Cell Biocatalyst Research Articles

The Kinetics of Interesterfication on Waste Cooking Oil (Sunflower Oil) for the Production of Fatty Acid Alkyl Esters using a Whole Cell Biocatalyst (Rhizopus oryzae) and Pure Lipase Enzyme

Recent research and technology have provided promising outcomes to rely on biodiesel as the alternative and conventional source of fuel. The use of renewable sources constitutes the main stream of research. Waste Cooking Oil (WCO) was used for biodiesel production in this study. Lipase enzyme producing fungi Rhizopus oryzae 262 and commercially available pure lipase enzyme were used for comparative study in the production of FAAE. The whole cell biocatalyst and pure enzyme were immobilized using calcium alginate beads. It was prepared by optimizing with different molar ratios of calcium chloride and different percent sodium alginate. Entrapment immobilization was done for whole cell biocatalyst. PE was also immobilized by entrapment for the transesterification reaction. Four different solvents methanol, ethanol, n-propanol, n-butanol were used as the acyl acceptors. The reaction parameters like temperature, molar ratio, reaction time, and amount of enzyme to be used were also optimized for methanol alone. The same parameters were adopted for the other acyl acceptors too. Among the different acyl acceptors, methanol whose reaction parameters were optimized showed maximum conversion of triglycerides to FAAE - 94% with PE and 84% with WCB. On the whole, PE showed better catalytic converting ability with all the acyl acceptor compared to WCB, further study, it was observed that three consecutive and reversible reactions occurred in the interesterification of triglycerides. So, a kinetic model based on Michaelis-Menten equation with competitive substrate inhibition was used to find the maximum reaction rate Vi for the four solvents using pure enzyme and WCB.

Autodisplay for the co-expression of lipase and foldase on the surface of E. coli: washing with designer bugs

BackgroundLipases including the lipase from Burkholderia cepacia are in a main focus in biotechnology research since many years because of their manifold possibilities for application in industrial processes. The application of Burkholderia cepacia lipase for these processes appears complicated because of the need for support by a chaperone, the lipase specific foldase. Purification and reconstitution protocols therefore interfere with an economic implementation of such enzymes in industry. Autodisplay is a convenient method to express a variety of passenger proteins on the surface of E. coli. This method makes subsequent purification steps to obtain the protein of interest unnecessary. If enzymes are used as passengers, the corresponding cells can simply be applied as whole cell biocatalysts. Furthermore, enzymes surface displayed in this manner often acquire stabilization by anchoring within the outer membrane of E. coli.ResultsThe lipase and its chaperone foldase from B. cepacia were co-expressed on the surface of E. coli via autodisplay. The whole cell biocatalyst obtained thereby exhibited an enzymatic activity of 2.73 mU mL-1 towards the substrate p-nitrophenyl palmitate when applied in an OD578 =1. Outer membrane fractions prepared from the same culture volume showed a lipase activity of 4.01 mU mL-1. The lipase-whole cell biocatalyst as well as outer membrane preparations thereof were used in a standardized laundry test, usually adopted to determine the power of washing agents. In this test, the lipase whole cell biocatalyst and the membrane preparation derived thereof exhibited the same lipolytic activity as the purified lipase from B. cepacia and a lipase preparation which is already applied in commercial washing agents.ConclusionsCo-expression of both the lipase and its chaperone foldase on the surface of E. coli yields a lipid degrading whole cell biocatalyst. Therefore the chaperone supported folding process, absolutely required for the lipolytic activity appears not to be hindered by surface display. Furthermore, the cells and the membrane preparations appeared to be stable enough to endure a European standard laundry test and show efficient fat removal properties herein.

Biotransformation of 3-cyanopyridine to nicotinic acid by free and immobilized cells of recombinant Escherichia coli

An efficient biocatalytic process for the production of nicotinic acid (niacin) from 3-cyanopyridine was developed using cells of recombinant Escherichia coli JM109 harboring the nitrilase gene from Alcaligenes faecalis MTCC 126. The freely suspended cells of the biocatalyst were found to withstand higher concentrations of the substrate and the product without any signs of substrate inhibition. Immobilization of the cells further enhanced their substrate tolerance, stability and reusability in repetitive cycles of nicotinic acid production. Under optimized conditions (37°C, 100mM Tris buffer, pH 7.5) for the immobilized cells, the recombinant biocatalyst achieved a 100% conversion of 1M 3-cyanopyridine to nicotinic acid within 5h at a cell mass concentration (fresh weight) of 500mg/mL. The high substrate/product tolerance and stability of the immobilized whole cell biocatalyst confers its potential industrial use.

