Guan Chiun Lee

Protein engineering and applications of Candida rugosa lipase isoforms

Commercial preparations of Candida rugosa lipase (CRL) are mixtures of lipase isoforms used for the hydrolysis and synthesis of various esters. The presence of variable isoforms and the amount of lipolytic protein in the crude lipase preparations lead to a lack of reproducibility of biocatalytic reactions. Purification of crude CRL improve their substrate specificity, enantioselectivity, stability, and specific activities. The expression of the isoforms is governed by culture or fermentation conditions. Unfortunately, the nonsporogenic yeast C. rugosa does not utilize the universal codon CTG for leucine; therefore, most of the CTG codons were converted to universal serine triplets by site-directed mutagenesis to gain expression of functional lipase in heterologous hosts. Recombinant expressions by multiple-site mutagenesis or complete synthesis of the lipase gene are other possible ways of obtaining pure and different CRL isoforms, in addition to culture engineering. Protein engineering of purified CRL isoforms allows the tailoring of enzyme function. This involves computer modeling based on available 3-D structures of lipase isoforms. Lid swapping and DNA shuffling techniques can be used to improve the enantioselectivity, thermostability, and substrate specificity of CRL isoforms and increase their biotechnological applications. Lid swapping can result in chimera proteins with new functions. The sequence of the lid can affect the activity and specificity of recombinant CRL isoforms. Candida rugosa lipase is toxicologically safe for food applications. Protein engineering through lid swapping and rationally designed site-directed mutagenesis will continue to lead to the production of CRL isoforms with improved catalytic power, thermostability, enantioselectivity, and substrate specificity, while providing evidence for the mechanisms of actions of the various isoforms.

Biocatalysis for the production of industrial products and functional foods from rice and other agricultural produce

Many industrial products and functional foods can be obtained from cheap and renewable raw agricultural materials. For example, starch can be converted to bioethanol as biofuel to reduce the current demand for petroleum or fossil fuel energy. On the other hand, starch can also be converted to useful functional ingredients, such as high fructose and high maltose syrups, wine, glucose, and trehalose. The conversion process involves fermentation by microorganisms and use of biocatalysts such as hydrolases of the amylase superfamily. Amylases catalyze the process of liquefaction and saccharification of starch. It is possible to perform complete hydrolysis of starch by using the fusion product of both linear and debranching thermostable enzymes. This will result in saving energy otherwise needed for cooling before the next enzyme can act on the substrate, if a sequential process is utilized. Recombinant enzyme technology, protein engineering, and enzyme immobilization are powerful tools available to enhance the activity of enzymes, lower the cost of enzyme through large scale production in a heterologous host, increase their thermostability, improve pH stability, enhance their productivity, and hence making it competitive with the chemical processes involved in starch hydrolysis and conversions. This review emphasizes the potential of using biocatalysis for the production of useful industrial products and functional foods from cheap agricultural produce and transgenic plants. Rice was selected as a typical example to illustrate many applications of biocatalysis in converting low-value agricultural produce to high-value commercial food and industrial products. The greatest advantages of using enzymes for food processing and for industrial production of biobased products are their environmental friendliness and consumer acceptance as being a natural process.

Multiple mutagenesis of non-universal serine codons of the Candida rugosa LIP2 gene and biochemical characterization of purified recombinant LIP2 lipase overexpressed in Pichia pastoris.

