Lipase and photodecarboxylase coexpression: A potential strategy for alkane-based biodiesel production from natural triglycerides

Alkane-based biodiesel is considered the next generation of biodiesel owing to its potential environmental benefits and the fact that it exhibits much higher specific caloric values than traditional biodiesel.However,the formidable obstacle impeding the commercialization of this cutting-edge fuel alternative lies in the cost associated with its production.In this study,an engineered strain Escherichia coli(E.coli)showcasing harmonized coexpression of a lipase(from Thermomyces lanuginosus lipase,TLL)and a fatty acid photodecarboxylase(from Chlorella variabilis,CvFAP)was first constructed to transform triglycerides into alkanes.The potential of E.coli BL21(DE3)/pRSFDuet-1-TLL-CvFAP for alkane synthesis was evaluated with tripalmitin as a model substrate under various process conditions.Following a comprehensive examination of the reaction parameters,the scope of the biotransformation was expanded to‘real’substrates(vegetable oils).The results showed that bioderived oils can be transformed into alkanes with high yields(0.80-10.20 mmol·L^(-1))under mild conditions(35℃,pH 8.0,and 36 h)and blue light illumination.The selected processes were performed on an increased lab scale(up to 100 ml)with up to 24.77 mmol·L^(-1) tripalmitin,leading to a yield of 18.89 mmol·L^(-1) pentadecane.With the employment of a method for efficiently producing alkanes under mild conditions and a simple procedure to isolate alkanes from the reaction system,the utilization of sustainable biomass as a fundamental feedstock emerges as the primary solution to lower the cost of alkane-based biodiesel.Thus,this study proposes a readily implementable and highly effective approach for alkane-based biodiesel production.

Enantioselective Conversion of Racemic Felodipine to S(-)-Felodipine by Aspergillus niger and Lipase AP6 Enzyme

The present study involves the enantioselective resolution of racemic Felodipine by using free and immobilized forms of microbial cultures as well as an enzyme (Lipase AP6). Among the microbial cultures employed in the present study, Aspergillus niger, Sphingomonas paucimobilis, Cunninghamella elegans, Escherichia coli, Pseudomonas putida and Cunninghamella blakesleeana were found to possess capability of enantioselective resolution of racemic Felodipine. The enantiomeric excess (ee%) of Felodipine after reaction catalyzed by whole-cell A. niger and S. paucimobilis was found as 81.59 and 71.67%, respectively. Immobilization enhanced the enantioselectivity (enantiomeric ratio (E)) of the biocatalysts and hence this led to enhanced enantiomeric purity of the drug. The ee% values were found to be enhanced in reactions catalyzed by A. niger and S. paucimobilis cultures after immobilization as 98.27 and 93.56%, respectively. Enantiomeric ratio (E) of the reactions catalyzed by all the biocatalysts has been improved after immobilization. E value of the reaction catalyzed by immobilized A. niger was found to be excellent (E > 100) and hence the drug showed high enantiomeric purity. In lipase AP6 catalyzed study, the enantioselectivity was enhanced after immobilization with excellent E value, which led to enhanced enantiomeric purity of the drug (99.21% ee%).

Lipase and pancreatic amylase activities in diagnosis of acute pancreatitis in patients with hyperamylasemia

