Biosynthesis method of high-activity recombinant human cholate activated lipase

Abstract

The invention belongs to the field of bioengineering, and particularly relates to a biosynthesis method of high-activity recombinant human cholate activated lipase. According to the biosynthesis method of the high-activity recombinant human cholate activated lipase provided by the invention, correct folding and stability of rBAL in a pichia pastoris system are greatly promoted through application of gene codon optimization and efficient secretion of signal peptide, especially a low-temperature fermentation control strategy in a protein induced expression stage, and the recombinant human cholate activated lipase has the advantages of high activity and high stability. Protein aggregation and degradation are effectively reduced, and finally an rBAL product with the specific activity far higher than that of a conventional method is obtained. Furthermore, the prepared rBAL is applied to food beneficial to improvement of fat digestion and absorption, especially infant formula milk powder, the digestion function of breast milk can be effectively simulated, the digestion and absorption rate of fat in the milk powder is remarkably increased, and the rBAL has important nutritional significance and market value.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering, specifically relating to a method for the biosynthesis of highly active recombinant human bile salt-activated lipase. Background Technology

[0002] Bile salt-activated lipase (BLA) is a key hydrolytic enzyme in the mammalian digestive system, especially abundant in breast milk. In the presence of bile salts, it efficiently hydrolyzes triglycerides, cholesterol esters, and fat-soluble vitamin esters, which is crucial for newborns (especially premature infants) whose fat digestion and absorption functions are not yet fully developed. BLA in breast milk effectively compensates for insufficient pancreatic secretion in infants, ensuring adequate absorption of fats and fat-soluble nutrients and promoting growth and development.

[0003] Currently, commercially available infant formula strives to mimic breast milk in terms of fat composition, but it is significantly lacking in digestive enzymes, especially active BAL. This is considered to be one of the important reasons why formula-fed infants have a lower fat absorption rate than breastfed infants.

[0004] Traditionally, BAL is extracted from animal tissues such as pig pancreas. This method has many inherent drawbacks, including: (1) limited sources and high costs: it depends on animal slaughter, the supply of raw materials is unstable, the extraction process is complex and the cost is high; (2) safety risks: there is a risk of contamination by animal pathogens (such as viruses and prions), which poses a potential threat to infants with weak immune systems; (3) quality and activity are difficult to control: the extraction process can easily lead to enzyme inactivation, and there are large differences in enzyme activity and purity between different batches, resulting in poor product quality uniformity; (4) immunogenicity: foreign proteins may cause allergic reactions.

[0005] Producing recombinant BAL (rBAL) using genetic engineering is an ideal way to solve the above problems. However, existing technologies face challenges in producing highly active rBAL suitable for the food industry. BAL is a structurally complex glycoprotein whose native conformation and activity are highly dependent on correct post-translational modifications. When expressed in prokaryotic systems such as E. coli, it easily forms inactive inclusion bodies due to the lack of glycosylation and other modification capabilities, making renaturation difficult. In some eukaryotic expression systems, although modification capabilities are present, problems such as low expression levels, protein degradation, or unsatisfactory glycosylation patterns often lead to insufficient activity and stability. Therefore, developing a biosynthetic method for rBAL that can achieve high expression, high activity, and good safety is of great significance for improving the quality of infant formula. Summary of the Invention

[0006] The problem the invention aims to solve

[0007] In response to the aforementioned needs of the prior art, this invention provides a biosynthetic method for recombinant human bile salt-activated lipase, as well as a highly active recombinant human bile salt-activated lipase preparation obtained using this biosynthetic method and its uses, which provides assistance for food preparation for people who need to improve fat digestion and absorption, especially for the preparation of infant formula.

