What Are Lipidoses?

Lipase (glyceride hydrolase) belongs to carboxyl ester hydrolase, which can gradually hydrolyze triglycerides into glycerol and fatty acids. Lipases are found in tissues of animals, plants, and microorganisms (such as molds, bacteria, etc.) that contain fat. Includes phosphatases, sterolase and carboxylesterase. Fatty acids are widely used in food, pharmaceuticals, leather, and daily chemicals.

Basic information on lipase

Chinese name: Lipase

Chinese alias:

English name: Lipase

English alias: Rizolipase [USAN: INN]; Accelerase; Allzyme Lipase; Amano N-AP; Butyrinase; Chirazyme L; EC3.1.1.3 .; Enzylon PF; Fetipase; Fluozim G 3Kh; Fungal lipase; GA 56 (Enzyme); GA-56; Glycerol ester hydrolase; Ilozyme; Lipase AP; Lipase, fungal; Lipase, triacylglycerol; Lipazin; Meito MY 30; Remzyme PL 600; Rizolipasa; Rizolipasa [INN-Spanish]; Rizolipase; Rizolipasum; Rizolipasum [INN-Latin] ; Steapsin; Takedo 1969-4-9; TheraCLEC-Lipase; Triacetinase; Triacylglycerol hydrolase; Triacylglycerol lipase; Tributyrase; Tributyrin esterase; Tributyrinase; Triglyceride hydrolase; Triglyceride lipase; Triolein hydrolase; Tween esterase; Tween esterase; Tween esterase; UNII-FQ3DRG0N5K; Lipase of Rhizopus arrhizus var. Delemar; Plant lipase; Animal lipase

Lipase source

Lipases are widely found in plants, animals and microorganisms. Plants containing more lipases are the seeds of oil crops, such as castor seeds and rapeseed. When oil seeds germinate, lipases can work in concert with other enzymes to catalyze the breakdown of oils and fats to produce sugars and provide seed rooting Nourishment and energy necessary for germination; the animal body contains more lipases, such as the pancreas and adipose tissue of higher animals, and a small amount of lipase in the intestinal fluid. The gastric juice contains a small amount of glyceryl butyrate. In animals, various types of lipases control processes such as digestion, absorption, fat reconstruction, and lipoprotein metabolism; bacteria, fungi, and yeast are richer in lipases (Pandey et al). Due to the variety of microorganisms, fast reproduction, and prone to genetic variation, it has a wider range of action than animals and plants, a range of action temperature and substrate specificity, and lipases derived from microorganisms are generally secreted extracellular enzymes. The main fermentation microorganisms are Aspergillus niger, Candida and so on. It is suitable for large-scale industrial production and obtaining high-purity samples. Therefore, microbial lipase is an important source of industrial lipase. Generally, the characteristics of lipase from different sources are different and have important significance in theoretical research.

Lipase classification

According to the substrate specificity of lipase, it can be divided into three categories: fatty acid specificity, position specificity and stereospecificity. According to the source of lipase, lipase can be divided into animal lipase, plant lipase and microbial lipase. Lipases from different sources can catalyze the same reaction, but when the reaction conditions are the same, the rate and specificity of the enzymatic reaction are not the same ^[1] .

Lipase properties

Lipases are a class of enzymes with a variety of catalytic capabilities, which can catalyze the hydrolysis, alcoholysis, esterification, transesterification and reverse synthesis of esters of triacylglycerols and other water-insoluble esters. It also shows the activity of other enzymes, such as phospholipase, lysophospholipase, cholesterol esterase, acyl peptide hydrolase activity (Hara; Schmid). The different activities of lipases depend on the characteristics of the reaction system, such as promoting ester hydrolysis at the oil-water interface, and enzymatic synthesis and transesterification in the organic phase.

