Surface display of active lipase in <Emphasis Type="Italic">Pichia pastoris</Emphasis> using Sed1 as an anchor protein

A Pichia pastoris cell-surface display system was constructed using the Sed1 anchor system that has been developed in Saccharomyces cerevisiae. Candida antarctica lipase B (CALB) was used as the model protein and was fused to an anchor that consisted of 338 amino acids of Sed1. The resulting fusion protein CALBSed1 was expressed under the control of the alcohol oxidase 1 promoter (pAOX1). Immunofluorescence microscopy of immunolabeled Pichia pastoris revealed that CALB was displayed on the cell surface. Western blot analysis showed that the fusion protein CALBSed1 was attached covalently to the cell wall and was highly glycosylated. The hydrolytic activity of the displayed CALB was more than 220 U/g dry cells after 120 h of culture. The displayed protein also exhibited a higher degree of thermostability than free CALB.     

Development of yeast cells displaying <Emphasis Type="Italic">Candida antarctica</Emphasis> lipase B and their application to ester synthesis reaction

We isolated the lipase B from Candida antarctica CBS 6678 (CALB CBS6678) and successfully constructed CALB-displaying yeast whole-cell biocatalysts using the Flo1p short (FS) anchor system. For the display of CALB on a yeast cell surface, the newly isolated CALB CBS6678 exhibited higher hydrolytic and ester synthesis activities than the well-known CALB, which is registered in GenBank (Z30645). A protease accessibility assay using papain as a protease showed that a large part of CALB, approximately 75%, was localized on an easily accessible part of the yeast cell surface. A comparison of the lipase hydrolytic activities of yeast whole cells displaying only mature CALB (CALB) and those displaying mature CALB with a Pro region (ProCALB) revealed that mature CALB is preferable for yeast cell surface display using the Flo1p anchor system. Lyophilized yeast whole cells displaying CALB were applied to an ester synthesis reaction at 60°C using adipic acid and n-butanol as substrates. The amount of dibutyl adipate (DBA) produced increased with the reaction time until 144 h. This indicated that CALB displayed on the yeast cell surface retained activity under the reaction conditions.     

Accurate analysis of fusion expression of <Emphasis Type="Italic">Pichia pastoris</Emphasis> glycosylphosphatidylinositol-modified cell wall proteins

Glycosylphosphatidylinositol (GPI)-anchored glycoproteins have diverse intrinsic functions in yeasts, and they also have different uses in vitro. The GPI-modified cell wall proteins GCW21, GCW51, and GCW61 of Pichia pastoris were chosen as anchoring proteins to construct co-expression strains in P. pastoris GS115. The hydrolytic activity and the amount of Candida antarctica lipase B (CALB) displayed on cell surface increased significantly following optimization of the fusion gene dosage and combination of the homogeneous or heterogeneous cell wall proteins. Maximum CALB hydrolytic activity was achieved at 4920 U/g dry cell weight in strain GS115/CALB-GCW (51 + 51 + 61 + 61) after 120 h of methanol induction. Changes in structural morphology and the properties of the cell surfaces caused by co-expression of fusion proteins were observed by transmission electron microscopy (TEM) and on plates containing cell-wall-destabilizing reagent. Our results suggested that both the outer and inner cell layers were significantly altered by overexpression of GPI-modified cell wall proteins. Interestingly, quantitative analysis of the inner layer components showed an increase in β-1,3-glucan, but no obvious changes in chitin in the strains overexpressing GPI-modified cell wall proteins.     

