence of several conversion routes resulting in a large number of products other than lactic acid. Copyright © 2011 Society of Chemical Industry     

Development of an ethanol/sorbitol process using a ceramic membrane cell recycle bioreactor

The properties of a ceramic microfiltration membrane unit have been investigated as a component of a cell recycle bioreactor used for the production of ethanol/sorbitol. The conversion is carried out by a glucose/fructose oxidoreductase (and bound co-factor NADP) using cells of Zymomonas mobilis and an equimolar mixture of glucose and fructose. With 100 g/l each of glucose and fructose, a product stream containing 40 g/l sorbitol and 75 g/l ethanol was obtained. The potential economic benefits of such a process are discussed.     

Enantioselective Biooxidation of Racemic trans‐Cyclic Vicinal Diols: One‐Pot Synthesis of Both Enantiopure (S,S)‐Cyclic Vicinal Diols and (R)‐α‐Hydroxy Ketones

Highly regio‐ and enantioselective alcohol dehydrogenases BDHA (2,3‐butanediol dehydrogenase from Bacillus subtilis BGSC1A1), CDDHPm (cyclic diol dehydrogenase from Pseudomonas medocina TA5), and CDDHRh (cyclic diol dehydrogenase from Rhodococcus sp. Moj‐3449) were discovered for the oxidation of racemic trans‐cyclic vicinal diols. Recombinant Escherichia coli expressing BDHA was engineered as an efficient whole‐cell biocatalyst for the oxidation of (±)‐1,2‐cyclopentanediol, 1,2‐cyclohexanediol, 1,2‐cycloheptane‐diol, and 1,2‐cyclooctanediol, respectively, to give the corresponding (R)‐α‐hydroxy ketones in >99% ee and (S,S)‐cyclic diols in >99% ee at 50% conversion in one pot. Escherichia coli (BDHA‐LDH) co‐expressing lactate dehydrogenase (LDH) for intracellular regeneration of NAD⁺ catalyzed the regio‐ and enantioselective oxidation of (±)‐1,2‐dihydroxy‐1,2,3,4‐tetrahydronaphthalene to produce the corresponding (R)‐α‐hydroxy ketone in >99% ee and (S,S)‐cyclic diol in 96% ee at 49% conversion. Preparative biotransformations were also demonstrated. Thus, a novel and useful method for the one‐pot synthesis of both vicinal diols and α‐hydroxy ketones in high ee was developed via highly regio‐ and enantioselective oxidations of the racemic vicinal diols.

     

Functional display of Rhizomucor miehei lipase on surface of Saccharomyces cerevisiae with higher activity and its practical properties

BACKGROUND: A display system, which can translate DNA to functional peptides or proteins, is used as a new protein expression system. In this system, peptides or proteins are displayed on the cell surface as a fusion form with some anchoring proteins. Yeast cells displaying lipases on their cell‐surface could be used as whole‐cell biocatalysts. This research focuses on the functional display of Rhizomucor miehei lipase (RML) on the surface of Saccharomyces cerevisiae with higher activity. RESULTS: The lipases (RML) from R.miehei 3.4960 were of active form. The RML‐α‐agglutinin fusion proteins produced were not secreted into the culture media and were mostly immobilized on the yeast cells. Cell surface displayed lipase showed the highest activity at 45 °C and pH 8.0. CONCLUSION: The gene encoding RML from R.miehei 3.4960 can be functionally expressed on the cell surface of S. cerevisiae MT8‐1 using a glycosylphosphatidylinositol (GPI) anchor with higher activity. Copyright © 2007 Society of Chemical Industry     

Aureobasidium subglaciale F134 is a bifunctional whole-cell biocatalyst for Baeyer–Villiger oxidation or selective carbonyl reduction controllable by temperature

The microbial production of either ester/lactones or enantio-enriched alcohols through Baeyer–Villiger oxidation or stereoselective reduction of ketones, respectively, is possible by using whole cells of A. subglaciale F134 as a bifunctional biocatalyst. The chemoselective pattern of acetophenone biotransformation catalyzed by these cells can be regulated through reaction temperature, directing the reaction either towards oxidation or reduction products. The Baeyer–Villiger oxidation activity of A. subglaciale F134 whole cells is particularly dependent on reaction temperature. Acetophenone was transformed efficiently to phenol via the primary Baeyer–Villiger product phenyl acetate at 20 °C after 48 h with 100% conversion. In contrast, at 35 °C, enantio-enriched (S)-1-phenylethanol was obtained as the sole product with 64% conversion and 89% ee. In addition, A. subglaciale F134 cells also catalyze the selective reduction of various structurally different aldehydes and ketones to alcohols with 40% to 100% yield, indicating broad substrate spectrum and good enantioselectivity in relevant cases. Our study provides a bifunctional biocatalyst system that can be used in Baeyer–Villiger oxidation as well as in asymmetric carbonyl reduction, setting the stage for future work concerning the identification and isolation of the respective enzymes.     

Photo-biohydrogen Production by Photosensitization with Biologically Precipitated Cadmium Sulfide in Hydrogen-Forming Recombinant Escherichia coli

An inorganic-biological hybrid system that integrates features of both stable and efficient semiconductors and selective and efficient enzymes is attractive for facilitating the conversion of solar energy to hydrogen. In this study, we aimed to develop a new photocatalytic hydrogen-production system based on Escherichia coli whole-cell genetically engineered as a biocatalysis for highly active hydrogen formation. The photocatalysis part was obtained by bacterial precipitation of cadmium sulfide (CdS), which is a visible-light-responsive semiconductor. The recombinant E. coli cells were sequentially subjected to CdS precipitation and heterologous [FeFe]-hydrogenase synthesis to yield a CdS@E. coli hybrid capable of light energy conversion and hydrogen formation in a single cell. The CdS@E. coli hybrid achieved photocatalytic hydrogen production with a sacrificial electron donor, thus demonstrating the feasibility of our system and expanding the current knowledge of photosensitization using a whole-cell biocatalyst with a bacterially precipitated semiconductor.     

A highly stable whole‐cell biocatalyst for the enantioselective synthesis of optically active alpha‐hydroxy acids

BACKGROUND: Biocatalysts have gained increasing attention because of their inherent advantages over chemical catalysts. However, the poor operational stability has always prevented their broad application. In this study, (R)‐mandelic acid was chosen as a model compound of alpha‐hydroxy acids. The objective was to obtain a new biocatalyst with desired operational stability for the preparation of (R)‐mandelic acid as well as other optically pure alpha‐hydroxy acids of pharmaceutical importance. RESULTS: Using a two‐step screening strategy, Saccharomyces ellipsoideus GIM2.105 was selected as an effective biocatalyst with high enantioselectivity and remarkable operational stability. After 20 cycles of reuse, whole cells of S. ellipsoideus GIM2.105 maintained its activity, and no obvious decrease in conversion or enantiomeric excess (ee) was observed. Furthermore, effects of various reaction parameters, including pH, temperature, co‐substrate (type, concentration), substrate concentration and reaction time, on the bioreduction were studied. Under optimal conditions, (R)‐mandelic acid and four substituted aromatic (R)‐alpha‐hydroxy acids were prepared in high ee (95–>99%) and good conversion (>90%). CONCLUSION: The high enantioselectivity, remarkable operational stability and mild reaction conditions showed S. ellipsoideus GIM2.105 to be an economical biocatalyst with great industrial application potential for the production of optically active alpha‐hydroxy acids. Copyright © 2009 Society of Chemical Industry