FATSOLUBLE VITAMINS AND SPORT

Vitamins are the cell biocatalysts, indispensable factors in performing the basic body functions. Fat-soluble vitamins are not involved in processes related to musscle contractions and energy expenditure, but they can affect physical performance indirectly because they are important for immune function (vitamin A, vitamin D, vitamin E), antioxidant function (vitamin A, vitamin E) or bone methabolism (vitamin D, vitamin K). Currently there are no clear recommendations for increase of fat-solubile vitamins intake in athletes, as well as evidence that athletic performance may be improved due to fatsolubile vitamins supplementation. In a small number of studies, it was shown that an antioxidant effect of beta carotene and vitamin E can prevent muscle damage and facilitate recovery after exercise. Also, athletes who perform the exercises in the halls should be informed about the necessity of sun exposure, as vitamin D is synthesised in the skin. Most athletes are not familiar with their needs for vitamins and trace elements, and take these compounds as supplements without consulting a nutritionist. It is important to emphasize that liposulubile vitamins are deposited in the body and can cause hypervitaminosis and toxic effects if taken in excess. It is indisputable that the lack of any fat-soluble vitamin cause problems in normal physiological processes, but supplementation is generally not required in athlets who have a well-balanced diet. Acta Medica Medianae 2013;52(4):63-68.

Pseudomonas sp. BCNU 154 유래의 유기용매 내성 리파아제

산업공단지역의 폐수에서 분리한 유기용매 내성 Pseudomonas sp. BCNU 154 리파아제의 최적조건은 <TEX>$37^{\circ}C$</TEX>, pH8로 조사되었다. BCNU 154의 crude 리파아제는 toluene에서 2시간 반응시 효소활성 약 6.01 U/ml (117.53%)로 가장 안정한 것으로 나타났다. 한편 <TEX>$Ca^{2+}$</TEX>, <TEX>$Mg^{2+}$</TEX>, <TEX>$NH_4{^+}$</TEX>, <TEX>$Na^+$</TEX> 이온 및 triton X-100은 효소를 활성화시킨 반면에 <TEX>$Ba^{2+}$</TEX>, <TEX>$Hg^{2+}$</TEX> 및 <TEX>$Zn^{2+}$</TEX> 이온은 효소활성을 억제하였다. Pseudomonas sp. BCNU 154 리파아제는 상용 고정화 효소인 Novozym 435와 비교해서도 안정한 활성을 보였다. 그러므로 유기용매 내성 리파아제는 별도의 고정화 처리없이도 화학산업공정에서 가능성 있는 whole cell 생물촉매로서 유용할 것으로 판단된다. An organic solvent-tolerant lipase of Pseudomonas sp. BCNU 154 that was isolated from wastewater in the industrial complex region had optimal activity at <TEX>$37^{\circ}C$</TEX> and pH 8. This crude extracellular lipase from BCNU 154 exhibited maximum stability in toluene, retaining about 6.01 U/ml (117.53%) activity for 2 h. <TEX>$Ca^{2+}$</TEX>, <TEX>$Mg^{2+}$</TEX>, <TEX>$NH_4{^+}$</TEX>, and <TEX>$Na^+$</TEX> ions and triton X-100 activated the enzymes, whereas <TEX>$Ba^{2+}$</TEX>, <TEX>$Hg^{2+}$</TEX>, and <TEX>$Zn^{2+}$</TEX> ions inhibited their activity. Pseudomonas sp. BCNU 154 lipase revealed stable activity comparable to that of the commercial immobilized Novozym 435. Thus, this organic solvent-tolerant lipase could have potential as a whole cell biocatalyst in industrial chemical processes without the use of immobilization.

A novel and robust recombinant Pichia pastoris yeast whole cell biocatalyst with intracellular overexpression of a Thermomyces lanuginosus lipase: Preparation, characterization and application in biodiesel production

A novel and robust recombinant Pichia pastoris yeast whole cell catalyst (WCC) with functional intracellular expression of Thermomyces lanuginosus lipase (Tll) was constructed and characterized for biodiesel production from waste cooking oils. This permeabilized WCC was able to convert waste cooking oils to biodiesel with 82% yield within 84 h at 6% dosage whole cells. The WCC showed two fold catalytic activity of 0.73 U/mg DCW compared to its commercial counterpart Lipozyme TLIM (immobilized Tll). Short chain alcohol tolerance of this WCC was significantly improved compared to Lipozyme TLIM. This beneficial property enabled it to catalyze biodiesel production efficiently with one step addition of methanol. The reusability of this biocatalyst retained 78% activity after three batch cycles. This easily prepared and cost-effective WCC showed better catalytic performance than Lipozyme TLIM with respect to biodiesel yield and productivity, thus suggesting a promising cost-effective biocatalyst for biodiesel production.

