Parameter optimization for thermostable lipase production and performance evaluation as prospective detergent additive

Abstract

Lipase based formulations has been a rising interest to laundry detergent industry for their eco-friendly property over phosphate-based counterparts and compatibility with chemical detergents ingredients. A thermo-stable Anoxybacillus sp. ARS-1 isolated from Taptapani Hotspring, India was characterized for optimum lipase production employing statistical model central composite design (CCD) under four independent variables (temperature, pH, % moisture and bio-surfactant) by solid substrate fermentation (SSF) using mustard cake. The output was utilized to find the effect of parameters and their interaction employing response surface methodology (RSM). A quadratic regression with R²?=?0.955 established the model to be statically best fitting and a predicted highest lipase production of 29.4?IU/g at an optimum temperature of 57.5?°C, pH 8.31, moisture 50% and 1.2?mg of bio-surfactant. Experimental production of 30.3?IU/g lipase at above conditions validated the fitness of model. Anoxybacillus sp. ARS-1 produced lipase was found to resist almost all chemical detergents as well as common laundry detergent, proving it to be a prospective additive for incorporation.     

Laccase production from oil palm industry solid waste: Statistical optimization of selected process parameters

Oil palm frond parenchyma tissue was used as a solid substrate for the production of laccase via solid‐state fermentation using the white rot fungus Pycnoporus sanguineus. With a rectangular aluminium tray as solid‐state fermentation bioreactor, process parameters such as bed height, moisture and supplemented nitrogen (as urea solution) levels were studied and optimized using a statistical design of experiment. The moisture level exerted a significant effect on the process. The interaction effect observed between bed height and supplemented nitrogen level suggested that uniform distribution of supplemented nitrogen into the substrate bed was important. The proposed regression model sufficiently predicted the process response over the experimental range tested. The optimum parameter combination for laccase production was a 3‐cm bed height, 72% w/w moisture and 0.21% w/v supplemented nitrogen. Laccase productivity remained constant when the tray size was increased from 1.4 to 3.4‐fold.     

Whole cell‐catalyzed transesterification of waste vegetable oil

Enzymatic transesterification of waste cooking oil, comprising fats, oil and grease (FOG), to produce fatty acid methyl esters (FAME) i.e. biodiesel, was investigated using a novel strain of the fungus Aspergillus niger, immobilized as a whole‐cell biocatalyst. Response surface methodology (RSM), with a five‐level‐three‐factor central composite rotatable design, was used to optimize the reaction and analyze the relationship of reaction variables and their coinfluent on the response i.e. FAME yield. Independent variables that affect the transesterification reaction include temperature, feedstock water content and enzyme amount. Using RSM, a second‐order polynomial equation was derived for FAME yield using multiple regression analysis. The second‐order polynomial regression model was highly significant (P<0.001) in predicting the actual relationship between the response and the variables, where a linear relationship was apparent between observed and predicted values (R²=0.9651). In addition, the predicted determination coefficient q² i.e. 0.7723, also proved that the model has a high predictive ability. The validation experiments, under optimized conditions, showed that the predicted value of maximum FAME yield (i.e. 91.3%) was in close agreement with the experimental value (i.e. 91.8%).     

Rhizopus sp.PW358菌脂肪酶固态发酵生产

研究了Rhizopus sp.PW358菌的固态生长和产脂肪酶条件。结果表明：黄豆饼粉为培养基的基本成分,用来生产脂肪酶。培养基中可加入淀粉和蛋白胨作为碳源和源,有利于脂肪酶的合成,培养基的含水量以及金属离子Ca^2 ,Mg^2 的浓度也影响Rhizopus sp.PW358菌和脂肪酶 产生。在优化条件下,12g豆粉中含1.0g淀粉及0.5g蛋白胨、15ml营养盐中Ca^2 ,Mg^2 离子浓度分别为8．0和4．0g/L,培养基含水量为55．6％,在接种后培养48h,酶活力可达最大值320IU／g干培养基。脂肪酶的基本性质研究表明,酶的最适反应温度和PH分别为35℃和7．0,酶的半失活温度为53．5℃,不同的PH环境中,30℃保温1h后酶在PH6．5－8．5范围内较为稳定。     

Bioprocess optimization for production of thermoalkali-stable protease from Bacillus subtilis K-1 under solid-state fermentation

