Discovery Of Bacillus Subtilis Esterase And Enzymatic Preparation Of L-Menthol

Menthol is an important flavoring chemical used extensively in pharmaceuticals, foods, tobacco products, cosmetics and oral products. The total annual production is in excess of 20,000 metric tons and the total value is as high as 0.5 billion US dollars. Due to affection of weather and region, and the recent rapid development of the global economy, the natural menthol has no longer satisfied the market demand. The biocatalytic processes for production of l-menthol have been attracting much attention in the flavor and fragrance industry. This is mainly because biocatalysis has the advantages of environmental friendliness, mild reaction conditions, less byproducts, high catalytic efficiency and often high stereo-, regio- or enantioselectivity.A new ideal biocatalyst, Bacillus subtilis ECU0554 with high substrate tolerance and excellent enantioselectivity towards dl-menthyl acetate, were successfully isolated from soil samples through the strageties of target reaction-oriented screening and the rapid analysis of TLC and GC. The reaction conditions of Bacillus subtilis esterase (BSE) were optimized in this work. The gene encoding BSE was cloned and overexpressed in E. coli, and then a scale-up reaction catalyzed by the recombintant Bacillus subtilis esterase (rBSE) for production of l-menthol was carried out. The immobilization of rBSE was also investigated. This dissertation is composed of four parts as follows:The first part of the dissertation is the target-oriented screening for novel biocatalysts. The study aimed to search for novel biocatalysts with excellent enantioselectivity and high substrate tolerance through the strageties of target reaction-oriented screening and a pressure of high substrate concentration. From soil samples,261 microbial strains were isolated through enrichment culture using dl-menthyl acetate as the sole carbon source, of which 98 was capable of hydrolyzing dl-menthyl acetate to produce l-menthol which was analyzed rapidly thin layer chromatography (TLC). The 81 strains of microorganisms with an obvious spot of menthol on TLC were marked for secondary screening by gas chromatography (GC). Using this method, the top 16 esterase-products with higher conversions of substrate and excellent optical purity (>90% ee) of product were selected. As far as industrial application is concerned, the substrate tolerance of these 16 candidate strains was investigated using an elevated concentration of dl-menthyl acetate. A strain number 0554 exhibited the best enantioselectivity and strong tolerance against a high substrate concentration among the top 16 strains. So it was selected as the best enzyme producer, and was subsequently identified as Bacillus subtilis based on its 16S rDNA sequence. It is hereafter named Bacillus subtilis ECU0554, and is currently deposited in China General Microbiological Culture Collection Center (Beijing, China), with an accession number of CGMCC 2548.The second part of the dissertation is the studies on catalytic properties of Bacillus subtilis esterase (BSE). The optimal substrate is dl-menthyl acetate and the optimal pH and temperature of reaction is 7.0 and 30℃, respectively. The enzymatic hydrolysis of dl-menthyl acetate could be effectively achived in high enantioselectivity(E>200) and>95% optical purity (eep) by BSE. Furthermore, BSE also exhibited strong tolerance against a high substrate concentration up to 100 g/L, indicating a promising biocatalyst for practical production of l-menthol in the future.The third part of the dissertation is cloning of the gene from Bacillus subtilis ECU0554, expression in E. coli BL21 (DE3) and the optimization of reaction catalyzed by recombinant Bacillus subtilis esterase (rBSE). The esterase production was significantly enhanced by overexpression. The esterase activity per liter of the fermentation borth was significantly improved by two orders of magnitude (up to 190 kU/L fermentation borth). The recombintant DNA technology enables the rBSE to be an inexpensive and readily available biocatalyst. The study of substrate specificity showed that the rBSE exhibited a marked activity for esters of short-chain fatty acids and a low hydrolytic activity for esters of long-chain fatty acids, confirming that rBSE is indeed an esterase. In contrast to the process catalyzed by wild-type BSE, the substrate loading was further increased from 100 g/L to 200 g/L, while the biocatalyst loading was reduced from 50 g/L to 2.5 g/L in the new process catalyzed by rBSE. The space-time yield (gprod/L/d) and specific production (gprod/gcat) were improved by ca. 4-fold and 75-fold, respectively, and the S/C-ratio was improved by 40 fold. A biocatalytic process with great potential for production of l-menthol was therefore developed using rBSE as the biocatalyst.The fourth part of the dissertation is the study on rBSE immobilization. A significantly stabilized biocatalyst of rBSE was developed by using a simple and effecient method of cross-linked enzyme aggregates (CLEAs). Thermal stability of the immobilized enzyme (CLA-rBSE) was significantly enhanced and was far superior to that of free BSE. Moreover, the CLA-rBSE also exhibited excellent operational stability, retaining 92% of its initial activity after 10 cycles of reuse. This indicates that CLA-rBSE is very promising as an immobilized biocatalyst for kinetic resolution of dl-menthyl acetate to produce l-menthol in industrial application.