BACKGROUND: Measurement of total serum amylase (AMY) is the most widely used biochemical test for the diagnosis of acute pancreatitis, but it is commonly considered a nonspecific marker. To improve the biochemical diagnosis of acute pancreatitis, lipase ( LIP ) and pancreatic amylase (PAMY) have been tested in recent years. The present study was designed to evaluate whether serum LIP and pancreatic PAMY tests could replace total amylase test to improve diagnostic efficiency in the evaluation of acute pancreatitis in patients with hyperamylasemia. METHODS: LIP and PAMY values were determined in serum samples from 92 patients with hyperamylasemia. Reference values for each enzyme were derived from serum samples of 147 healthy subjects. The activities of LIP and PAMY in patients with various diseases were shown directly by the boxplot graph. The diagnostic accuracy of LIP and PAMY was defined as the area under the receiver operating characteristic (ROC) curve. Their sensitivity and specificity in detecting acute pancreatitis at varying cutoff points were shown by the curve, and the best cutoff value for each enzyme was shown by the modified ROC curve. The diagnostic values of LIP, PAMY and LIP + AMY with each upper limit of reference range (ULR) were compared with the corresponding best cutoff values. RESULTS: The references values of LIP and PAMY were 12.2-47.6 U/L and 28-95 U/L, respectively. These values in patients with acute pancreatitis were higher than those patients with other diseases. The areas under the ROC curve ( AUC) of LIP and PAMY were 0. 799 and 0. 792, respectively, With the best diagnostic cutoff point of maximum (sensitivity + specificity) -100%, we obtained values of 97.9 U/L(LIP97.9 =2. 06 × ULR) for LIP and 209 U/L (PAMY209 =2.20 ×ULR) for PAMY. The best cutoff values for LIP, PAMY and LIP +AMY demonstrated the specificity, positive predictive value, and diagnostic efficiency higher than the corresponding ULRs. CONCLUSIONS: Serum LIP and PAMY are specific for the pancreas and might replace total amylase for the diagnosis of acute pancreatitis in hyperamylasemia patients. LIP97.9 is more efficient than PAMY209 in the diagnosis of acute pancreatitis. A combined test of both enzymes is not superior to single test of either enzyme in diagnostic accuracy.

Enzymatic Glycerolysis of Chinese Vegetable Tallow Fraction by Lipase and Study of the Mechanism

Glycerolysis of Chinese vegetable tallow (CVT) fraction was investigated using a 1,3-specific lipase from Rhizopus arrhizus as catalyst. Based upon a binary gradient HPLC with an evaporative light-scattering detector (ELSD), the contents of free fatty acids (FFA), monoglycerides (MG), diglycerides(DG) and triglycerides (TG)with their positional isomers during the glycerolysis were determined. The effects of water content and the ratio of glycerol to oil on the product distribution of glycerolysis were studied. Under the optimum reactant conditions:250 units lipase per gram oil at 37 ℃ with 1:2 molar ratio of oil to glycerol in a solvent-free system, after 24 h reaction, the product consisted of 7.2% TG, 25.6% MG, 56.1% DG and 4.9% FFA (all by mass). Furthermore, the mechanism of glycerolysis was discussed in detail.

Reduced lysosomal acid lipase activity: A new marker of liver disease severity across the clinical continuum of non-alcoholic fatty liver disease?

Lysosomal acid lipase(LAL)plays a key role in intracellular lipid metabolism.Reduced LAL activity promotes increased multi-organ lysosomal cholesterol ester storage,as observed in two recessive autosomal genetic diseases,Wolman disease and Cholesterol ester storage disease.Severe liver steatosis and accelerated liver fibrosis are common features in patients with genetic LAL deficiency.By contrast,few reliable data are available on the modulation of LAL activity in vivo and on the epigenetic and metabolic factors capable of regulating its activity in subjects without homozygous mutations of the Lipase A gene.In the last few years,a less severe and non-genetic reduction of LAL activity was reported in children and adults with non-alcoholic fatty liver disease(NAFLD),suggesting a possible role of LAL reduction in the pathogenesis and progression of the disease.Patients with NAFLD show a significant,progressive reduction of LAL activity from simple steatosis to non-alcoholic steatohepatitis and cryptogenic cirrhosis.Among cirrhosis of different etiologies,those with cryptogenic cirrhosis show the most significant reductions of LAL activity.These findings suggest that the modulation of LAL activity may become a possible new therapeutic target for patients with more advanced forms of NAFLD.Moreover,the measurement of LAL activity may represent a possible new marker of disease severity in this clinical setting.

Effect of Homogenization Temperature and Pressure on Lipoprotein Lipase Activity and Free Fatty Acids Accumulation in Milk

This study demonstrated that homogenization did not increase the activity of lipoprotein lipase (LPL) in spite of a fast accumulation of free fatty acids (FFA). Two homogenization pressures (100 and 170 bar) and two temperatures (40℃and 50℃) were examined. The activity of LPL was analyzed and the formation of FFA was measured with two different methods, the B.D.I.-method and a nonesterified fatty acids (NEFA) method. A homogenization temperature of 50℃ resulted in a decreased LPL activity compared to 40℃. No effect of homogenization pressure was found. Analyzing FFA concentration with the B.D.I.-method resulted in significant effect of homogenization temperature and no effect of pressure. The largest formation of FFA was found in milk homogenized at 40℃. Using the NEFA method, another result was obtained, indicating no effect of homogenization temperature and a larger FFA accumulation at 100 bar than at 170 bar. Both analytic methods demonstrated significant production of FFA during 60 min incubation at homogenization temperature after treatment. The level of FFA in the milk samples immediately after homogenization was very high, demonstrating that LPL cleaves the triglycerides very rapidly when the native membrane was damaged. The regression between the B.D.I.-method and the NEFA was fair in the interval between 4 and 14 mmol/100 g fat, whereas at higher concentrations, the correlation was poor.