[0008] Solution for solving the problem

[0009] [1]. A method for the biosynthesis of recombinant human bile salt-activated lipase, wherein the method comprises the following steps:

[0010] S1: Constructing a Pichia pastoris recombinant host cell containing a codon-optimized human bile salt-activated lipase encoding gene;

[0011] S2: Fermentation culture of the recombinant host cells of Pichia pastoris and obtaining fermentation supernatant; the fermentation culture includes a cell growth stage with glycerol as carbon source and a protein induction expression stage with methanol as carbon source and inducer; wherein, the fermentation temperature of the protein induction expression stage is 20℃-25℃;

[0012] S3: Separate and purify the recombinant human bile salt-activated lipase from the fermentation supernatant.

[0013] [2]. According to the biosynthesis method described in [1], in S1, the codon-optimized human bile salt-activated lipase encoding gene is fused with the Saccharomyces cerevisiae α-factor signal peptide encoding gene.

[0014] [3]. According to the biosynthesis method described in [1] or [2], in S2, glycerol is added by fed-batch method during the cell growth stage. When the cell concentration reaches 150 g / L-200 g / L on a wet weight basis, the feeding of glycerol is stopped. After the glycerol is exhausted, methanol is added by fed-batch method, and the concentration of methanol is controlled at 0.5% (v / v)-1.0% (v / v) to carry out the protein induction expression stage.

[0015] [4]. The biosynthesis method according to any one of [1]-[3], wherein in S2, the fermentation temperature of the cell growth stage is 28℃-30℃ and the fermentation pH is 5.0-6.0.

[0016] [5]. The biosynthesis method according to any one of [1]-[4], wherein in S2, the fermentation pH of the protein induction expression stage is 5.5-6.5 and the fermentation time is 60h-96h.

[0017] [6]. The biosynthesis method according to any one of [1]-[5], wherein, in S2, the protein induction expression stage sequentially includes a protein expression initiation stage at a fermentation temperature of 24℃-25℃ and a protein expression folding stage at a fermentation temperature of 20℃-21℃.

[0018] [7]. The biosynthesis method according to any one of [1]-[6], wherein in S3, the fermentation supernatant is first concentrated by ultrafiltration, and then separated and purified by ion exchange chromatography and hydrophobic interaction chromatography in sequence.

[0019] [8]. A recombinant human bile salt activated lipase preparation, wherein the recombinant human bile salt activated lipase preparation is prepared by any one of the biosynthetic methods described in [1]-[7], and the specific activity of the recombinant human bile salt activated lipase preparation is greater than 8000 U / mg.

[0020] [9]. Application of the recombinant human bile salt-activated lipase preparation according to [8] in the preparation of food for improving fat digestion and absorption, wherein the food is infant food, children's food, adult food, maternal food, and / or elderly food.

[0021]

[10] . An infant formula food, wherein the infant formula food comprises a recombinant human bile salt activated lipase preparation according to [8].

[0022] The effects of the invention

[0023] This invention provides a method for the biosynthesis of recombinant human bile salt-activated lipase (rBAL), comprising the steps of constructing recombinant Pichia pastoris host cells, fermenting and culturing them, and isolating and purifying the recombinant human rBAL. By optimizing the codons of the rBAL encoding gene and fusing it with a highly efficient secretion signal peptide, particularly through the application of a low-temperature fermentation control strategy during the protein induction expression stage, the correct folding and stability of rBAL in the Pichia pastoris system are greatly promoted, effectively reducing protein aggregation and degradation. Ultimately, an rBAL product with a specific activity far exceeding that of conventional methods is obtained. Simultaneously, this invention optimizes the high-density fermentation process, combined with precise carbon source switching and methanol feed control, achieving high-density cell growth and efficient secretory expression of the target protein, thus improving the yield per unit volume. This invention uses the recognized safe Pichia pastoris as the production host, eliminating the risk of animal-derived pathogen contamination. Furthermore, the humanized gene sequence reduces the risk of immunogenicity. The product is particularly suitable for foods for populations requiring improved fat digestion and absorption, especially infant formula.