Cost-effective production of proteases, which are robust enough to function under harsh process conditions, is always sought after due to their wide industrial application spectra. Solid-state production of enzymes using agro-industrial wastes as substrates is an environment-friendly approach, and it has several advantages such as high productivity, cost-effectiveness, being less labor-intensive, and less effluent production, among others. In the current study, different agro-wastes were employed for thermoalkali-stable protease production from Bacillus subtilis K-1 under solid-state fermentation. Agricultural residues such as cotton seed cake supported maximum protease production (728?U?ml^?1), which was followed by gram husk (714?U?ml^?1), mustard cake (680?U?ml^?1), and soybean meal (653?U?ml^?1). Plackett–Burman design of experiment showed that peptone, moisture content, temperature, phosphates, and inoculum size were the significant variables that influenced the protease production. Furthermore, statistical optimization of three variables, namely peptone, moisture content, and incubation temperature, by response surface methodology resulted in 40% enhanced protease production as compared to that under unoptimized conditions (from initial 728 to 1020?U?ml^?1). Thus, solid-state fermentation coupled with design of experiment tools represents a cost-effective strategy for production of industrial enzymes.     

Response surface optimization of mixed substrate solid state fermentation for the production of lovastatin by Monascus purpureus

The objective of this work is to enhance the production of lovastatin using Monascus purpureus MTCC 369 in mixed substrate solid state fermentation using various solid substrates and to optimize the combination of the solid substrates by response surface methodology. Solid state fermentation was conducted in a 250 mL Erlenmeyer flask at 30°C for 14 days with initial moisture content of 40% and inoculum size of 10% active culture. Barley, long grain rice and sago starch were found to be the suitable substrates producing maximum lovastatin of 193.7 mg, 190.2 mg and 180.9 mg/g of dry solids. These substrates were further used in various combinations as designed by the central composite design for enhancing the lovastatin production using Monascus purpureus. To the best of our knowledge this is the first report on the production of lovastatin using a mixed substrate solid state fermentation using Monascus purpureus.     

高产纤维素酶的拟康宁木霉菌株8985固态发酵条件优化

木霉是重要的产纤维素酶真菌,在其可利用性评价筛选过程中,获得了一株在实验室条件下高产纤维素酶的拟康宁木霉菌株8985.采用响应面法对8985产纤维素酶的固态发酵条件进行了研究,以滤纸酶活为响应值,通过Plackett-Burman设计对11个因素进行了筛选,包括温度、湿度、发酵时间、K2HPO4、(NH4)2SO4、T...     

Cloning and expression of keratinase gene in Bacillus megaterium and optimization of fermentation conditions for the production of keratinase by recombinant strain

AIMS: Cloning and expression of keratinase gene in Bacillus megaterium and optimization of fermentation conditions for the production of keratinase by recombinant strain. METHODS AND RESULTS: The keratinase gene with and without leader sequence from the chromosomal DNA of Bacillus licheniformis MKU3 was amplified by PCR and cloned into pET30b and transferred into Escherichia coli BL21. The ker gene without leader sequence only expressed in E. coli and the recombinant strain produced an intracellular keratinase activity of 74.3 U ml(-1). The ker gene was further subcloned into E. coli-Bacillus shuttle vector, pWH1520. Bacillus megaterium ATCC 14945 carrying the recombinant plasmid pWHK3 expressed the ker gene placed under xylA promoter and produced an extracellular keratinase activity of 95 U ml(-1). Response surface methodology (RSM) was employed to optimize the fermentation condition and to improve the level of keratinase production by the recombinant strain. A maximum keratinolytic activity of 166.2 U ml(-1) (specific activity, 33.25 U mg(-1)) was obtained in 18 h of the fermentation carried out with an initial inoculum of 0.4 OD600 nm and xylose concentration of 0.75% w/v. CONCLUSIONS: Bacillus licheniformis keratinase was cloned and successfully expressed using T7 promoter in E. coli and xylose inducible expression system in B. megaterium. Response surface methodology was employed to optimize the process parameters, which resulted in a three-fold higher level of keratinase production by the recombinant B. megaterium (pWHK3) than the wild type strain B. licheniformis MKU3. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that B. megaterium is a suitable host for the expression of cloned genes from heterologous origin. Optimization of fermentation conditions improved the keratinase production by B. megaterium (pWHK3) and suggested that this recombinant strain could be used for the production of keratinase.     

Lucrative pectinase production by novel strain Pseudozyma sp. SPJ with statistical optimization techniques using agro-industrial residues

Production of high titers of an alkaline, extracellular and thermo-tolerant pectinase by a newly isolated yeast Pseudozyma sp. SPJ was carried out under solid state fermentation. Citrus peel, the inexpensive agro-industrial residue used as substrate, was experienced to be unsurpassed. Response surface methodology was conducted to optimize the culture conditions for Pseudozyma sp. SPJ for hyper production of pectinase. Plackett Burman design was applied to identify the most effective culture variables. Out of nine variables studied, incubation time, moisture content and ammonium sulfate were detected as most important. A full factorial Central Composite Design was used to optimize the levels of these variables, which resulted in 17-fold increase （71.19 IU/g to 1215.66 IU/g dry substrate） in the enzyme yield. The results of analysis of variance and multiple regression analysis implies that the effect of incubation time （p 〈 0.000） and moisture content （p 〈 0.018） is more than ammonium sulfate. And also the interaction of moisture content with ammonium sulfate （p 〈 0.002） is more significant.     