Effects of Different Dietary Lipid Contents on Growth and Lipase Activity of Eriocheir sinensis Larvae

The effects of different dietary lipid content on the growth and lipase activity ofEriocheir sinensis larvae were studied in the paper. The results showed that the survival, metamorphic rate and weight gain of E. sinensis larvae at different stages of growth all varied significantly with lipid content （P〈0.05）. Further, the survival and metamorphosis rates were the highest during the larval phases Z3 to Z4, and the weight gain was the highest during the larval phases Z5 to M. During the first 20h after metamorphosis of every larval stage, the lipase activity increased over time at Z1, Z2, Z3 and M and declined at Z4 and Zs, and was influenced significantly by lipid content （P〈0.05）. In addition, lipase activity at each larval stage began to respond to dietary lipid contents 4h after the larvae were fed, and tended to be stable after 12 h. The diets with higher lipase activity and lower lipid content were selected to give the suitable recipe of lipid requirements at each larval stage. It was concluded that the suitable lipid requirements at Zb Z2, Z3, Z4, Z5 and M were 6%, 4% 6%, 8%, 8% and 10%, respectively.

Effects of long-term ethanol consumption on jejunal lipase and disaccharidase activities in male and female rats

AIM: To study the effect of long-term ethanol consumption on jejunal lipase and disaccharidase (sucrase, maltase,and lactase) activities in rats and its gender difference. METHODS: Age-matched male and female Wistar rats were fed control or ethanol-containing liquid diets for 12 wk following the Lieber-DeCarli model. According to both theplasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities, 40 rats were divided into four groups as follows: male control group (MC), male ethanol group (ME), female control group (FC), and female ethanol group (FE).RESULTS: After ethanol feeding for 12 wk, the results revealed that plasma AST and ALT activities of group MEwere significantly increased by 58% and 92%, respectively,than those of group MC (P＜0.05). Similarly, plasma AST and ALT activities of group FE were also significantly increased by 61% and 188%, respectively, than those of group FC (P＜0.05). Fat accumulation was observed in both ethanol treated groups, while fatty changes were more severe in group FE than those in group ME. The induction of hepatic microsomal cytochrome P450 2E1 (CYP2E1) was obviously seen in group ME and group FE, but was not detected in group MC and group FC. Jejunal lipase activity of group ME was significantly increased by 1.25-fold than that of group MC (P＜0.05). In contrast to, sucrase, maltase, and lactase activities of group ME were significantly decreased by 63%, 62% and 67%, respectively, than those of group MC (P＜0.05). Similarly, activities of these three enzymes of group FE were also significantly decreased by 43%, 46% and 52%, respectively, than those of group FC (P＜0.05).There were no significant epithelial changes of the duodenal mucosa in any group.CONCLUSION: Long-term ethanol consumption significantly can increase jejunal lipase and decrease jejunal disaccharidase activities in both male and female rats.

Combined Use of Co-immobilized Lysozyme and Lipase and Chemical Inhibitors in Circulating Cooling Water System

In order to improve the stability and corrosion inhibition performance of bioenzyme, lipase and lsozyme were co-immobilized on the mesoporous molecular sieve MCM-41 by the adsorption method. Then the immobilized enzymes were combined with amino trimethylene phosphonic acid and polyaspartic acid to prevent corrosion caused by circulating cooling water. The weight-loss method and electrochemical techniques were used to evaluate the performance of composite inhibitors. The co-immobilized lysozyme and lipase achieved good inhibition effects. After they were combined with amino trimethylene phosphonic acid and polyaspartic acid, the corrosion inhibition properties were further improved. The inhibition efficiency was promoted to 94.4%. During the corrosion inhibition process, the immobilized enzymes played an important role. The addition of corrosion inhibitor could inhibit the anodic dissolution and cathodic hydrogen evolution process of carbon steel at the same time. The adsorption of co-immobilized lysozyme and lipase composite inhibitor on the steel surface was a joint action involving physical adsorption and chemical adsorption.