[0024] Furthermore, the present invention applies the prepared rBAL to infant formula milk powder, which can effectively simulate the digestive function of breast milk and significantly improve the digestibility and absorption rate of fat in milk powder, which has important nutritional significance and market value. Attached Figure Description

[0025] Figure 1 Example 1 illustrates the biosynthesis method of recombinant human bile salt-activated lipase. Detailed Implementation

[0026] The following describes embodiments of the present invention, but the present invention is not limited thereto. The present invention is not limited to the various configurations described below, and various modifications can be made within the scope of the claims. Embodiments and examples obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention.

[0027] In this invention, the terms "comprising," "having," "including," or "containing" can mean included or open-ended, and do not exclude additional, uncited elements or method steps. At the same time, "comprising," "having," "including," or "containing" can also mean closed-ended, excluding additional, uncited elements or method steps.

[0028] In this invention, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0029] In this invention, "optional" or "optionally" means that certain substances, components, execution steps, application conditions, etc., are used or not used.

[0030] In this invention, the numerical range referred to as “value A ~ value B”, “value A - value B”, or “value A above / below” refers to the range that includes the endpoint values ​​A and B.

[0031] In this invention, the term "about" is used to define that the numerical ranges and parameters of this invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. Unless otherwise explicitly stated, it should be understood that all ranges, quantities, values, and percentages used in this invention are modified by "about". Here, "about" generally means that the actual value is within ±5%, ±3%, ±1%, or ±0.5% of a specific value or range. Furthermore, the values ​​and ranges appearing in this invention should be understood to include systematic errors that are unavoidable in industrial production.

[0032] In this invention, the terms "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to the described embodiment that are included in at least one of the embodiments described herein, and may or may not be present in other embodiments. Furthermore, it should be understood that the elements may be combined in any suitable manner in various embodiments.

[0033] In this invention, "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of the protein encoded by the inserted polynucleotide, the vector is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to, plasmids, viruses, bacteriophages, Coase plasmids, transposons, and artificial chromosomes.

[0034] In this invention, "host cell" refers to a cell into which the aforementioned vector can be introduced. Host cells may include bacterial, fungal, plant, or animal cells. Examples of suitable bacteria include, but are not limited to, members of the Enterobacteriaceae family, such as strains of *Escherichia coli*. Examples of suitable fungi include, but are not limited to, *Saccharomyces cerevisiae* and *Pichia pastoris*. Examples of suitable animal host cell lines include, but are not limited to, CHO (Chinese hamster ovary cell line) and NSO cells.

[0035] In this invention, "codon optimization" refers to configuring the nucleotide sequence encoding a polypeptide to contain codons preferred by the host cell or organism in order to improve gene expression and translation efficiency in the host cell or organism.

[0036] Unless otherwise defined, other technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0037] I. Biosynthetic method for recombinant human bile salt-activated lipase

[0038] This invention provides a method for the biosynthesis of recombinant human bile salt-activated lipase, the method comprising the following steps:

[0039] S1: Constructing a Pichia pastoris recombinant host cell containing a codon-optimized human bile salt-activated lipase encoding gene;

[0040] S2: Ferment and culture the above-mentioned Pichia pastoris recombinant host cells and obtain fermentation supernatant; the fermentation culture includes a cell growth stage using glycerol as a carbon source and a protein induction expression stage using methanol as a carbon source and inducer; wherein, the fermentation temperature of the protein induction expression stage is 20℃-25℃;

[0041] S3: Separate and purify the recombinant human bile salt-activated lipase from the fermentation supernatant.

[0042] In some implementations, in S1, the coding gene sequence of human bile salt-activated lipase is artificially designed and optimized according to the codon preference of the target organism (Pichia pastoris) to obtain an optimized nucleotide sequence, which is then cloned into a vector to construct a recombinant expression vector. The vector is then transformed into a host cell (Pichia pastoris) to obtain a recombinant host cell.