The 17 non-universal serine codons (CTG) in the Candida rugosa LIP2 gene have been converted into universal serine codons (TCT) by overlap extension PCR-based multiple site-directed mutagenesis. An active recombinant LIP2 lipase was overexpressed in Pichia pastoris and secreted into the culture medium. The recombinant LIP2 showed distinguishing catalytic activities when compared with recombinant LIP4 and commercial C. rugosa lipase. The purified enzyme showed optimum activity at pH 7 and a broad temperature optimum in the range 30-50 degrees C. The enzyme retained 80% of residual activity after being heated at 70 degrees C for 10 min. Recombinant LIP2 demonstrated high esterase activity towards long-chain (C12-C16) p-nitrophenyl esters. Tributyrin was the preferred substrate among all triacylglycerols tested for lipolysis. Among cholesteryl esters, LIP2 showed highest lipolytic activity towards cholesteryl laurate. The esterification of myristic acid with alcohols of various chain lengths showed that the long-chain n-octadecanol (C18) was the preferred substrate. In contrast, the esterification of n-propanol with fatty acids of various chain lengths showed that the short-chain butyric acid was the best substrate. From comparative modelling analysis, it appears that several amino acid substitutions resulting in greater hydrophobicity in the substrate-binding site might play an important role in the substrate specificity of LIP2.

Multiple mutagenesis of the Candida rugosa LIP1 gene and optimum production of recombinant LIP1 expressed in Pichia pastoris

Candida rugosa lipase, a significant catalyst, had been widely employed to catalyze various chemical reactions such as non-specific, stereo-specific hydrolysis and esterification for industrial biocatalytic applications. Several isozymes encoded by the lip gene family, namely lip1 to lip7, possess distinct thermal stability and substrate specificity, among which the recombinant LIP1 showed a distinguished catalytic characterization. In this study, we utilized PCR to remove an unnecessary linker of pGAPZαC vector and used overlap extension PCR-based multiple site-directed mutagenesis to convert the 19 non-universal CTG-serine codons into universal TCT-serine codons and successfully express a highly active recombinant C. rugosa LIP1 in the Pichia expression system. Response surface methodology and 4-factor-5-level central composite rotatable design were adopted to evaluate the effects of growth parameters, such as temperature (21.6–38.4°C), glucose concentration (0.3–3.7%), yeast extract (0.16–1.84%), and pH (5.3–8.7) on the lipolytic activity of LIP1 and biomass of P. pastoris. Based on ridge max analysis, the optimum LIP1 production conditions were temperature, 24.1°C; glucose concentration, 2.6%; yeast extract, 1.4%; and pH 7.6. The predicted value of lipolytic activity was 246.9±39.7 U/ml, and the actual value was 253.3±18.8 U/ml. The lipolytic activity of the recombinant LIP1 resulting from the present work is twofold higher than that achieved by a methanol induction system.

Conversion of crude Jatropha curcas seed oil into biodiesel using liquid recombinant Candida rugosa lipase isozymes

The versatile Candida rugosa lipase (CRL) has been widely used in biotechnological applications. However, there have not been feasibility reports on the transesterification of non-edible oils to produce biodiesel using the commercial CRL preparations, mixtures of isozymes. In the present study, four liquid recombinant CRL isozymes (CRL1-CRL4) were investigated to convert various non-edible oils into biodiesel. The results showed that recombinant CRL2 and CRL4 exhibited superior catalytic efficiencies for producing fatty acid methyl ester (FAME) from Jatropha curcas seed oil. A maximum 95.3% FAME yield was achieved using CRL2 under the optimal conditions (50 wt% water, an initial 1 equivalent of methanol feeding, and an additional 0.5 equivalents of methanol feeding at 24h for a total reaction time of 48 h at 37 °C). We concluded that specific recombinant CRL isozymes could be excellent biocatalysts for the biodiesel production from low-cost crude Jatropha oil.