The study of the properties of lipase mainly includes the optimal temperature and pH, temperature and pH stability, and substrate specificity. So far, a large number of microbial lipases have been isolated and purified, and their properties have been studied. They differ in molecular weight, optimum pH, optimum temperature, pH and thermal stability, isoelectric point and other biochemical properties (Veeraragavan et al. ). In general, microbial lipases have a wider range of action pH, temperature range, high stability and activity than animal and plant lipases, and are specific for substrates (Schmid et al .; Kazlauskas et al.).

The catalytic properties of lipase are: its catalytic activity is the largest at the oil-water interface. This phenomenon was discovered by Sarda and Desnnelv as early as 1958. Water-soluble enzymes act on water-insoluble substrates, and the reaction proceeds at the interface between two completely different phases separated from each other. This is a feature that distinguishes lipase from esterase. Esterase (E C3.1.1.1) is a water-soluble substrate, and the most suitable substrate is an ester formed from short-chain fatty acids (C8).

Lipase is one of the important varieties of industrial enzyme preparations, which can catalyze reactions such as lipolysis, transesterification, and ester synthesis. It is widely used in oil processing, food, medicine, and daily chemical industries. Lipases from different sources have different catalytic characteristics and catalytic activities. The large-scale production of lipases with transesterification or esterification functions for organic phase synthesis is of great significance for the enzyme-catalyzed synthesis of fine chemicals and chiral compounds.

Lipase is a special ester bond hydrolase that acts on the ester bonds of triglycerides to degrade triglycerides into diglycerides, monoglycerides, glycerol, and fatty acids.

An enzyme is an active protein. Therefore, all factors that affect protein activity affect enzyme activity. The activity of enzymes and substrates is affected by many factors such as temperature, pH value, enzyme solution concentration, substrate concentration, enzyme activator or inhibitor, and so on.

Lipases are widely distributed among microorganisms, and their producing bacteria are mainly molds and bacteria. There are 33 different types of lipases that have been published for triglyceride processing, 18 of which are from molds and 7 from bacteria.

Lipase can hydrolyze glycerides (oil, fat) and release fatty acids, diglycerides, monoglycerides and glycerol at different stages. Fatty acids produced by hydrolysis can be titrated with a standard alkaline solution, and the titer value indicates the enzyme activity.

The reaction formula is: RCOOH + NaOH RCOONa + H2O

Lipase Lipase

ATGL fat triglyceride lipase, HSL hormone sensitive lipase, and monolipase lipase, where ATGL only hydrolyzes TG, while HSL can hydrolyze TG and DG, and MGL only hydrolyzes monoglyceride.

Main uses of lipase

Microbial-derived lipases can be used to enhance the flavor of cheese products. Limited hydrolysis of fats in milk can be used in the production of chocolate milk. Lipase can make food form a special milk flavor.

Lipase can prevent the taste of baked goods through the release of monoglycerides and diglycerides. The degreasing of bones during gelatin production needs to be performed under mild conditions. Lipase-catalyzed hydrolysis can accelerate the degreasing process ^[2] .

Lipase Catalytic Mechanism

Lipase has the affinity of the oil-water interface, and can catalyze the hydrolysis of water-insoluble lipids at a high rate on the oil-water interface. Lipase acts on the hydrophilic-hydrophobic interface layer of the system, which is also different from esterase. A feature.

Lipases from different sources may have large differences in amino acid sequences, but their tertiary structures are very similar. Lipase active site residues consist of serine, aspartic acid, and histidine, which belong to serine proteases. The catalytic site of lipase is buried in the molecule, and the surface is covered with a spiral cap-like structure formed by relatively hydrophobic amino acid residues (also known as "cap"), which protects the catalytic site of the triplet. The amphiphilicity of the alpha-helix in the "lid" will affect the ability of the lipase to bind to the substrate at the oil-water interface, and its reduced amphiphilicity will lead to a reduction in lipase activity. The outer surface of the "lid" is relatively hydrophilic, while the inner surface facing the inside is relatively hydrophobic. Due to the association between lipase and the oil-water interface, the "lid" is opened and the active site is exposed, which enhances the binding ability of the substrate to the lipase. The substrate easily enters the hydrophobic channel and combines with the active site. Enzyme-substrate complex. Interfacial activation can increase the hydrophobicity near the catalytic site, leading to -helix reorientation, thereby exposing the catalytic site; the presence of the interface can also cause the enzyme to form an incomplete hydration layer, which is beneficial to the aliphatic substrate of the aliphatic The side chain folds onto the surface of the enzyme molecule, making enzyme catalysis easy ^[1] .