Potential of agroindustrial waste from olive oil industry for fuel ethanol production

Olive pulp (OP) is a highly polluting semi-solid residue generated from the two-stage extraction processing of olives and is a major environmental issue in Southern Europe, where 80% of the world olive oil is produced. At present, OP is either discarded to the environment or combusted with low calorific value. In this work, utilization of OP as a potential substrate for production of bioethanol was studied. Enzymatic hydrolysis and subsequent glucose fermentation by baker's yeast were evaluated for OP from 10% to 30% dry matter (i.e., undiluted). Enzymatic hydrolysis resulted in an increase in glucose concentration by 75%, giving final glucose yields near 70%. Fermentation of undiluted OP hydrolysate (OPH) resulted in the maximum ethanol produced (11.2 g/L) with productivity of 2.1 g/L/h. Ethanol yields were similar for all tested OPH concentrations and were in the range of 0.49-0.51 g/g. Results showed that yeast could effectively ferment OPH even without nutrient addition, revealing the tolerance of yeast to OP toxicity. Because of low xylan (12.4%) and glucan (16%) content in OP, this specific type of OP is not a suitable material for producing only ethanol and thus, bioethanol production should be integrated with production of other value-added products.     

Biodiesel production from waste cooking oil by immobilized lipase on superparamagnetic Fe3O4 hollow sub-microspheres

Superparamagnetic Fe₃O₄ hollow sub-microspheres (FHSM) with strong response to an external magnet were prepared via a solvothermal method, followed by acid etching. Lipase from Candida sp. 99–125 was directly immobilized onto the amino-functional FHSM by simple adsorption, without glutaraldehyde linkage. The immobilized lipase was used to catalyze the esterification/transesterification of waste cooking oil with methanol to produce fatty acid methyl ester (FAME), a major source of biodiesel. FAME yield exceeded 93.4% over a wide range of temperatures from 10 to 40?°C. Notably, stability was clearly improved at the lower temperatures, in particular, giving a FAME yield of 89.6% after eight cycles of use at 10?°C.     

Optimization of mycophenolic acid production in solid state fermentation using response surface methodology

Mycophenolic acid (MPA) can be produced in solid state fermentation. An isolate of Penicillium brevi-compactum ATCC 16024 grown on moist wheat bran produced a titre of 425 mg per kg of wheat bran. Central composite rotatable design and response surface methodology were employed to derive a statistical model for media optimization towards production of mycophenolic acid. Five levels with a five factorial design were adopted. The correlation coefficient was 0.82, ensuring a satisfactory adjustment of the model to the experimental values. This statistical design was very effective in improving the titre of mycophenolic acid up to 3286 mg per kg of wheat bran. Received 24 July 1998/ Accepted in revised form 4 December 1998     

Pediocin production by Pediococcus acidilactici in solid state culture on a waste medium: process simulation and experimental results

The production of pediocin by Pediococcus acidilactici was comparatively studied in submerged and solid-state culture, using polyurethane foam particles soaked in commercial (MRS) and waste media with various supplements, where product concentrations were 15 times higher in MRS medium. For the solid state analysis, cultures were treated by successive compression and refilling of tubular minireactors equipped with a piston, without the need for reinoculation. This method was found to be simple, reproducible, and easily controllable, allowing culture productivity to be maintained for long periods of time without alterations in the basic properties of the system. In addition, yields were found to be superior compared to those from submerged culture. The system kinetics were modeled on the basis of widely accepted assumptions with a good fit to the experimental results and observed biomass fluctuations less evident than those predicted by the kinetic model.     

Utilization of prawn waste for chitinase production by the marine fungus Beauveria bassiana by solid state fermentation

Prawn waste, a chitinous solid waste of the shellfish processing industry, was used as a substrate for chitinase production by the marine fungus Beauveria bassiana BTMF S10, in a solid state fermentation (SSF) culture. Theprocess parameters influencing SSF were optimized. A maximum chitinase yield of 248.0 units/g initial dry substrate (U/gIDS) was obtained in a medium containing a 5:1 ratio (w/v) of prawn waste/sea water, 1% (w/w) NaCl,2.5% (w/w) KH2PO4, 425–600m substrate particle size at 27°C, initial pH 9.5, and after 5 days of incubation. The presence of yeast extract reduced chitinase yield. The results indicate scope for the utilization of shellfish processing (prawn) waste for the industrial production of chitinase by using solid state fermentation.     