Display of Candida antarctica lipase B on Pichia pastoris and its application to flavor ester synthesis

Two alternative cell-surface display systems were developed in Pichia pastoris using the α-agglutinin and Flo1p (FS) anchor systems, respectively. Both the anchor cell wall proteins were obtained originally from Saccharomyces cerevisiae. Candida antarctica lipase B (CALB) was displayed functionally on the cell surface of P. pastoris using the anchor proteins α-agglutinin and FS. The activity of CALB displayed on P. pastoris was tenfold higher than that of S. cerevisiae. The hydrolytic and synthetic activities of CALB fused with α-agglutinin and FS anchored on P. pastoris were investigated. The hydrolytic activities of both lipases displayed on yeast cells surface were more than 200 U/g dry cell after 120 h of culture (200 and 270 U/g dry cell, respectively). However, the synthetic activity of CALB fused with α-agglutinin on P. pastoris was threefold higher than that of the FS fusion protein when applied to the synthesis of ethyl caproate. Similarly, the CALB displayed on P. pastoris using α-agglutinin had a higher catalytic efficiency with respect to the synthesis of other short-chain flavor esters than that displayed using the FS anchor. Interestingly, for some short-chain esters, the synthetic activity of displaying CALB fused with α-agglutinin on P. pastoris was even higher than that of the commercial CALB Novozyme 435.     

Preparation of a whole-cell biocatalyst of mutated <Emphasis Type="Italic">Candida antarctica</Emphasis> lipase B (mCALB) by a yeast molecular display system and its practical properties

To prepare a whole-cell biocatalyst of a stable lipase at a low price, mutated Candida antarctica lipase B (mCALB) constructed on the basis of the primary sequences of CALBs from C. antarctica CBS 6678 strain and from C. antarctica LF 058 strain was displayed on a yeast cell surface by α-agglutinin as the anchor protein for easy handling and stability of the enzyme. When mCALB was displayed on the yeast cell surface, it showed a preference for short chain fatty acids, an advantage for producing flavors; although when Rhizopus oryzae lipase (ROL) was displayed, the substrate specificity was for middle chain lengths. When the thermal stability of mCALB on the cell surface was compared with that of ROL on a cell surface, T _1/2, the temperature required to give a residual activity of 50% for heat treatment of 30 min, was 60°C for mCALB and 44°C for ROL indicating that mCALB displayed on cell surface has a higher thermal stability. Furthermore, the activity of the displayed mCALB against p-nitrophenyl butyrate was 25-fold higher than that of soluble CALB, as reported previously. These findings suggest that mCALB-displaying yeast is more practical for industrial use as the whole-cell biocatalyst.     

Biodiesel fuel production by Aspergillus niger whole-cell biocatalyst in optimized medium

Methanolysis of sunflower oil catalyzed by immobilized Aspergillus niger mycelium was studied in a packed-bed reactor. The optimal cultivation parameters for A. niger were determined using full factorial and steepest ascent experimental designs. Sunflower oil, yeast extract and soybean meal were selected as the best carbon and nitrogen sources and were used in the subsequent experiments. Intracellular lipase activity and cell mass concentration were respectively 3.2 and 2.4 times greater and cultivation period decreased by 24 h compared with the initial medium. The optimum values of these most significant parameters were as follows: sunflower oil (13.2 g/L), yeast extract (6.2 g/L), soybean meal (7.4 g/L) and incubation period (72 h) at 30 °C. With A. niger biocatalyst grown in optimized conditions, the biodiesel fuel yield reached 23.1% after sixth pass of recycled reaction mixture through the reactor.     

南极假丝酵母脂肪酶B的酿酒酵母表面展示及其催化己酸乙酯的合成

从南极假丝酵母(Candida antarctica)基因组克隆得到南极假丝酵母脂肪酶B(Candida antarctica Lipase B, CALB)全基因片段, 利用连接肽celA Linker将CALB与酿酒酵母细胞表面展示蛋白a-凝集素的C端连接融合, 构建表面展示载体pICAS-celAL-CALB, 转化酵母后获得重组酵母菌Saccharomyces cerevisiae pICAS-celAL-CALB。该重组酵母菌经葡萄糖诱导表达及分析, 表明CALB已在酿酒酵母细胞表面成功展示, 水解活力达26.26 u/(g·dry cell)。重组酵母菌经冻干能有效地实现在非水相中全细胞催化己酸和乙醇酯化合成己酸乙酯。反应物己酸与乙醇的摩尔比为1:1.25, 己酸乙酯的产率为98.0%, 具有较好的操作稳定性。     