Simultaneously improving stability and specificity of cell surface displayed glucose dehydrogenase mutants to construct whole-cell biocatalyst for glucose biosensor application

The improved stability and substrate specificity of cell surface displayed glucose dehydrogenase (GDH) mutants by replacing four amino acids from Bacillus subtilis by using site-directed mutagenesis was systematically investigated. A series of mutated GDHs including E170R/Q252L, V149K/E170R/Q252L, E170R/Q252L/G259A and V149K/E170R/Q252L/G259A, were fused to the ice nucleation protein for displaying on cell surface of Eschericia coli. Q252L/E170R/V149K, Q252L/E170R/G259A and Q252L/E170R/V149K/G259A variants were found stable at a wide pH range and shown excellent thermostability. Especially, the Q252L/E170R/V149K/G259A mutant showed half-life of ~3.8days at 70 °C. Q252L/E170R/V149K/G259A variant exhibited the narrowest substrate specificity for d-glucose. The whole cell displayed GDH mutant could be cultured in a large scale with excellent enzyme activity and productivity. In addition, a sensitive and stable electrochemical glucose biosensor can be prepared using the GDH-mutant bacteria modified electrode. Thus, the whole cell biocatalysts are promising candidates for exploitation in a wide range of industrial applications.

Synthesis of ω‐hydroxy dodecanoic acid based on an engineered CYP153A fusion construct

A bacterial P450 monooxygenase-based whole cell biocatalyst using Escherichia coli has been applied in the production of ω-hydroxy dodecanoic acid from dodecanoic acid (C12-FA) or the corresponding methyl ester. We have constructed and purified a chimeric protein where the fusion of the monooxygenase CYP153A from Marinobacter aquaeloei to the reductase domain of P450 BM3 from Bacillus megaterium ensures optimal protein expression and efficient electron coupling. The chimera was demonstrated to be functional and three times more efficient than other sets of redox components evaluated. The established fusion protein (CYP153AM. aq.-CPR) was used for the hydroxylation of C12-FA in in vivo studies. These experiments yielded 1.2 g l–1 ω-hydroxy dodecanoic from 10 g l–1 C12-FA with high regioselectivity (> 95%) for the terminal position. As a second strategy, we utilized C12-FA methyl ester as substrate in a two-phase system (5:1 aqueous/organic phase) configuration to overcome low substrate solubility and product toxicity by continuous extraction. The biocatalytic system was further improved with the coexpression of an additional outer membrane transport system (AlkL) to increase the substrate transfer into the cell, resulting in the production of 4 g l–1 ω-hydroxy dodecanoic acid. We further summarized the most important aspects of the whole-cell process and thereupon discuss the limits of the applied oxygenation reactions referring to hydrogen peroxide, acetate and P450 concentrations that impact the efficiency of the production host negatively.

Cell surface display of Staphylococcus haemolyticus L62 lipase in Escherichia coli and its application as a whole cell biocatalyst for biodiesel production

• Plasmid pPELB-Tu::L62 was constructed to display SHL62 lipase on the surface of Escherichia coli using EstAβ8 domain as an anchoring motif. • SHL62 lipase was successfully displayed on the cell surface of E. coli . • The displayed SHL62 lipase was used as a whole cell biocatalyst for biodiesel production. Staphylococcus haemolyticus L62 (SHL62) lipase was displayed on the cell surface of Escherichia coli using an autotransporter protein of Pseudomonas putida EstAβ8 as an anchoring motif. Localization of the SHL62 lipase on the cell surface of E. coli was confirmed by immunofluorescence microscopy, flow cytometry analysis, Western blot, protease accessibility, and whole-cell enzyme activity assays. SHL62 lipase activity of whole cells reached 168 U/g dry cell weight toward p -nitrophenyl caprylate after being induced by 0.1 mM IPTG for 24 h. The optimum temperature and pH of the displayed SHL62 lipase was 40–45 °C and pH 10, respectively. The displayed SHL62 was stable up to 45 °C, at which it had >80% of maximum activity. The displayed SHL62 lipase showed a preference for medium chain fatty acids of p -nitrophenyl esters. Moreover, the displayed SHL62 lipase was used as a whole cell catalyst to produce biodiesel that was obtained at a yield of nearly 89.4% after a 96 h reaction at 30 °C. These results suggest that the displayed SHL62 lipase on the cell surface of E. coli using an EstAβ8 anchoring motif can be used for biocatalytic applications.