[0043] In some embodiments, the codon-optimized human bile salt-activated lipase encoding gene is fused with a Saccharomyces cerevisiae α-factor signal peptide encoding gene. This invention does not impose any particular limitation on the codon-optimized human bile salt-activated lipase encoding gene, as long as it can encode human bile salt-activated lipase (GenBank: AAA63211.1).

[0044] In some embodiments, the recombinant expression vector includes control elements, which are promoters, terminators, and / or enhancers.

[0045] In some embodiments, the recombinant expression vector is a plasmid vector containing the codon-optimized human bile salt-activated lipase encoding gene sequence; a preferred plasmid vector is pPICZα A.

[0046] In some specific implementations, the Pichia pastoris is strain GS115.

[0047] In some embodiments, in S2, during the bacterial growth phase, glycerol is added in a fed-batch manner. When the bacterial concentration reaches 150 g / L-200 g / L (e.g., 150 g / L, 160 g / L, 170 g / L, 180 g / L, 190 g / L, 200 g / L, etc.) on a wet weight basis, the feeding of glycerol is stopped. After the glycerol is depleted, methanol is added in a fed-batch manner, and the concentration of methanol is controlled at 0.5% (v / v)-1.0% (v / v) (e.g., 0.5% (v / v), 0.6% (v / v), 0.7% (v / v), 0.8% (v / v), 0.9% (v / v), 1.0% (v / v), etc.) to carry out the protein induction expression phase.

[0048] In some embodiments, in S2, the fermentation during the cell growth phase can be carried out in a basal salt medium supplemented with glycerol, or in a BMGY medium supplemented with glycerol.

[0049] In some embodiments, in S2, the protein induction expression phase can be carried out in a basal salt medium supplemented with methanol or in a BMMY medium supplemented with methanol.

[0050] In some embodiments, in S2, the fermentation temperature during the cell growth stage is 28°C-30°C (e.g., 28°C, 29°C, 30°C, etc.), and the fermentation pH is 5.0-6.0 (e.g., 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, etc.).

[0051] In some embodiments, in S2, the fermentation pH of the protein induction expression phase is 5.5-6.5 (e.g., 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, etc.), and the fermentation time is 60h-96h (e.g., 60h, 72h, 84h, 96h, etc.).

[0052] In some embodiments, in S2, the protein induction expression stage sequentially includes a protein expression initiation stage at a fermentation temperature of 24℃-25℃ and a protein expression folding stage at a fermentation temperature of 20℃-21℃; preferably, the protein expression initiation stage lasts for 11h-13h, more preferably 12h.

[0053] In some implementations, in S3, the fermentation supernatant is first concentrated by ultrafiltration, and then separated and purified by ion exchange chromatography and hydrophobic interaction chromatography in sequence.

[0054] In some embodiments, the ion exchange chromatography primarily utilizes the difference in charge between recombinant human bile salt-activated lipase and other proteins at a specific pH for preliminary capture and purification. At pH 7.5, rBAL (whose isoelectric point pI is expected to be below 7.5) carries a negative charge and will bind to the positively charged chromatographic medium.

[0055] In some embodiments, the hydrophobic interaction chromatography primarily utilizes the difference in hydrophobicity between recombinant human bile salt-activated lipase and other proteins for fine purification. At high salt concentrations, hydrophobic regions on the protein surface are exposed and bind to a hydrophobic medium (phenyl). Through a decreasing salt gradient, the least hydrophobic proteins are eluted first, thus achieving separation.

[0056] In some implementations, the ion exchange chromatography can be performed using the DEAE Sepharose medium.

[0057] In some implementations, the hydrophobic interaction chromatography can be performed using Phenyl Sepharose or Butyl Sepharose High Performance media.

[0058] In some embodiments, after step S3, a step of freeze-drying the product obtained in S3 is further included; preferably, a step of adding a freeze-drying protectant to the product obtained in S3 is included before freeze-drying; more preferably, the freeze-drying protectant is trehalose, and the amount added is preferably 5%.

