Shu-Wei Chang

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 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.

Application of solvent engineering to optimize lipase-catalyzed 1,3-diglyacylcerols by mixture response surface methodology

Abstract1,3-Diacylglycerol has been introduced in Japan as a cooking oil under the trade name of Econa to reduce body fat accumulation. Solvent engineering was applied to determine the optimum solvent mixtures for the lipase-catalyzed synthesis of 1,3-DAG by mixture response surface methodology. n-Hexane was required to maintain the lipase activity and the product selectivity could be adjusted by changing the hydrophobicity of reaction medium. The optimum yield (∼40%) of 1,3-DAG synthesis was obtained with n-hexane/octane (1:1, v/v).

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 (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.

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.