English

The main obstacle to using lipase as a catalyst in industrial scale biodiesel production is the cost and availability of the enzyme. To overcome this obstacle, the potential of using a whole cell biocatalyst (for at least partial in situ lipase production) was evaluated as a means to reduce the cost of the lipase. The reaction conditions for biodiesel production via transesterification between Jatropha curcas (physic nut) oil and methanol when catalyzed in the presence of lipase-producing Aureobasidium pullulans yeast cells was investigated. The appropriate conditions for optimal biodiesel production were found to be 1:3 oil:methanol molar ratio at 30°C with constant stirring at 250 rpm. Under these conditions a maximum fatty acid methyl ester (biodiesel) production level of 71.8% was obtained after 72 h. Keywords: Lipase, Aureobasidium pullulans , physic nut oil, biodiesel, green energy African Journal of Biotechnology Vol. 12(27), pp. 4380-4386

Effect of organic solvents and ionic liquids on resolution of 2‐epoxyhexane by whole cells of Rhodotorula glutinis in a two‐liquid phase system

AbstractBACKGROUNDEnantiopure epoxides and diols have attracted much attention as important building blocks for the production of flavorings, agrochemicals and pharmaceutical products. Enantioselective hydrolysis reaction of rac‐epoxide in a biphasic system using whole cell catalysts is one of the promising methods to obtain enantiopure compounds.RESULTSThe kinetic resolution of racemic 1,2‐epoxyhexane was carried out using whole cell biocatalyst in a hydrophobic solvent/buffer biphasic system and the effect of organic solvents and ionic liquids on the initial reaction rate of (R)‐enantiomer, rR0 and enantiomeric ratio, E was examined. In the case of using hydrophobic hydrocarbons, rR0 increased with the hydrophobicity of hydrocarbon (log PO/W), and a linear correlation was observed between rR0 and log PO/W. On the other hand, there were no correlations between rR0 and log PO/W of ionic liquids. Moreover, effects on E were small in both solvents. However, the addition of 1‐heptanol to dodecane was very effective at increasing E value. The kinetic data correlated well with the reaction kinetic model, considering the distribution between two liquid phases and competitive product inhibition.CONCLUSIONThe high enantiomeric ratio of (R)‐diol (E&gt;100) without significant decrease in the reactivity was accomplished by adding 1‐heptanol in minute amounts to dodecane. © 2013 Society of Chemical Industry

A highly selective whole cell biocatalysis method for the production of two major bioactive conjugated linoleic acid isomers

Abstract Conjugated linoleic acid (CLA) is well known potential anticarcinogenic, antiatherogenic and a number of other therapeutic qualities bearing substance. In the present study we have isolated Lactobacillus plantarum from local dairy buffalo milk and used as a whole cell biocatalyst, for selective CLA isomer production from linoleic acid (LA). Quantitative analysis revealed that the cis-9, trans-11 CLA comprised of 51% (w/w), while trans-10, cis-12 CLA accounted for 49% (w/w) of total CLA produced by L. plantarum. The product formation and quantitation was monitored by the spectrophotometric method and the individual isomers separation was done with GC–FID. After optimization of biotransformation conditions such as pH, LA concentration, effect of preincubation, fermentation time; the highest CLA production (19.07% conversion) was achieved at 120 h in the presence of 1.62 mg ml−1 of LA. The results revealed that the bacterium is an efficient biocatalyst for the production of two major bioactive CLA isomers using LA as a substrate.