[0059] II. Recombinant human bile salt-activated lipase preparations

[0060] This invention provides a recombinant human bile salt-activated lipase preparation prepared using the above-described biosynthesis method. The specific enzyme activity of this preparation is greater than 8000 U / mg, and can reach 8500 U / mg.

[0061] The present invention does not impose any special limitation on the form of the recombinant human bile salt activated lipase preparation, which can be a solid powder preparation or a liquid preparation.

[0062] III. Uses of recombinant human bile salt-activated lipase preparations

[0063] This invention provides the application of the above-mentioned recombinant human bile salt-activated lipase preparation in the preparation of food for improving fat digestion and absorption.

[0064] In some implementations, the food is infant food, children's food, adult food, maternal food, and / or elderly food.

[0065] In some implementations, the infant food is infant formula milk powder.

[0066] IV. Infant Formula

[0067] This invention provides an infant formula food containing the above-mentioned recombinant human bile salt-activated lipase preparation.

[0068] In some implementations, the infant formula food is infant formula milk powder.

[0069] In some embodiments, the amount of recombinant human bile salt-activated lipase preparation added to the infant formula is: when the formula is prepared into a reconstituted milk liquid that can be directly consumed by infants, the enzyme activity of recombinant human bile salt-activated lipase in each liter of reconstituted milk liquid is 1000U-5000U.

[0070] Example

[0071] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0072] Example 1

[0073] Reference Figure 1 A biosynthesis method for highly active recombinant bile salt-activated lipase, comprising the following steps.

[0074] (1) Construction of genetically engineered bacteria

[0075] Based on the codon bias of *Pichia pastoris*, the coding sequence of the human bile salt-activated lipase (GenBank: AAA63211.1) gene was optimized to eliminate rare codons and improve translation efficiency. The optimized gene was then fused with the coding sequence of a highly efficient secretion signal peptide (*Saccharomyces cerevisiae* α-factor signal peptide) to ensure efficient secretion of the recombinant protein into the extracellular culture medium. This fusion gene fragment was cloned into a *Pichia pastoris*-specific integrative expression vector (pPICZα A) to construct a recombinant expression plasmid. Subsequently, the linearized recombinant plasmid was integrated into the genome of a *Pichia pastoris* host strain (e.g., GS115) via electroporation to obtain the recombinant genetically engineered strain.

[0076] (2) Screening of high-yielding strains

[0077] Preliminary antibiotic resistance screening was performed using antibiotic resistance markers carried on the expression vector, and gene integration was further verified by colony PCR. Positive clones were then subjected to small-scale shake-flask fermentation to express the target protein under methanol induction. The fermentation supernatant was collected, and enzyme activity assays (such as a colorimetric method using p-nitrophenol palmitate as a substrate) were used to screen for single colonies with the highest rBAL (recombinant bile salt-activated lipase) expression and strongest enzyme activity, which were then used as high-yield seed strains for subsequent fermentation production.

[0078] The steps are as follows:

[0079] Fifty single colonies were randomly selected from the positive clones and inoculated into BMGY medium (Buffered Glycerol-complex Medium), and cultured at 30°C and 250 rpm with shaking until the late logarithmic growth stage.

[0080] The bacterial cells were collected by centrifugation, resuspended in BMMY medium (Buffered Methanol-complex Medium), and 0.5% (v / v) methanol was added. The cells were induced to express at 28-40℃ and 250 rpm. Methanol was added every 24 hours for a total of 96 hours.

[0081] After induction, the supernatant was collected by centrifugation. Using p-nitrophenol palmitate (p-NPP) as a substrate, the lipase activity of each supernatant was measured at 37°C in Tris-HCl buffer (pH 7.0) containing 4 mM sodium cholate.

[0082] The strain with the highest enzyme activity was selected.