Efficient yeast cell-surface display of an endoglucanase of <Emphasis Type="Italic">Aspergillus flavus</Emphasis> and functional characterization of the whole-cell enzyme

The endoglucanase gene endo753 from Aspergillus flavus NRRL3357 strains was cloned, and the recombinant Endo753 was displayed on the cell surface of Saccharomyces cerevisiae EBY100 strain by the C-terminal fusion using Aga2p protein as anchor attachment tag. The results of indirect immunofluorescence and Western blot confirmed the expression and localization of Endo753 on the yeast cell surface. The hydrolytic activity test of the whole-cell enzyme revealed that Endo753 immobilized on the yeast cell surface had high endoglucanase activity. The functional characterization of the whole-cell enzyme was investigated, and the whole-cell enzyme displayed the maximum activity at pH 8 and 50 °C. The enzyme was stable in a pH range of 7.0–10.0. Furthermore, the whole-cell enzyme displayed high thermostability below 50 °C and moderate stability between 50 and 70 °C. These properties make endo753 a good candidate in bioethanol production from lignocellulosic materials after displaying on the yeast cell surface.     

Multicomponent synthesis of dihydropyrano[2,3-c]pyrazoles catalyzed by lipase from Aspergillus niger

Lipase from Aspergillus niger (ANL) was found to be an extremely effective catalyst for four-component synthesis of dihydropyrano[2,3-c]pyrazoles from a stoichiometric mixture of ethyl acetoacetate, hydrazine hydrate, aldehyde/ketone, and malononitrile in ethanol. The lipase ANL showed a broad range of enzymatic promiscuity toward various aromatic and aliphatic aldehydes as well as ketones. Use of environmentally benign biocatalyst, reusability of the catalyst, room temperature reaction conditions, no hazardous solvent, and excellent yields are some of the important features of this protocol.     

Display of Bacterial Lipase on the Escherichia coli Cell Surface by Using FadL as an Anchoring Motif and Use of the Enzyme in Enantioselective Biocatalysis

We have developed a novel cell surface display system by employing FadL as an anchoring motif, which is an outer membrane protein involved in long-chain fatty acid transport in Escherichia coli. A thermostable Bacillus sp. strain TG43 lipase (44.5 kDa) could be successfully displayed on the cell surface of E. coli in an active form by C-terminal deletion-fusion of lipase at the ninth external loop of FadL. The localization of the truncated FadL-lipase fusion protein on the cell surface was confirmed by confocal microscopy and Western blot analysis. Lipase activity was mainly detected with whole cells, but not with the culture supernatant, suggesting that cell lysis was not a problem. The activity of cell surface-displayed lipase was examined at different temperatures and pHs and was found to be the highest at 50°C and pH 9 to 10. Cell surface-displayed lipase was quite stable, even at 60 and 70°C, and retained over 90% of the full activity after incubation at 50°C for a week. As a potential application, cell surface-displayed lipase was used as a whole-cell catalyst for kinetic resolution of racemic methyl mandelate. In 36 h of reaction, (S)-mandelic acid could be produced with the enantiomeric excess of 99% and the enantiomeric ratio of 292, which are remarkably higher than values obtained with crude lipase or cross-linked lipase crystal. These results suggest that FadL may be a useful anchoring motif for displaying enzymes on the cell surface of E. coli for whole-cell biocatalysis.     

Display of lipase on the cell surface of Escherichia coli using OprF as an anchor and its application to enantioselective resolution in organic solvent

We have developed a new cell surface display system using a major outer membrane protein of Pseudomonas aeruginosa OprF as an anchoring motif. Pseudomonas fluorescens SIK W1 lipase gene was fused to the truncated oprF gene by C-terminal deletion fusion strategy. The truncated OprF-lipase fusion protein was successfully displayed on the surface of Escherichia coli. Localization of the truncated OprF-lipase fusion protein was confirmed by western blot analysis, immunofluorescence microscopy, and whole-cell lipase activity. To examine the enzymatic characteristics of the cell surface displayed lipase, the whole-cell enzyme activity and stability were determined under various conditions. Cell surface displayed lipase showed the highest activity at 37 degrees C and pH 8.0. It retained over 80% of initial activity after incubation for a week in both aqueous solution and organic solvent. When the E. coli cells displaying lipases were used for enantioselective resolution of racemic 1-phenylethanol in hexane, (R)-phenyl ethyl acetate was successfully obtained with the enantiomeric excess of greater than 96% in 36 h of reaction. These results suggest that E. coli cells displaying lipases using OprF as an anchoring motif can be employed for various biotechnological applications both in aqueous and nonaqueous phases.     