Production of ginsenoside aglycons and Rb1 deglycosylation pathway profiling by HPLC and ESI-MS/MS using Sphingobacterium multivorum GIN723

Using enrichment culture, Sphingobacterium multivorum GIN723 (KCCM80060) was isolated as having activity for deglycosylation of compound K and ginsenoside F1 to produce ginsenoside aglycons such as S-protopanaxadiol (PPD(S)) and S-protopanaxatriol (PPT(S)). Through BLAST search, purified enzyme from S. multivorum GIN723 was revealed to be the outer membrane protein. The purified enzyme from S. multivorum GIN723 has unique specificity for the glucose moiety. However, it has activity with PPD and PPT group ginsenosides such as ginsenosides Rb1, Rb2, Rb3, Rc, F2, CK, Rh2, Re, and F1. From these results, it was predicted that the enzyme has activity on several ginsenosides. Therefore, the biotransformation pathway from Rb1, which is a major, highly glycosylated compound of ginseng, was analyzed using high-performance liquid chromatography and electrospray ionization mass spectrometry/mass spectrometry. The dominant biotransformation pathway from Rb1 to PPD(S) was determined to be Rb1 → Gp-XVII → Gp-LXXV → CK → PPD(S). S. multivorum GIN723 can be used as a whole cell biocatalyst because its activity as whole cells is nine times higher than its activity as cell extracts. The specific activity of whole cells is 2.89nmol/mg/min in the production of PPD(S). On the other hand, the specific activity of cell extracts is 0.32nmol/mg/min. The productivity of this enzyme in whole cell form is 500mg/1l of cultured cell. Its optimum reaction condition is 10mM of calcium ions added to a phosphate buffer with a pH of 8.5.

Producing biodiesel from cottonseed oil using Rhizopus oryzae ATCC #34612 whole cell biocatalysts: Culture media and cultivation period optimization

The effect of culture medium composition and cultivation time on biodiesel production by Rhizopus oryzae ATCC #34612 whole cell catalysts, immobilized on novel rigid polyethylene biomass supports, was investigated. Supplementation of the medium with carbon sources led to higher lipase activity and increased the biomass immobilized on the BSPs. Statistical analysis indicates that a cultivation period of 72h in a basal medium supplemented with both cottonseed oil and glucose is optimal for biodiesel production by R. oryzae, resulting in a fatty acid methyl ester (FAME) yield of 27.9wt.% (228.2g/L).

One-step magnetic modification of yeast cells by microwave-synthesized iron oxide microparticles

Baker's yeast (Saccharomyces cerevisiae) cells were magnetically modified with magnetic iron oxide particles prepared by microwave irradiation of iron(II) sulfate at high pH. The modification procedure was very simple and fast. Both non-cross-linked and glutaraldehyde cross-linked magnetic cells enabled efficient sucrose conversion into glucose and fructose, due to the presence of active intracellular invertase. The prepared magnetic whole-cell biocatalyst was stable; almost the same catalytic activity was observed after 1-month storage at 4°C. Simple magnetic separation and stability of the developed biocatalyst enabled its reusability without significant loss of enzyme activity. Magnetic whole yeast cell biocatalyst containing intracellular invertase in its natural environment has been prepared. Magnetic properties enable its easy separation from reaction mixture. Magnetically modified Saccharomyces cerevisiae cells have been used for invert sugar production, hydrolysing sucrose into glucose and fructose. The described magnetization procedure employing microwave-synthesized iron oxide microparticles is a low-cost and easy-to-perform alternative to already existing magnetization techniques.

Saccharomyces cerevisiae catalyzed one pot synthesis of isoindolo[2,1-a]quinazoline performed under ultrasonication

An efficient and simple one pot method has been developed for the synthesis of isoindolo[2,1-a]quinazolines using Saccharomyces cerevisiae (baker's yeast) as a whole cell biocatalyst at room temperature. The synergetic effect of baker's yeast and ultrasound irradiation has been discussed.

The influence of microbial physiology on biocatalyst activity and efficiency in the terminal hydroxylation of n-octane using Escherichia coli expressing the alkane hydroxylase, CYP153A6

BackgroundBiocatalyst improvement through molecular and recombinant means should be complemented with efficient process design to facilitate process feasibility and improve process economics. This study focused on understanding the bioprocess limitations to identify factors that impact the expression of the terminal hydroxylase CYP153A6 and also influence the biocatalytic transformation of n–octane to 1-octanol using resting whole cells of recombinant E. coli expressing the CYP153A6 operon which includes the ferredoxin (Fdx) and the ferredoxin reductase (FdR).ResultsSpecific hydroxylation activity decreased with increasing protein expression showing that the concentration of active biocatalyst is not the sole determinant of optimum process efficiency. Process physiological conditions including the medium composition, temperature, glucose metabolism and product toxicity were investigated. A fed-batch system with intermittent glucose feeding was necessary to ease overflow metabolism and improve process efficiency while the introduction of a product sink (BEHP) was required to alleviate octanol toxicity. Resting cells cultivated on complex LB and glucose-based defined medium with similar CYP level (0.20 μmol gDCW-1) showed different biocatalyst activity and efficiency in the hydroxylation of octane over a period of 120 h. This was influenced by differing glucose uptake rate which is directly coupled to cofactor regeneration and cell energy in whole cell biocatalysis. The maximum activity and biocatalyst efficiency achieved presents a significant improvement in the use of CYP153A6 for alkane activation. This biocatalyst system shows potential to improve productivity if substrate transfer limitation across the cell membrane and enzyme stability can be addressed especially at higher temperature.ConclusionThis study emphasises that the overall process efficiency is primarily dependent on the interaction between the whole cell biocatalyst and bioprocess conditions.