Hawthorn Nectar Enhances Gastrointestinal Motility as Well as Stimulates Intestinal Am-ylase and Lipase Activities in Mice

Gastrointestinal (GI) digestion, which facilitates the decomposition of ingested food into absorbable small molecules for further utilization in the body, necessitates both neural- and hormonal-regulated coordination of GI motility and secretion of digestive enzymes in the GI tract. The dysregulation of such coordination is likely associated with a wide range of disorders in the digestive system. Hawthorn Nectar (HN) is a health supplement for improving the wellness of the gastrointestinal digestive system in humans. The ingredients of HN, which include hawthorn, citrus, germinated barley and honeysuckle, are commonly prescribed to increase appetite and to treat digestive disorders in the practice of traditional Chinese medicine. Pharmacological studies have also shown that these herbs can produce beneficial effects on the GI digestive system. In the present study, HN was first examined for its effects on gastric emptying and postprandial intestinal motility in mice. The activities of digestive enzymes in gastric and pancreatic juice were also measured in HN-pretreated mice. Our results showed that long-term HN treatment increased the extents of gastric emptying and postprandial intestinal motility in mice. HN pretreatment also stimulated the activities of intestinal amylase and lipase in mice, while gastric pepsin and intestinal chymotrypsin activities remained unchanged. However, intestinal trypsin activity was suppressed by HN pretreatment. In conclusion, long-term HN consumption may produce beneficial effect on GI digestive function in humans.

The effects of steaming and roasting treatments on lipase activity and nutritional components of “oat rice” (OR): the peeled naked oat (Avena nuda) kernels

Peeled naked oat kernels, named “oat rice” (OR) by Chinese food scientists and processors, are novel oat products in China. This study exam-ined the effects of steaming and roasting treat-ments on the enzyme activities, nutritional con-tents, and flour pasting properties of OR kernels. Results showed that a peeling time of 20 s caused 16.13% β-glucan loss, while a peeling time 25 s caused 34.29% β-glucan loss in the kernels. OR kernels with a 20 s peeling treatment demonstrated significantly higher starch levels and kernel whiteness compared with normal oat kernels (P<0.01). It was also found that normal pressure steaming, autoclaved steaming and infrared roasting treatments could exterminate lipase activities in the OR kernels, and provide the OR kernels with significantly lower final viscosities and setback values than normal kernels (P<0.01).

Surfactant-coated Candida rugosa Lipase as Catalyst for Hydrolysis of Olive Oil in Solvent-Free Two-Phase System

The surfactant-coated Candida rugosa lipase was used as catalyst for hydrolysis of olive oil in two-phase system consisting of olive oil and phosphate buffer without organic solvent. For both the coated and native lipases,the optimal buffer/oil volume ratio of 1.0, aqueous pH 6.8 and reaction temperature 30℃ were determined. The maximum activity of the coated lipase was ca 1.3 times than that of the native lipase. The half-life of the coated lipase in olive oil and the native lipase in phosphate buffer was ca 9 h and 12 h, and the final residual activity was 27% and 20% of their initial values, respectively. The final substrate conversion by the coated lipase was ca 20% higher than that of the native lipase.

Enzymatic reaction of ethanol and oleic acid by lipase and lignin peroxidase in rhamnolipid(RL) reversed micelles

An environment friendly bio-surfactant of rhamnolipid(RL) was used as a solvent. The enzymatic reaction of oleic acid catalyzed by lipase and lignin peroxidase(lip) was evaluated. The optimum conditions of enzymatic reaction catalyzed by lipase(lip) were water to amphiphile molar ratio of 30(20), RL of 60(60) critical micelle concentration(CMC), pH of 7.0(3.0) and temperature of 40(30) °C, respectively. The change of enzyme conformation indicates that, for catalytic of lipase, water content is the most important factor of the enzymatic reaction of oleic acid, and p H for lip. With individual optimum conditions, the enzymatic efficiency of oleic acid catalyzed by lipase is higher than that by lip. In the presence of ethanol, the enzymatic reaction of oleic acid catalyzed by lipase suits Ping-Pong Bi-Bi mechanism. As an alternative to chemical reversed micelles, the RL reversed micelles are promising methods to enzymatic reaction of oleic acid.