Immobilized CALB Catalyzed Transesterification of Soybean Oil and Phytosterol

Candida antarctica lipase B (CALB) was immobilized on Fe₃O₄/SiO_x-g-P(GMA) polymer carrier to catalyzed the transesterification of soybean oil and phytosterol. The enzyme loading of the obtained particles was 98.7 mg/g supports and the enzyme activity was 1226.5 U/g. The average particle size was 100.5?±?1.30 nm and the magnetization was 15.80 emu/g. The immobilized enzyme showed higher activities at a wider range of pH and temperatures. Its optimum reaction temperature was up to 50 °C; increased by 5 °C compared to the free enzyme. The obtained magnetic immobilized Fe₃O₄/SiO_x-g-P(GMA) lipase was nanoscale. First-grade soybean oils were used as a substrate. System pH was adjusted to 7.0. The optimal reaction temperature was 50 °C and the reaction time was 3 h. The phytosterol concentration of 5% and immobilized CALB of 2% were obtained. The conversion rate of transesterification reaction between soybean oil and phytosterol was 86.2%. The use of magnets can quickly separate the immobilized enzymes from the substrates. The relative activity of the immobilized enzymes was 83.0% when reused seven times. The prepared immobilized CALB can improve efficiently enzyme activity and reutilization.     

Surface Display of Bacterial Laccase CotA on <Emphasis Type="Italic">Escherichia coli</Emphasis> Cells and its Application in Industrial Dye Decolorization

Laccase CotA from Bacillus subtilis 168 was successfully displayed on the membrane of Escherichia coli cells using poly-γ-glutamate synthetase A protein (PgsA) from B. subtilis as an anchoring matrix. Further analyses demonstrated that the fusion protein PgsA/CotA efficiently translocates to the cell surface of E. coli with an enzymatic activity of 65 U/10⁸ cells. Surface-displayed CotA was shown to possess improved enzymatic properties compared with those of the wild-type CotA, including higher thermal stability (above 90% activity at 70 °C and nearly 40% activity at 90 °C after 5-h incubation) and stronger inhibitor tolerance (approximately 80 and 65% activity when incubated with 200 and 400 mM NaCl, respectively). Furthermore, the whole-cell system was demonstrated to have high enzymatic activity against anthraquinone dye, Acid Blue 62, triphenylmethane dye, Malachite Green, and azo dye, Methyl Orange with the decolorization percentages of 91, 45, and 75%, after 5-h incubation, respectively.     

Immobilization of <Emphasis Type="Italic">Candida antarctica</Emphasis> lipase B onto Purolite<Superscript>®</Superscript> MN102 and its application in solvent-free and organic media esterification

The aim of this study was to develop simple and efficient method for immobilization of Candida antarctica lipase B onto hydrophobic anion exchange resin Purolite^® MN102 and to apply immobilized catalyst for the enzymatic synthesis of two valuable esters—isoamyl acetate and l-ascorbyl oleate. At optimized conditions (1 M phosphate buffer pH = 7, 7 h at 25 °C, and 18.75 mg of offered proteins g^?1 of support), immobilized lipase with hydrolytic activity of 888.4 p-nitrophenyl butyrate units g^?1 was obtained. Afterwards, preparation was applied for the solvent-free synthesis of isoamyl acetate from triacetin and isoamyl alcohol. At 75 °C, 1 M of isoamyl alcohol, and 6 mg ml^?1 of enzyme 100 % yield was achieved in 6 h, while at prolonged reaction times, complete conversion was enabled even at lower temperatures, lower lipase loadings, and higher substrate concentrations. After 15 consecutive reuses (60 h), activity of catalyst dropped to 50 % of its initial value and total amount of 1.31 mol (170.55 g) of ester with 1 g of biocatalyst was produced. Even higher operational stability of lipase (25 % loss of activity in 200 h) was observed in the synthesis of l-ascorbyl oleate performed in organic solvent (t-butanol). Multiple use of one batch of immobilized biocatalyst in both cases led to a significant process cost reduction and substantial increment of corresponding productivities.     