[0083] (3) Optimization of high-density fermentation and induced expression

[0084] The selected high-yield seed strains were inoculated into fermenters for high-density fermentation. A staged fermentation strategy was adopted.

[0085] Cell growth stage: In the basal salt medium, glycerol is used as both a carbon and energy source, and its concentration is controlled at a non-inhibitory level using a fed-batch method. During this stage, the fermentation temperature is controlled at 28-30℃, the pH at 5.0-6.0, and the dissolved oxygen saturation is maintained at 20%-40% by adjusting the stirring speed and aeration rate to promote rapid cell growth and high-density accumulation.

[0086] Induction phase: When the cell concentration (wet weight) reaches 150-200 g / L, glycerol feeding is stopped. After the residual glycerol is depleted (usually indicated by a sharp rise in dissolved oxygen), methanol is fed in as the sole carbon source and potent inducer. During this phase, the fermentation temperature is significantly reduced to 20-25°C, the pH is maintained at 5.5-6.5, and the methanol concentration is precisely controlled within the range of 0.5%-1.0% (v / v) by controlling the flow rate. This low-temperature induction strategy helps slow down protein synthesis, promotes proper folding of rBAL, reduces protein degradation and misfolded aggregation, thereby significantly increasing the yield of active protein. This induction phase lasts 60-96 hours.

[0087] (4) Downstream separation and purification

[0088] After fermentation, the bacterial cells were removed by centrifugation or tangential flow microfiltration, and the clarified fermentation supernatant was collected. The supernatant was first concentrated and buffer-replaced using an ultrafiltration membrane. Subsequently, a two-step chromatography method was used for fine purification:

[0089] Step 1: Ion exchange chromatography: Using anion exchange medium (such as DEAE Sepharose), capture is performed under near-neutral pH conditions (such as 20 mM Tris-HCl, pH 7.5), followed by elution using a salt gradient to initially separate and enrich rBAL.

[0090] The second step is hydrophobic chromatography: After adjusting the salt concentration of the active elution peak of ion exchange, the sample is loaded onto a hydrophobic chromatography medium (such as Phenyl Sepharose) and eluted using a decreasing salt gradient, which can effectively remove host proteins and pigments to obtain high-purity rBAL.

[0091] (5) Enzyme preparation

[0092] The specific activity of purified rBAL was determined. Then, a food-grade preservative (such as 5% trehalose) was added to the purified rBAL solution, and after thorough mixing, it was freeze-dried to prepare a solid enzyme powder with good flowability and high stability, which is convenient for storage, transportation and subsequent application.

[0093] Example 2

[0094] Based on Example 1 above, a method for preparing recombinant bile salt-activated lipase is disclosed, the steps of which are as follows.

[0095] (1) Construction of genetically engineered bacteria

[0096] Same as Example 1.

[0097] (2) Screening of high-yielding strains

[0098] Similar to Example 1, a high-yielding strain GS115 / BAL-H7 was screened.

[0099] (3) High-density fermentation and induced expression

[0100] The GS115 / BAL-H7 strain was subjected to high-density fermentation in a 5L fermenter. A staged fermentation strategy was adopted.

[0101] Cell growth stage: 50% (v / v) glycerol was added using a fed-batch method, and the fermentation temperature was controlled at 28℃ and the pH at 5.5.

[0102] Induction expression phase: When the wet weight of the bacterial cells reaches 180 g / L, stop the glycerol feeding and wait for it to be consumed. Then start the methanol feeding for induction, controlling the methanol concentration in the range of 0.5%-1.0% (v / v). During the induction phase, the temperature is reduced to 22℃, the pH is maintained at 6.0, and the induction time is 72 hours.

[0103] (4) Separation, purification and formulation

[0104] Same as Example 1. The final rBAL specific activity reached 8500 U / mg. 5% trehalose was added as a protective agent, and the mixture was freeze-dried to produce enzyme powder.