Lipoprotein lipase and obesity

Obesity is one of the fast-growing major diseases in developed and developing countries. As has been persuasively argued, long-term imbalance between intake and expenditure of fat is a central factor in the etiology of obesity. Obesity aggravates insulin resistance and promotes cardiovascular diseases and atherosclerosis. We hypothesized that elevating lipoprOtein lipase (LPL) activity in skeletal muscle would cause an improvement of obesity. To test this hypothesis, we studied the effects of the LPL activator NO-1886 inobese animals. NO-1886 elevated LPL activity in skeletal muscle, and improved obesity as well as insulin resistance in obese rats. Furthermore, NO-1886 mitigated body weight gain induced by pioglitazone without suppressive effect on the adiponectin-increasing action of pioglitazone. LPL activators hold a lot of promise of curing several diseases shown above in clinical scene.

Steeping of Whole Dried Maize Seeds in Buffers Altered Fatty Acid Ionization State, Composition, and Lipase Activity

Steeping is a simple model of studying the activation and modulation of the physiological pathways involved in seed germination. In this study, steeping of grains of the ‘obatanpa’ maize variety in buffers at different pH was monitored through the measurements of lipase activity, oil yield, fatty acid component and unsaturation, and germination capacity. Lipase activity of grains steeped for four days decreased in the order: pH 3 > pH 5 > pH 7 > pH 9 > pH 11. Decreasing lipase activity was corroborated with decreasing free fatty acid components, protein concentrations and oil yields. The unsaturation components of the oil fractions only marginally increased with increasing steeping media pH. Three major components were detected by TLC in all oil fractions. The unique components were confirmed by their uniform UV-absorption spectra converging at an isosbestic point of 290 nm. Germination capacity was much reduced for seeds steeped in buffered media for 24 hours compared with seeds steeped in portable water though the pattern of germination, which was monitored for five days, did not change. This study has demonstrated the use of pH changes of steeping medium to modulate physicochemical properties and germination of seeds. The physicochemical changes were observed after seeds have been submerged under steeping buffer for four days. Practical application: With proliferation of specialty maize hybrids, the study provides an insight into the development of experimental protocols for the selection of types of maize grain for preparation of foods and beverages in terms of general characterization and lipolytic activity, which have implications for flavor, taste and odor of the final products. The imminence of this in some traditional ways of preparing malted and fermented maize foods and beverages, which go through days of steeping, cannot be overemphasized. This study therefore provides another dimension to the manipulation of the steeping stage to develop varieties of maize-based product.

Lipase and Phospholipase in the Hydrolysis of Lipids in Wastewater from Swine Slaughterhouse and Subsequent Biological Treatment Study

Wastewaters from slaughterhouses are characterized by a high concentration of oils and greases that can cause problems in conventional treatments. The degradation of difficult pollutants in more easily degradable products can be mediated by enzymes. Enzymatic hydrolysis facilitates the biodegradation because it makes the organic material is made available as compounds of lower molecular weight, the lipids being made available in the form of free fatty acids. The application of enzymes as processing aids in wastewater treatment has some advantages, such as the specificity that allows control of the products, which leads to increased income generation for the non-toxic byproducts and moderate conditions of operation. The objective of this work was optimizing the conditions for lipids enzymatic hydrolysis present in swine slaughterhouse wastewaters using lipolytic enzymes and subsequent biological treatment study. Temperature, pH and enzyme concentration were the variables tested. The enzymes showed good activity and could therefore be used for the hydrolysis process proposed here. The optimized conditions to maximize the release of fatty acids were： temperature of 36 ℃, pH 8.5 and enzyme concentration of 1.1%, yielding fatty acids in the order of 31.50μmol/mL for lipase and 31.13 μmol/mL for phospholipase. All variables influenced the release of fatty acids. The maximum yield of biogas was 89.65 mL in the reactor added the sludge, raw wastewater and phospholipase in the conditions optimized the hydrolysis step, obtaining a chemical oxygen demand （COD） reduction of 90.01% in relation to the value of the COD of the raw wastewater.