Quantitative evaluation of Candia antarctica lipase B displayed on the cell surface of a Pichia pastoris based on an FS anchor system

A new approach is described to quantify the number of enzyme molecules, such as Candia antarctica lipase B, that are displayed on the cell surface of Pichia pastoris. Enhanced green fluorescent protein (EGFP) and Candida antarctica lipase B (CALB) were fused and displayed on the surface of P. pastoris by linking to the anchor flocculation functional domain of FLO1p from Saccharomyces cerevisiae. Confocal laser scanning microscopy, flow cytometry, and fluorescence spectrophotometry were used to monitor the fluorescence intensity of fused EGFP. Combined with the corresponding protein concentration detected in the medium, a standard curve describing the relationship between the fusion protein concentration and fluorescence intensity were obtained and could be used to number CALB displayed on the cell surface. The results showed that approx. 10⁴ molecules of CALB molecules were immobilized on the single P. pastoris cell wall based on FS anchor system.     

Production of glucoamylase in pyruvate decarboxylase deletion mutants of the yeast Kluyveromyces lactis

We have developed a novel Escherichia coli cell surface display system by employing PgsA as an anchoring motif. In our display system, C-terminal fusion to PgsA anchor protein from Bacillus subtilis was used. The enzymes selected for display were α-amylase (AmyA) from Streptococcus bovis 148 and lipase B (CALB) from Candida antarctica. The molecular mass values of AmyA and CALB are approximately 77 and 34 kDa, respectively. The enzymes were displayed on the surface as a fusion protein with a FLAG peptide tag at the C terminus. Both the PgsA-AmyA-FLAG and PgsA-CALB-FLAG fusion proteins were shown to be displayed by immunofluorescence labeling using anti-FLAG antibody. The displayed enzymes were active forms, and AmyA and CALB activities reached 990 U/g (dry cell weight) and 4.6 U/g (dry cell weight), respectively. AmyA-displaying E. coli cells grew utilizing cornstarch as the sole carbon source, while CALB-displaying E. coli cells catalyzed enantioselective transesterification, indicating that they are effective whole-cell biocatalysts. Since a target enzyme with a size of 77 kDa and an industrially useful lipase have been successfully displayed on the cell surface of E. coli for the first time, PgsA protein is probably a useful anchoring motif to display various enzymes.     

Efficient synthesis of enantiomeric ethyl lactate by <Emphasis Type="Italic">Candida antarctica</Emphasis> lipase B (CALB)-displaying yeasts

The whole-cell biocatalyst displaying Candida antarctica lipase B (CALB) on the yeast cell surface with α-agglutinin as the anchor protein was easy to handle and possessed high stability. The lyophilized CALB-displaying yeasts showed their original hydrolytic activity and were applied to an ester synthesis using ethanol and l-lactic acid as substrates. In water-saturated heptane, CALB-displaying yeasts catalyzed ethyl lactate synthesis. The synthesis efficiency increased depending on temperature and reached approximately 74% at 50°C. The amount of l-ethyl lactate increased gradually. l-Ethyl lactate synthesis stopped at 200 h and restarted after adding of l-lactic acid at 253 h. It indicated that CALB-displaying yeasts retained their synthetic activity under such reaction conditions. In addition, CALB-displaying yeasts were able to recognize l-lactic acid and d-lactic acid as substrates. l-Ethyl lactate was prepared from l-lactic acid and d-ethyl lactate was prepared from d-lactic acid using the same CALB-displaying whole-cell biocatalyst. These findings suggest that CALB-displaying yeasts can supply the enantiomeric lactic esters for preparation of useful and improved biopolymers of lactic acid.     