[0105] Example 3

[0106] Based on the above-described Example 1, a fermentation process is disclosed.

[0107] 1. Fermentation process

[0108] The high-yielding strain GS115 / BAL-H7, obtained from screening in Example 2, was used. High-density fermentation was carried out in a 30-liter fermenter. A staged fermentation strategy was adopted.

[0109] Cell growth stage: 50% (v / v) glycerol was added using an exponential fed-batch strategy, the fermentation temperature was controlled at 28±0.5℃, the pH was controlled at 5.5±0.1, and the specific growth rate was strictly controlled so that the cell wet weight reached 190g / L within 18 hours.

[0110] Induction phase: Glycerol feeding was stopped and allowed to deplete before methanol feeding was initiated, maintaining a methanol concentration of 0.5%-1.0% (v / v); pH was maintained at 6.0; for the initial 12 hours, the temperature was set to 25°C to rapidly initiate protein expression; after 12 hours, the temperature was lowered to 20°C to slow protein synthesis, promote proper folding, and reduce degradation. The entire induction phase lasted 84 hours.

[0111] During the induction expression phase, an additional 0.5% casein amino acid hydrolysate was added as a nitrogen source and amino acid supplement to alleviate metabolic stress and increase protein production.

[0112] 2. Large-scale downstream processing

[0113] The fermentation broth was continuously centrifuged using a disc centrifuge with a processing capacity of 100 L / h to obtain a clarified fermentation broth.

[0114] A hollow fiber column ultrafiltration system (10kDa MWCO) was used to rapidly concentrate and replace the fermentation supernatant, reducing its volume to 1 / 20 of the original volume.

[0115] The purification steps were performed using an automated chromatography system (AKTA pilot). Ion exchange chromatography was performed using DEAESepharose XL media, and hydrophobic chromatography was performed using Butyl Sepharose High Performance media. Elution was carried out using an optimized linear gradient, which improved separation efficiency and recovery rate.

[0116] The final product yield was increased by about 15% compared to the 5-liter scale, and the specific activity remained stable at around 8500 U / mg, demonstrating the scalability of the process.

[0117] Example 4

[0118] Based on the above Example 2, the preparation and efficacy verification of an infant formula milk powder containing rBAL are disclosed, and the steps are as follows.

[0119] The rBAL enzyme powder obtained in Example 2 was thoroughly mixed with the spray-dried infant formula base powder in a dry mixing device. The mixing environment was controlled at low temperature (<15°C) and low humidity (relative humidity <30%). The amount of rBAL added was controlled to be 280,000 units of enzyme activity per 100 grams of formula powder. Accordingly, after reconstitution at a 14% ratio, the enzyme activity per liter of formula was approximately 2,800 units.

[0120] In vitro simulated gastrointestinal digestion experiments showed that milk powder supplemented with the rBAL of this invention had a triglyceride hydrolysis rate that was more than 45% higher than that of ordinary milk powder without the rBAL. Animal feeding experiments (young mice) also confirmed that the experimental group fed with milk powder supplemented with rBAL had a significantly lower fecal fat excretion rate, and their weight gain and fat-soluble vitamin levels were better than those of the control group.

[0121] Animal experiments:

[0122] Sprague-Dawley juveniles (14 days old, n=20) were randomly divided into a formula milk group, a formula milk + rBAL group, and a breast milk group, and fed continuously for 7 days.

[0123] result:

[0124] (1) Fat absorption rate: 86.2±2.1% in the formula milk group, 93.5±1.8% in the formula milk + rBAL group (P<0.01 vs formula milk), and 94.1±1.5% in the breastfeeding group;

[0125] (2) Triglyceride content in feces: The formula milk + rBAL group showed a 42% reduction;

[0126] (3) Weight gain: The formula milk + rBAL group increased by 11.3%, which was not different from the breast milk group.