The Effectiveness of Watermelon Endocarp Extract in Inhibiting Lipase Activity Relative to the Hypolipidemic Drugs

In the effort to prove the effectiveness of watermelon endocarp extract as a hypolipidemic, the authors have done an initial experiment of lipase inhibition with the extract. The puposes of this study are to evaluate： （1） the optimum condition of lipase in the pankreoflat tablet, （2） the effect of watermelon endocarp extract in inhibition the lipase activity, and （3） the effectiveness of watermelon endocarp extract as lipase inhibitor relative to the hypolipidemic drugs （orlistat）. Watermelon endocarp was extracted by blender, squeezed and filtered. As a source of lipase has been used pankreoflat （Kimia Farina） tablet, inhibition test was performed by mixing 35 mL with substrate （olive oil） that has been coupled with one tablet （0.25 g） ofpankreoflat and extract of 50 g watermelon endocarp and then incubated at 37℃, pH 7.5 for 25 minutes. Lipase activity is indicated by the amount ofNaOH titrant which was used to neutralize the free fatty acid from hydrolysis of olive oil. The results showed that 50 g of watermelon endocarp can produce 26 mL extract and decrease the lipase activity by 70.34%, or equivalent to 85% of the effectiveness of one tablet orlistat （120 mg）, one of the hypolipidemic drugs.

Microencapsulation of Lipase and Savinase Enzymes by Spray Drying Using Arabic Gum as Wall Material

Enzymes have been used in detergents over the years. They can improve the detergent’s efficiency due to their activities against hard stains. Nevertheless, enzymes cannot maintain their properties indefinitely, since they are exposed to stress factors, like temperature, pH, mechanical processes and others. Consequently, enzymes lose their structure and they are not functional. For this reason, microencapsulating these proteins is a feasible solution to improve their use in industrial processes and commercial products. Spray drying technology has been selected because a lot of scientific literature proved its useful application in a variety of industries. In particular, savinase and lipase are the two encapsulated enzymes in this work. Savinase attacks proteins and lipase removes fats, so they are suitable enzymes for detergent industry. Arabic gum has been used as wall material. Morphology, size and activity of the obtained microcapsules have been analyzed in order to find the best conditions to produce them. In conclusion, useful microcapsules of lipase and savinase can be obtained with the mentioned technology.

Study on the Determination Conditions of Lipase Activity and the Effects of Commercial Detergent Products

The activity of lipase was determined by alkaline titration method and the conditions for the determination of lipase activity were studied. The results showed that the lipase activity was stable when the volume fraction of olive oil was 25% and the temperature was at 40℃ with PH=7.5. Under the proper conditions, the effects of anionic and nonionic surfactants were investigated. The effect of nonionic surfactants on the activity of lipase was less than that of anionic surfactants. Finally, the analysis method was used to determine the activity of the lipase in the commercial detergent products. The results showed that the method can be used for the determination of lipase activity in both liquid detergent and laundry powder. However, the temperature and pH value of the detergent solution should be controlled strictly during the conduction of the determination.

Bound lipase:an important form of lipase in rice bran (Oryza sativa)

Rice bran residue possessed a steady lipase activity((26.68 ± 3.69)%)after its endogenous lipase was extracted continuously by phosphate buffer solution(PBS)for 24 h. T herefore, the aim of this research was to explore whether there exist any bound lipases in rice bran(Oryza sativa). Three physical treatments(grinding, homogenizing and ultrasound crush)and 6 enzymatic treatments(cellulase, hemicellulase, pectinase, complex cellulase, glucoamylase and α-amylase)were applied to rice bran in order to investigate this bound lipase. The relative catalytic activities of extraction supernatant and residue for pectinase group were(437.63 ± 22.54)% and(159.26 ± 2.12)%, respectively, which were significantly higher(P < 0.05)than other groups. This phenomenon demonstrated that lipase was the most likely to combine with pectin. Molecular simulation proved that pectin could combine with two rice bran lipases(lipase 315 and lipase 308)and cover the catalytic centers so as to prevent the lipases from encountering the substrate and inhibiting their catalytic activities. During combination, pectin could make the lipases more compact and reduce the solvent accessible surface area of lipases, which would make the lipases inactive to molecular interaction. In summary, part of rice bran lipase was proved to exist in bound form and combined with the pectin.