Improvement of cell-bound lipase from Rhodotorula mucilaginosa P11I89 for use as a methanol-tolerant, whole-cell biocatalyst for production of palm-oil biodiesel

Rhodotorula mucilaginosa P11I89, isolated from oil-contaminated soil, was effectively used as the methanol-tolerant, whole-cell lipase for the synthesis of fatty acid methyl ester (FAME) via transesterification reaction in the presence of palm oil and methanol substrates at a 1:6 mole ratio. A combination of Taguchi experimental design and response surface methodology (RSM) were applied to systemically enhance transesterification activity of the whole-cell lipase or cell-bound lipase (CBL) from R. mucilaginosa P11I89 in a solvent-free system. The significant impacts of four factors including carbon sources, nitrogen sources, surfactants and pH on hydrolysis activity of extracellular and cell-bound lipases, and on the transesterification activity of CBL were evaluated using Taguchi design. Gum Arabic was the most significant component for high transesterification activity, whereas soybean oil was the most influential factor for the hydrolysis activity. Maximal CBL production of 272.72 U/L was obtained in the cultivation medium containing 2.1 % palm oil, 0.2 % NH₄NO₃ , and 0.45 % Gum Arabic, with initial pH 5.0 under shaking speed of 200 rpm at a temperature of 30?±?2 °C after 60 h incubation using Central Composite Design (CCD). Yeast cells grown under such conditions increased FAME yield from 84.0 to 92.98 % when the transesterification reaction was carried out, in comparison to those cultivated in the initial medium.     

Partial characterization of a crude cold-active lipase from Rhodococcus cercidiphylli BZ22

Cold-active lipase production by the psychrophilic strain Rhodococcus cercidiphylli BZ22 isolated from hydrocarbon-contaminated alpine soil was investigated. Depending on the medium composition, high cell densities were observed at a temperature range of 1–10 °C in Luria–Bertani (LB) broth or 1–30 °C in Reasoner’s 2A (R2A). Maximum enzyme production was achieved at a cultivation temperature of 1–10 °C in LB medium. About 70–80 % of the secreted enzyme was bound to the cell and was highly active as a cell-immobilized lipase which exhibited good reusability; more than 60 % of the initial lipase activity was retained after five-fold reuse. The properties of the lipase produced by the investigated strain were compared with those of a mesophilic porcine pancreatic lipase (PPL). The thermal stability of the cell-immobilized bacterial lipase was higher than that of the extracellular enzyme. Highest activity was detected at 30 °C for the cell-immobilized enzyme and for PPL, while the extracellular enzyme displayed highest activity at 10–20 °C. The bacterial lipase hydrolyzed p-nitrophenyl (p-NP) esters with different acyl chain lengths (C₂–C₁₈). The highest hydrolytic activity was obtained with p-NP-butyrate (C₄) as substrate, while the highest substrate affinity was obtained with p-NP-dodecanoate (C₁₂) as substrate, indicating a clear preference of the enzyme for medium acyl chain lengths.     

Production of flavor esters catalyzed by CALB-displaying Pichia pastoris whole-cells in a batch reactor

Candida antarctica lipase B (CALB) has been employed as an efficient catalyst in the preparation of many flavor esters. A CALB-displaying yeast whole-cell biocatalyst could be an attractive alternative to commercial immobilized CALB because of its low-cost preparation and high enzymatic activity. We investigated the potential application of CALB-displaying Pichia pastoris cells for the production of flavor esters. The optimal conditions for flavor esters synthesis by this biocatalyst were determined in 50-ml shake flasks. Under optimized conditions, the synthesis of 12 kinds flavor esters were scaled up in a 5-l batch stirred reactor. Among these, the mole conversions of 10 exceeded 95% after reactions for 4h. In addition, this biocatalyst showed good tolerance for high substrates concentration and excellent operational stability. Repeated use of the cells in 10 batches resulted in an activity loss of less than 10%. Thus, CALB-displaying P. pastoris whole cells are robust biocatalysts with potential commercial application in the large-scale production of flavor esters in non-aqueous media.