产脂肪酶重组枯草芽胞杆菌的发酵优化

笔者所在实验室前期筛选到1株产脂肪酶粘质沙雷氏菌,克隆其脂肪酶基因,构建重组枯草芽胞杆菌Bacillus subtilis 168/pMA5-lipA,成功实现了来源于粘质沙雷氏菌的脂肪酶基因在枯草芽胞杆菌中的表达。基于以上工作基础上,对B.subtilis 168/pMA5-lipA进行了摇瓶水平上的产酶发酵优化。首先通过单因素和正交试验确定了有利于产脂肪酶的最佳培养基成分,并对发酵条件进行了优化。结果表明:优化后的培养基组分为蔗糖35 g/L,玉米浆27.5 g/L,(NH4)2SO41.25 g/L,CaCl24 g/L,pH 7.0。在最优发酵培养基的条件下,37℃、160 r/min摇床培养33 h,每毫升发酵液中重组菌脂肪酶酶活可达98.6 U,是优化前的3倍。     

Phytase production by solid-state fermentation of groundnut oil cake by Aspergillus niger: A bioprocess optimization study for animal feedstock applications

This investigation deals with the use of agro-industrial waste, namely groundnut oil cake (GOC), for phytase production by the fungi Aspergillus niger NCIM 563. Plackett–Burman design (PBD) was used to evaluate the effect of 11 process variables and studies here showed that phytase production was significantly influenced by glucose, dextrin, distilled water, and MgSO₄ · 7H₂O. The use of response surface methodology (RSM) by Box–Behnken design (BBD) of experiments further enhanced the production by a remarkable 36.67-fold from the original finding of 15 IU/gds (grams of dry substrate) to 550 IU/gds. This is the highest solid-state fermentation (SSF) phytase production reported when compared to other microorganisms and in fact betters the best known by a factor of 2. Experiments carried out using dried fermented koji for phosphorus and mineral release and also thermal stability have shown the phytase to be as efficient as the liquid enzyme extract. Also, the enzyme, while exhibiting optimal activity under acidic conditions, was found to have significant activity in a broad range of pH values (1.5–6.5). The studies suggest the suitability of the koji supplemented with phytase produced in an SSF process by the “generally regarded as safe” (GRAS) microorganism A. niger as a cost-effective value-added livestock feed when compared to that obtained by submerged fermentation (SmF).     

黑曲霉固态发酵生产单宁酶的条件优化

研究采用响应面法优化黑曲霉固态发酵生产单宁酶的培养条件。应用Plackett—Burman试验筛选出重要影响因子：五倍子粉含量、（NH4）2SO4浓度以及接种孢子量,最陡爬坡试验逼近最大响应区域。应用Box．Behnken响应面试验对重要影响因子进一步优化。得到最佳培养条件：每250mL三角瓶中装入1．0g五倍子粉、4．4g稻壳和0．5g麸皮、液固比（mL／g）2：1且营养盐溶液组成为（NH4）2s0421g/L、MgSO4·7H2O1g／L、NaCl1g／L,培养基pH自然,接种5．7×10^7个孢子后在30℃温度下培养4d。在此条件下,单宁酶产量从40U／g提高到114U／g,3次重复验证性试验平均值为115U／g,验证了模型的可靠性。     

假丝酵母99-125脂肪酶喷雾干燥工艺及催化性质的研究

利用响应面法对假丝酵母脂肪酶喷雾干燥工艺条件进行优化,考察进口温度、雾化速度、保护剂含量对脂肪酶活力收率的影响。确定了最佳喷雾条件:保护剂为10～15 g/L的阿拉伯胶,进口温度115～120℃,雾化速度0.4 L/h,可得到收率最高为60.5%的脂肪酶酶粉。制得的固定化酶用于手性拆分(R,S)-1-苯乙醇,光学产率最高可达到53.6%;用于催化合成生物柴油,转化率最高可达到90.2%。在4、30℃下密封保存,半衰期可分别达到15个月、3个月。     

Utilisation of fruits waste for citric acid production by solid state fermentation

A solid state fermentation method was used to utilise pineapple, mixed fruit and maosmi waste as substrates for citric acid production using Aspergillus niger DS 1. Experiments were carried out in the presence and absence of methanol at different moisture levels. In the absence of methanol the maximum citric acid was obtained at 60% moisture level whereas in the presence of methanol the maximum citric acid was obtained at 70% moisture level. The stimulating effect of methanol was less at lower moisture level. The inhibitory effect of metal ions was also not observed and maximum citric acid yield of 51.4, 46.5 and 50% (based on sugar consumed) was obtained from pineapple, mixed fruit and maosmi residues, respectively.