Expression of lipase activity

At a certain temperature and a certain pH, the amount of enzyme that hydrolyzes triglycerides to produce 1 mol fatty acid per minute is an international unit, expressed in u / ml or u / g.

Normal Lipase

Titration method: The enzymatic reaction is 0.06 to 0.89 U / ml for 4 hours, and the enzymatic reaction is 0.2 to 1.5 U / ml for 16 to 24 hours.

Turbidimetric method: Positively skewed distribution, the lowest is OU, and the upper limit of 95% on one side is 7.9U.

Clinical significance of lipase

The pancreas is the main source of human LPS. Increased serum LPS is common in acute pancreatitis and pancreatic cancer, and occasionally in chronic pancreatitis. In acute pancreatitis, the increase in serum amylase is short, and the increase in serum LPS activity can last for 10 to 15 days. When mumps does not affect the pancreas, LPS is usually in the normal range. In addition, common bile duct stones or cancer, intestinal obstruction, duodenal perforation, etc. may sometimes increase.

Determination of lipase activity

method:

Preparation of crude enzyme solution

Crude lipase 0.010 g, 0.020 g, and 0.030 g were weighed with an electronic balance, dissolved in distilled water and made up to 100 mL, to prepare crude enzyme solutions with concentrations of 0.01%, 0.02%, and 0.03%, respectively.

Experimental design

In this experiment, 10 mL salad oil was used as the substrate, and the optimum conditions for hydrolysis were selected by measuring the acid value with the amount of enzyme, hydrolysis temperature, and reaction time as factors.

Determination of osmium acid value

Acid value is the number of milligrams of NaOH needed to neutralize 1 mol of free fatty acid. It is used to measure the degree of hydrolysis of fats and oils. In the experiment, the acid value of the hydrolysate was determined by acid-base titration. With reference to the method used in [3, 4], 1 mL of a 95% ethanol solution was added dropwise to the obtained hydrolysate, and the mixture was shaken to stop the reaction, and added Two drops of phenolphthalein indicator were quickly titrated with a 0.05 mol / L NaOH solution until the solution was reddish, and the end point did not disappear within 30 s. The number of milliliters (V) of the NaOH solution consumed was recorded. The blank value was determined by the same method. Each test was repeated twice, and the average value was used as the measurement result.

The acid value is calculated as follows:

X = C (V-V0) × 40 / M

In the formula: X oil acid value (mgNaOH / g oil)

CNaOH standard solution concentration (mol / L)

M mass of the sample (g)

40NaOH in mmol (mg / mmol)

4 Determination of crude lipase activity

Under the best hydrolysis conditions, the acid values X1 and X2 of crude lipase and standard lipase hydrolysate were measured, and the activity of crude lipase was obtained according to the formula U1 / U = X1 / X2, where U1 is the activity of crude lipase , U is the standard lipase activity.

5 Preparation of standard lipase solution

According to the optimal conditions of the experiment, prepare a standard lipase solution. A related literature on the determination of crude lipase activity.pdf

Answer: The experimental design. This experiment uses 10 mL salad oil as the substrate, and uses the amount of enzyme, hydrolysis temperature, and reaction time as factors. The optimal conditions for hydrolysis are selected by measuring the acid value. It is not appropriate to use salad oil as a substrate, usually olive oil, chemically pure.