Non-linear kinetic modelling of reversible bioconversions: Application to the transaminase catalyzed synthesis of chiral amino-alcohols

This work describes the establishment of a full kinetic model, including values of apparent kinetic parameters, for the whole cell E. coli mediated synthesis of the chiral amino-alcohol (2S,3R)-2-amino-1,3,4-butanetriol (ABT), using (S)-(−)-α-methylbenzylamine (MBA) as amino donor. The whole cell biocatalyst expressed the CV2025 ω-transaminase from Chromobacterium violaceum. Establishment of the most suitable reaction mechanism and determination of the complete forward and reverse kinetic parameter values for the reversible bioconversion where obtained using a hybrid methodology. This combined traditional initial rate experiments to identify a solution in the vicinity of the global minimum, with nonlinear regression methods to determine the exact location of the solution. The systematic procedure included selection and statistical evaluation of different kinetic models that best described the measured reaction rates and which ultimately provided new insights into the reaction mechanism; in particular the possible formation of a dead end complex between the amino donor and the cofactor enzyme complex. The hybrid methodology was combined with a microscale experimental platform, to significantly reduce both the number of experiments required as well as the time and material required for full kinetic parameter estimation. The equilibrium constant was determined to be 849, and the forward and reverse rate constants were found to be 97 and 13min−1, respectively, which greatly favoured the asymmetric synthesis of chiral ABT. Using the established kinetic model, the asymmetric synthesis of ABT was simulated, and excellent agreement was found between the experimental and predicted data over a range of reaction conditions. A sensitivity analysis combined with various simulations suggested the crucial bottleneck of the reaction was the second half reaction of the ping pong bi–bi mechanism, in part due to the low Michaelis constant of substrate l-erythrulose (ERY). The toxicity of MBA towards the transaminase was identified as another major bottleneck. The kinetic model was useful to give early insights into the most appropriate bioconversion conditions, which can improve the rate and yield of ABT formation, as well as minimizing the toxicity and inhibition effects of the substrates and products. The systematic methodology developed here is considered to be generic and useful in regard to speeding up bioconversion process design and optimization.

Investigation of spore coat display of Bacillus subtilis β-galactosidase for developing of whole cell biocatalyst

The production of highly efficient, recyclable and cost-effective enzymes is one of the most important goals in industrial biotechnology. Bacterial spores are highly resistant to harsh environmental conditions, easy to produce and are suitable for manipulation of genetic materials. These features make them a very efficient tool for biotechnology. Here, we show the use bacterial spores for presentation of functional enzyme. Spore coat display was used to produce a biocatalyst, which expresses β-galactiosidase (LacA). This enzyme is commonly used to produce lactose-free milk for lactose intolerant individuals. The lacA gene from Bacillus subtilis strain 168 was expressed on the surface of B. subtilis RH101(ΔcotC) spores using CotC as protein carrier. Presence of LacA protein is verified by western blotting. Results of β-galactiosidase assay show that the expressed enzyme retained its activity in condition of freezing and drying, as well as after recovery from the reaction's mixture.

Expressing Xylanases in Escherichia Coli by Cell Surface Display

In this research, xylan was utilized by a recombinant whole cell biocatalyst, which was developed by expressing three xylanases — β-xylosidase, endoxylanase, and α-arabinofuranosidase — on the surface of the E. coli BL21 (DE3). The xylanases were displayed on the surface of the cells by fusing with anchor proteins, Blc. The assimilation of xylan by cell surface display was the first step in the consolidated bioprocessing (CBP). This result shows that the engineering strains could be endowed with the ability to assimilate xylan. The co-display engineering strains utilized xylan and expressed less metabolic burden than the engineering strains secreting extracellular xylanases.