Role of the CCAAT-Binding Protein NFY in SCA17 Pathogenesis

Spinocerebellar ataxia 17 (SCA17) is caused by expansion of the polyglutamine (polyQ) tract in human TATA-box binding protein (TBP) that is ubiquitously expressed in both central nervous system and peripheral tissues. The spectrum of SCA17 clinical presentation is broad. The precise pathogenic mechanism in SCA17 remains unclear. Previously proteomics study using a cellular model of SCA17 has revealed reduced expression of heat shock 70 kDa protein 5 (HSPA5) and heat shock 70 kDa protein 8 (HSPA8), suggesting that impaired protein folding may contribute to the cell dysfunction of SCA17 (Lee et al., 2009). In lymphoblastoid cells, HSPA5 and HSPA8 expression levels in cells with mutant TBP were also significantly lower than that of the control cells (Chen et al., 2010). As nuclear transcription factor Y (NFY) has been reported to regulate HSPA5 transcription, we focused on if NFY activity and HSPA5 expression in SCA17 cells are altered. Here, we show that TBP interacts with NFY subunit A (NFYA) in HEK-293 cells and NFYA incorporated into mutant TBP aggregates. In both HEK-293 and SH-SY5Y cells expressing TBP/Q(61~79), the level of soluble NFYA was significantly reduced. In vitro binding assay revealed that the interaction between TBP and NFYA is direct. HSPA5 luciferase reporter assay and endogenous HSPA5 expression analysis in NFYA cDNA and siRNA transfection cells further clarified the important role of NFYA in regulating HSPA5 transcription. In SCA17 cells, HSPA5 promoter activity was activated as a compensatory response before aggregate formation. NFYA dysfunction was indicated in SCA17 cells as HSPA5 promoter activity reduced along with TBP aggregate formation. Because essential roles of HSPA5 in protection from neuronal apoptosis have been shown in a mouse model, NFYA could be a target of mutant TBP in SCA17.

Structure of the Alkalohyperthermophilic Archaeoglobus fulgidus Lipase Contains a Unique C-Terminal Domain Essential for Long-Chain Substrate Binding

Several crystal structures of AFL, a novel lipase from the archaeon Archaeoglobus fulgidus, complexed with various ligands, have been determined at about 1.8 A resolution. This enzyme has optimal activity in the temperature range of 70-90 degrees C and pH 10-11. AFL consists of an N-terminal alpha/beta-hydrolase fold domain, a small lid domain, and a C-terminal beta-barrel domain. The N-terminal catalytic domain consists of a 6-stranded beta-sheet flanked by seven alpha-helices, four on one side and three on the other side. The C-terminal lipid binding domain consists of a beta-sheet of 14 strands and a substrate covering motif on top of the highly hydrophobic substrate binding site. The catalytic triad residues (Ser136, Asp163, and His210) and the residues forming the oxyanion hole (Leu31 and Met137) are in positions similar to those of other lipases. Long-chain lipid is located across the two domains in the AFL-substrate complex. Structural comparison of the catalytic domain of AFL with a homologous lipase from Bacillus subtilis reveals an opposite substrate binding orientation in the two enzymes. AFL has a higher preference toward long-chain substrates whose binding site is provided by a hydrophobic tunnel in the C-terminal domain. The unusually large interacting surface area between the two domains may contribute to thermostability of the enzyme. Two amino acids, Asp61 and Lys101, are identified as hinge residues regulating movement of the lid domain. The hydrogen-bonding pattern associated with these two residues is pH dependent, which may account for the optimal enzyme activity at high pH. Further engineering of this novel lipase with high temperature and alkaline stability will find its use in industrial applications.

Altered expression of HSPA5, HSPA8 and PARK7 in spinocerebellar ataxia type 17 identified by 2-dimensional fluorescence difference in gel electrophoresis

