Studies of optimum conditions for covalent immobilization of Candida rugosa lipase on poly(gamma-glutamic acid) by RSM.

Poly(gamma-glutamic acid) (gamma-PGA) is a material of polymer. Immobilization of Candida rugosa lipase (Lipase AY-30) by covalent binding on gamma-PGA led to a markedly improved performance of the enzyme. Response surface methodology (RSM) and 3-level-3-factor fractional factorial design were employed to evaluate the effects of immobilization parameters, such as immobilization time (2-6h), immobilization temperature (0-26 degrees C), and enzyme/support ratio (0.1-0.5, w/w). Based on the analysis of ridge max, the optimum immobilization conditions were as follows: immobilization time 2.3h, immobilization temperature 13.3 degrees C, and enzyme/support ratio 0.41 (w/w); the highest lipase activity obtained was 1196 U/mg-protein.

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 pGAPZalphaC 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 degrees 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 degrees 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.

Optimization of lipase-catalyzed biodiesel by response surface methodology.

Biodiesel prepared by catalyzed mild transesterification has become of much current interest for bioenergy. The ability of a commercial immobilized lipase (Novo Industries--Bagsvaerd, Denmark) from Rhizomucor miehei (Lipozyme IM-77) to catalyze the transesterification of soybean oil and methanol was investigated in this study. Response surface methodology and 5-level-5-factor central composite rotatable design were employed to evaluate the effects on reaction time, temperature, enzyme amount, molar ratio of methanol to soybean oil, and added water content on percentage weight conversion to soybean oil methyl ester by transesterification. Based on ridge max analysis, the optimum synthesis conditions giving 92.2% weight conversion were: reaction time 6.3 h, temperature 36.5 degrees C, enzyme amount 0.9 BAUN (Batch Acidolysis Units NOVO), substrate molar ratio 3.4:1, and added water 5.8%.

Formulation of physical properties of methyl glucoside polyester by mixture response surface methodology.

Methyl glucoside polyester (MGPE), consisting of a methyl glucoside molecule esterified with four fatty acids, is a potential fat substitute. A mixture response surface methodology was employed to model the physical properties (melting point, density, and viscosity) of MGPE prepared from composite blends of fatty acid methyl esters (FAME). The measured physical properties were similar to those of soybean oil, except for a higher viscosity. The physical properties correlated significantly with the degree of saturation of their composite fatty acids. Results showed that the physical properties of MGPE prepared from a FAME mixture derived from high oleic acid safflower oil and soybean oil (molar ratio 1:1) was similar to those of soybean oil. The physical properties of MGPE can be adjusted by modifying the saturated fatty acids of synthesized MGPE to simulate the physical properties of soybean oil.

Optimized synthesis of lipase-catalyzed hexyl acetate in n-hexane by response surface methodology.

Hexyl acetate, a short-chain ester with fruity odor, is a significant green note flavor compound and widely used in the food industry. The ability for immobilized lipase from Mucor miehei (Lipozyme IM-77) to catalyze the transesterification of hexanol with triacetin was investigated in this study. Response surface methodology and five-level-five-factor central composite rotatable design were adopted to evaluate the effects of synthesis variables, such as reaction time (2-10 h), temperature (25-65 degrees C), enzyme amount (10-50%; 0.024-0.118 BAUN), substrate molar ratio of triacetin to hexanol (1:1 to 3:1), and added water content (0-20%) on percentage molar conversion of hexyl acetate. The results showed that reaction temperature and substrate molar ratio were the most important parameters and that added water content had less of an effect on percent molar conversion. On the basis of canonical analysis, optimum synthesis conditions were as follows: reaction time, 7.7 h; temperature, 52.6 degrees C; enzyme amount, 37.1% (0.089 BAUN); substrate molar ratio, 2.7:1; and added water, 12.5%. The predicted value was 88.9% molar conversion, and the actual experimental value was 86.6% molar conversion.

Application of response surface methodology to the study of methyl glucoside polyester synthesis parameters in a solvent-free system.