[0127] Safety: A 28-day repeated-dose toxicity study (100 times the clinically intended dose) showed no abnormal clinical signs, hematological and histopathological changes; allergen assessment (Bioinformatics + serum IgE binding assay) showed no homologous sequences with known allergens (E-value < 0.001) and no cross-reactivity.

[0128] Example 5

[0129] Based on the above Examples 1, 2, and 4, the application of recombinant bile salt-activated lipase in infant formula is disclosed. The specific infant formula is infant formula milk powder. The steps are as follows.

[0130] The prepared rBAL enzyme preparation was added to infant formula in precise doses.

[0131] The addition method can be: adding liquid enzyme solution to the liquid milk base before spray drying, or uniformly mixing solid enzyme powder with milk powder base powder during the dry production process of milk powder.

[0132] The amount of rBAL added to the final milk powder product should ensure that: when the milk powder is prepared into reconstituted milk according to the product instructions, the rBAL enzyme activity contained in each liter of reconstituted milk is between 1,000 and 5,000 units; preferably, the amount added is 2,000 to 4,000 units per liter of reconstituted milk.

Claims

1. A method for the biosynthesis of recombinant human bile salt-activated lipase, characterized in that, The method includes the following steps: S1: Constructing a Pichia pastoris recombinant host cell containing a codon-optimized human bile salt-activated lipase encoding gene; S2: Fermentation culture of the recombinant host cells of Pichia pastoris and obtaining fermentation supernatant; the fermentation culture includes a cell growth stage with glycerol as carbon source and a protein induction expression stage with methanol as carbon source and inducer; wherein, the fermentation temperature of the protein induction expression stage is 20℃-25℃; S3: Separate and purify the recombinant human bile salt-activated lipase from the fermentation supernatant.

2. The biosynthesis method according to claim 1, characterized in that, In S1, the codon-optimized human bile salt-activated lipase encoding gene is fused with the Saccharomyces cerevisiae α-factor signal peptide encoding gene.

3. The biosynthesis method according to claim 1 or 2, characterized in that, In S2, during the bacterial growth phase, glycerol is added by fed-batch feeding. When the bacterial concentration reaches 150 g / L-200 g / L on a wet weight basis, the feeding of glycerol is stopped. After the glycerol is depleted, methanol is added by fed-batch feeding, and the concentration of methanol is controlled at 0.5% (v / v)-1.0% (v / v) to carry out the protein induction expression phase.

4. The biosynthesis method according to any one of claims 1-3, characterized in that, In S2, the fermentation temperature during the cell growth stage is 28℃-30℃, and the fermentation pH is 5.0-6.

0.

5. The biosynthesis method according to any one of claims 1-4, characterized in that, In S2, the fermentation pH during the protein induction expression phase is 5.5-6.5, and the fermentation time is 60-96 hours.

6. The biosynthesis method according to any one of claims 1-5, characterized in that, In S2, the protein induction expression stage sequentially includes a protein expression initiation stage at a fermentation temperature of 24℃-25℃ and a protein expression folding stage at a fermentation temperature of 20℃-21℃.

7. The biosynthesis method according to any one of claims 1-6, characterized in that, In S3, the fermentation supernatant is first concentrated by ultrafiltration, and then separated and purified by ion exchange chromatography and hydrophobic interaction chromatography in sequence.

8. A recombinant human bile salt-activated lipase preparation, characterized in that, The recombinant human bile salt activated lipase preparation is prepared by the biosynthetic method according to any one of claims 1-7, and the specific activity of the recombinant human bile salt activated lipase preparation is greater than 8000 U / mg.

9. The application of the recombinant human bile salt-activated lipase preparation according to claim 8 in the preparation of food for improving fat digestion and absorption, characterized in that, The food products mentioned are infant food, children's food, adult food, food for pregnant and postpartum women, and / or food for the elderly.

10. An infant formula food, characterized in that, The infant formula contains the recombinant human bile salt-activated lipase preparation according to claim 8.

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