BACKGROUND Expansion of the CAG repeat of the TATA-box binding protein (TBP) gene has been identified as the causative mutations in spinocerebellar ataxia 17 (SCA17). TBP is ubiquitously expressed in both central nervous system and peripheral tissues. The underlying molecular changes of SCA17 are rarely explored. METHODS To study the molecular mechanisms underlying SCA17, we generated stably induced isogenic 293 cells expressing normal TBP-Q(36) and expanded TBP-Q(61) and analyzed the expressed proteins using two-dimensional difference in gel electrophoresis (2D-DIGE), followed by mass spectrometry and immunoblotting. RESULTS Upon induction with doxycycline, the expanded TBP-Q(61) formed aggregates with significant increase in the cell population at subG1 phase and cleaved caspase-3. Proteomics study identified a total of 16 proteins with expression changes greater than 1.5 fold. Among the 16 proteins, PARK7, GLRX3, HNRNPA1, GINS1, ENO1, HNRPK and NPM1 are increased, and SERPINA5, HSPA5, VCL, KHSRP, HSPA8, HNRPH1, IMMT, VCP and HNRNPL are decreased in cells expressing TBP-Q(61) compared with those expressing TBP-Q(36). The altered expression of HSPA5, HSPA8 and PARK7 were further validated by 2D and Western immunoblot analyses. CONCLUSIONS The results illustrate the utility of proteomics to identify alterations of proteins which underlie pathogenesis of SCA17, and may serve as potential therapeutic targets.

Genes and biochemical characterization of three novel chlorophyllase isozymes from Brassica oleracea.

Three full length cDNAs (BoCLH1, 1140 bp; BoCLH2, 1104 bp; BoCLH3, 884 bp) encoding putative chlorophyllases were cloned from the cDNA pools of broccoli (Brassica oleracea) florets and characterized. The amino acid sequence analysis indicated that these three BoCLHs contained a highly conserved lipase motif (GXSXG). However, only BoCLH3 lacked the His residue which is the component of the catalytic triad (Ser-His-Asp). N-terminal sequences of BoCLH1 and BoCLH2 were predicted to have typical signal sequences for the chloroplast, whereas the plasma membrane-targeting sequence was identified in BoCLH3. The predicted molecular masses of BoCLH1, 2, and 3 were 34.7, 35.3, and 23.5 kDa, respectively. The recombinant BoCLHs were successfully expressed in Escherichia coli for the biochemical characterization. The recombinant BoCLH3 showed very low chlorophyllase activity possibly due to its incomplete catalytic triad. BoCLH1 and BoCLH2 showed significant differences in biochemical properties such as pH stability and temperature optimum. Kinetic analysis revealed that BoCLH1 preferably hydrolyzed Mg-free chlorophyll, while BoCLH2 hydrolyzed both chlorophyll and Mg-free chlorophyll at a similar level. Different characteristics between BoCLH1 and BoCLH2 implied that they may have different physiological functions in broccoli. The catalytic triad of recombinant BoCLH2 was identified as Ser141, His247, and Asp170 by site-directed mutagenesis. It suggested that the three broccoli chlorophyllase isozymes were serine hydrolases.

Site-Specific Saturation Mutagenesis on Residues 132 and 450 of Candida rugosa LIP2 Enhances Catalytic Efficiency and Alters Substrate Specificity in Various Chain Lengths of Triglycerides and Esters

The catalytic versatility of recombinant Candida rugosa LIP2 has been known to have potential applications in industry. In this study, site-specific saturation mutagenesis on residues L132 and G450 of recombinant LIP2 has been employed to investigate the impact of both residues on substrate specificity of LIP2. Point mutations on L132 and G450 were done separately using mutagenic degenerate primer sets containing 32 codons to generate two libraries of mutants in Pichia pastoris . Replacements of amino acid on these mutants were identified as L132A, L132I, G450S, and G450A. In lipase activity assay, L132A and L132I mutants showed a shift of preference from short- to medium-chain triglyceride, whereas G450S and G450A mutants retained preferences as compared to wild-type LIP2. Among mutants, G450A has the highest activity on tributyrin. However, hydrolysis of p-nitrophenyl (p-NP) esters with L132A, L132I, and G450S did not show differences of preferences over medium- to long-chain esters except in G450A, which prefers only medium-chain ester as compared to wild-type LIP2. All mutants showed an enhanced catalytic activity and higher optimal temperature and pH stability as compared to wild-type LIP2.