Response surface methodology (RSM) and 3-level-3-factor fractional factorial design were used to evaluate the effects of synthesis parameters, including reaction time (4 to 8 h), temperature (110 to 130 degrees C), and substrate molar ratio of fatty acid methyl esters (FAME) from soybean oil to methyl glucoside (4:1 to 6:1) on the percent molar conversion to methyl glucoside polyester (MGPE), utilizing 15 g of methyl glucoside as the reactant in a solvent-free system. All synthesis variables (reaction time, temperature, and substrate molar ratio) exhibited significant effects on percent molar conversion to MPGE in the experimental range. Optimization of the synthesis reaction was suggested by ridge max analysis to compute the estimated ridge of optimum response for increasing radii from the center of the original design. Based on the ridge max analysis, optimum conditions were: reaction time 6.3 h, synthesis temperature 123.8 degrees C, and substrate molar ratio 5.9:1. The predicted molar conversion was 55.68% (i.e., 15 g methyl glucoside yielded 56.5 g MGPE) at the optimum point.

Optimized enzymatic synthesis of geranyl butyrate with lipase AY from Candida rugosa.

Response surface methodology (RSM) and five-level, five-variable central composite rotatable design (CCRD) were used to evaluate the effects of synthetic variables, such as reaction time (1-9 h), temperature (25-65 degrees C), enzyme amount (10-50%), substrate molar ratio of geraniol to tributyrin (1:0.33-1:1), and added water amount (0-20%) on molar percent yield of geranyl butyrate, using lipase AY from Candida rugosa. Reaction time and temperature were the most important variables and substrate molar ratio had no effect on percent molar conversion. Based on contour plots, optimum conditions were: reaction time 9 h, temperature 35 degrees C, enzyme amount 50%, substrate molar ratio 1:0.33, and added water 10%. The predicted value was 100% and actual experimental value was 96.8% molar conversion. (c) 1996 John Wiley & Sons, Inc.

Rectovaginal fistula: a review of 11 years experience.

The records of 22 patients at Temple University Hospital with a diagnosis of rectovaginal fistula, from January 1970 to December 1980, were reviewed. The age at the time of diagnosis ranged from 18 to 75 years with an average of 47.3 years. Most had symptoms and signs which included fecal incontinence, flatus passage per vagina, tenesmus, distressing diarrhea and urinary tract infection. The majority of the fistulas were present for more than a year before the corrective operative procedure was attempted. The patients were divided into two groups: postirradiation fistulas and non-irradiation fistulas. The former resulted from the treatment of carcinoma of the cervix, urinary bladder, urethra, or rectum. The latter resulted from inflammatory bowel diseases, diverticulitis, or trauma from obstetric procedures. The former group were usually treated conservatively, either no operation or defunctioning colostomy. The latter group were treated more aggressively, either by direct repair or resection of the diseased segment.

Primary carcinoma of the gallbladder: a review of a 16-year experience at the Waterbury Hospital Health Center.

The charts of 48 cases of primary carcinoma of the gallbladder seen at the Waterbury Hospital Health Center over the past 16 years were reviewed. In 47 patients, the diagnosis was confirmed pathologically. In one patient, the diagnosis was made only on clinical and radiologic grounds. This condition is more common in females and in the elderly (mean 71.7 years). Symptoms and signs were nonspecific, and duration of symptoms was extremely variable (range: one day to 21 years). There were 16.6% patients who had an associated primary tumor elsewhere. In 89.3% of the carcinomas of the gallbladder, the histologic type was adenocarcinoma. Two thirds of the patients had associated cholelithiasis. Overall five-year survival rate was 12.5%. In more than 80% of the patients, resection was made impossible by local invasion of liver, biliary ducts, and adjacent structures. Patients were divided into three groups according to the degree of invasion of the tumor. Group A had a survival rate of 66.6% at five years. There were no survivors in Group B and Group C. The different factors in each group are compared and discussed. Chemotherapy appeared to have no beneficial effect in the management of carcinoma of the gallbladder. Only one patient survived more than 12 months when tumor invaded the liver or the nodes along the biliary ducts. The postoperative complications and causes of death are also discussed.