Jei-Fu Shaw

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.

Extended flower longevity of Petunia hybrida plants transformed with boers, a mutated ERS gene of Brassica oleracea

Petunia x hybridaHort.Vilm.-Andr. was transformed with boers, a mutatedallele of BOERS, an ethylene receptor sensor gene ofBrassicaoleracea.boers was obtained by removing anEcoRI cutting site with a silent mutation at Gly-521 andintroducing a point mutation at Ile-62, replacing isoleucine withphenylalanine. Transformation was Agrobacterium tumefaciens mediated.Hygromycin resistant regenerants were tentatively confirmed as transformants byPCRs for HPH and boers and moredefinitively by Southern hybridization of genomic DNA with pBOERS4421. Flowersof transgenic plants retained turgidity and pigmentation considerably longerthan those of untransformed controls, whether left undisturbed on plants orexcised and placed in water. Furthermore, flowers were unaffected by exposureto exogenous ethylene. Excised shoots of transgenic plants released considerablymore ethylene than those of untransformed plants. Transformed plants alsoproduced apparently larger flowers. Unexpectedly higher mortality was observed,suggesting that the ethylene insensitive petunia plants were also lower indisease resistance.

Expression of a bi-functional and thermostable amylopullulanase in transgenic rice seeds leads to autohydrolysis and altered composition of starch

Overexpression of bacterial-derived starch metabolic enzymes in plant starch storage organs represents a valuable strategy for improving starch quality, bioprocessing and nutritional value. Transgenic rice seeds producing a thermostable and bifunctional starch hydrolase, amylopullulanase (APU) from Thermoanaerobacter ethanolicus 39E, were generated. Starch in these seeds could be hydrolyzed with optimal temperatures between 85 and 95xa0°C, which resulted in complete conversion of starch into soluble sugars and production of protein-enriched flour within a few hours. By expressing various levels of APU, rice seeds containing reduced amounts of amylose, which is an important factor affecting starch quality, were obtained without a significant impact on grain yield. Elevation in granule-bound pullulanase activity correlates with the reduction of amylose in developing APU-containing rice seeds. APU was found to be localized within amyloplasts and in cell walls, which could be the result of overexpression of APU with a signal peptide. This study establishes novel approaches to alter starch properties, accelerate bioprocessing of starch and production of protein-enriched flour from rice seeds, and could significantly impact the industrial and food uses of cereals.

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.

Optimized enzymatic synthesis of propylene glycol monolaurate by direct esterification

Abstract Propylene glycol monoesters with low hydrophile–lipophile balance value are widely used in the food industry. The ability of immobilized lipase, from Rhizomucor miehei (Lipozyme IM-77), to catalyze the direct esterification of propylene glycol with lauric acid was investigated in this study. Response surface methodology (RSM) and 3-level-4-factor fractional factorial design were adopted to evaluate the effects of synthesis variables, such as reaction time (3–9 h), temperature (25–65xa0°C), enzyme amount (15–45%) and substrate molar ratio of lauric acid to propylene glycol (1:1 to 3:1), on the percentage molar conversion to propylene glycol monolaurate. Results showed that reaction temperature and reaction time were the most important parameters and enzyme amount had less effect on percent molar conversion. Based on ridge max analysis, optimum synthesis conditions were: reaction time 7.6 h, temperature 37.6xa0°C, enzyme amount 37.1% and substrate molar ratio 2.6:1 with 100% molar conversion in this study. An actual experimental value of 96% molar conversion was obtained.

Optimal lipase-catalyzed formation of hexyl laurate

A medium-chain ester, hexyl laurate, with a fruity flavor is primarily used in personal care formulations as an important emollient for cosmetic applications. In order to conform to the “natural” interests of consumers, the ability for immobilized lipase from Rhizomucor miehei n (Lipozyme IM-77) to catalyze the direct esterification of hexanol and lauric acid was investigated in this study. Response surface methodology (RSM) and 4-factor-5-level central composite rotatable design (CCRD) were employed to evaluate the effects of synthesis parameters, such as reaction time (20 to 100 min), temperature (25 to 65 °C), enzyme amount (10 to 50%), and substrate molar ratio of hexanol to lauric acid (1 : 1 to 3 : 1) on percentage molar conversion of hexyl laurate by direct esterification. Reaction time and enzyme amount had significant effects on percent molar conversion. Based on ridge max analysis, the optimum conditions for synthesis were: reaction time 74.8 min, temperature 47.5 °C, enzyme amount 45.5%, and substrate molar ratio 1 : 1.5. The predicted value was 90.0% and the actual experimental value 92.2% molar conversion.

Ethylene insensitive and post-harvest yellowing retardation in mutant ethylene response sensor (boers) gene transformed broccoli (Brassica olercea var. italica)

A mutant broccoli ers (ethylene-response-sensor, boers) gene was obtained through site directed mutagenesis by replacing the isoleucine with phenylanine at the 62th residue. Two plasmids were constructed with this mutant gene regulated by the CaMV 35S promoter together with the nptII (kanamycin resistance gene) coding sequence and hpt (hygromycin resistance gene), respectively, for the pBI-mERSI62F and pSM1H-mERSI62F plasmids. Genetic transformation of the above two constructs via A. tumefaciens has been conducted to evaluate their effects on floret yellowing of harvested broccoli. Over a hundred transformants have been obtained on the selected cotyledon and hypocotyl explants. PCR and Southern analysis demonstrated integration of the transgenes in the transformants. However, through Southern hybridization, we determined that multi-site integration and DNA rearrangements had occurred in most transformants. Morphological and characteristic alternation such as slower plant growth, shorter plant height, easy branching, late bolting, and relative higher mortality in comparison with other transgenes were noted in some transformants. Transgenic lines showing delayed senescence in leaves and floral heads were obtained. The expression of transgene was confirmed by Northern blot analysis. The transformed progenies also showed ethylene insensitivity in seed germination, detached leaves and harvested florets. Nevertheless, in most lines, the yellowing was only delayed 1–2 days.

Optimized Growth Kinetics of Pichia pastoris and Recombinant Candida rugosa LIP1 Production by RSM

A predictive model for Pichia pastoris expression of highly active recombinant Candida rugosa LIP1 was developed by combining the Gompertz function and response surface methodology (RSM) to evaluate the effect of yeast extract concentration, glucose concentration, temperature, and pH on specific responses. Each of the responses (maximum population densities, specific growth rate (µmax), protein concentration, and minimum lag phase duration) was determined using the modified Gompertz function. RSM and 4-factor-5-level central composite rotatable design (CCRD) 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 responses of P. pastoris growth kinetics.Based on ridge maximum analysis, the optimum population density conditions were: temperature 24.4°C, glucose concentration 2.0%, yeast extract 1.5%, and pH 7.6. The optimum specific growth rate conditions were: temperature 28.9°C, glucose concentration 2.0%, yeast extract 1.1%, and pH 6.9. The optimum protein concentration conditions were: temperature 24.2°C, glucose concentration 1.9%, yeast extract 1.5%, and pH 7.6. Based on ridge minimum analysis, the minimal lag phase conditions were: temperature 32.3°C, glucose concentration 2.1%, yeast extract 1.1%, and pH 5.4. For the predicted value, the maximum population density, specific growth rate, protein concentration, and minimum lag phase duration were 15.7 mg/ml, 3.4 h–1, 0.78 mg/ml, and 4.2 h, and the actual values were 14.3 ± 3.5 mg/ml, 3.6 ± 0.6 h–1, 0.72 ± 0.2 mg/ml, and 4.4 ± 1.6 h, respectively.