A method for preparing a recombinant human bile salt-activated lipase

Abstract

This invention belongs to the field of bioengineering, specifically relating to a method for preparing recombinant human bile salt-activated lipase. The method comprises the following steps: S1: constructing a recombinant expression vector containing a codon-optimized human bile salt-activated lipase encoding gene; S2: introducing the recombinant expression vector into *Pichia pastoris* strain KM71 to obtain recombinant host cells; S3: fermenting the recombinant host cells and obtaining a fermentation supernatant; the fermentation includes a stage I using glycerol as a carbon source and a stage II using methanol and sorbitol as carbon sources; S4: isolating and purifying the recombinant human bile salt-activated lipase from the fermentation supernatant. This invention significantly improves the secretion efficiency and product yield of recombinant human bile salt-activated lipase and constructs a linearly scalable fermentation process platform.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering, specifically relating to the efficient expression of human bile salt-activated lipase in Pichia pastoris, and more specifically to a method for preparing recombinant human bile salt-activated lipase. Background Technology

[0002] Bile salt-stimulated lipase (BSSL) is an important lipase in the vertebrate gut (cited in reference 1), playing a crucial role in the complete digestion and intestinal absorption of dietary fats, as well as the absorption of fat-soluble vitamins (cited in reference 2). BSSL catalyzes the hydrolysis of various substrates, including cholesterol esters, phospholipids, lysophospholipids, triglycerides, and hydroxy fatty acid esters (cited in references 2-5). BSSL is mainly found in the acinar tissue cells of the pancreas (cited in reference 6) and is secreted into the intestine through the exocrine glands of the pancreas (cited in reference 7). In the intestinal lumen, it plays a vital role in the hydrolysis of dietary fats such as fat-soluble vitamins and cholesterol esters (cited in reference 8). In addition, during lactation, the mammary glands of some higher mammals (such as humans, gorillas, mice, ferrets, and seals) also produce BSSL (cited in reference 7), promoting efficient metabolism of neonatal milk fat (cited in reference 9). Notably, BSSL is undetectable in the breast milk of mammals such as cattle, goats, pigs, rats, and rhesus monkeys (cited in reference 10).

[0003] Breast milk remains the only natural source of BSSL for human infants, as they cannot obtain this nutrient from cow's milk or infant formula. Triglycerides in breast milk account for 50% of an infant's dietary energy (citation 10). However, newborns, especially premature infants, have underdeveloped pancreatic function and low lipase secretion (citation 11), which hinders their independent digestion and absorption of dietary fat (citation 12). Therefore, obtaining additional lipase from breast milk is crucial for enhancing infants' digestion and absorption of milk fat. Previous studies have shown that limited fat digestion can hinder infant weight gain and adversely affect infant development (citation 13). Partially digested fat may be oxidized and accumulate in the distal intestine (citations 13-14), potentially triggering neonatal intestinal inflammation and accelerating the onset of diseases such as necrotizing enterocolitis (NEC) (citation 15). However, supplementing breastfed infants with secreted BSSL to address their lack of endogenous lipase can significantly improve the efficient utilization of milk fat (citations 16-19).

[0004] Adding hBSSL to formula or breast milk is a key strategy to ensure optimal fat digestion and absorption in infants, thereby promoting their healthy growth. Efficient methods for expressing and purifying hBSSL have become a research focus. Currently, it has been successfully expressed in various genetically modified organisms, including *Escherichia coli* (cited in reference 20), mammalian cell lines (cited in references 1, 21), *Pichia pastoris* (cited in references 22-23), transgenic mice (cited in references 24-26), transgenic sheep (cited in reference 27), and transgenic cattle (cited in reference 28).

[0005] Some researchers have attempted to produce hBSSL using transgenic animals. In transgenic mice, hBSSL expressed targets the mammary gland, with expression levels in milk ranging from 0.5 to 1 g / L. In transgenic sheep, genomic BSSL regulated by the betalactoglobuline (BLG) promoter resulted in hBSSL expression levels exceeding 3 g / L in transgenic sheep milk. Furthermore, in milk from transgenic cattle using the pBAC-hLF-hBSSL vector, hBSSL expression levels reached 9.8 g / L.

[0006] Most current methods involve purifying hBSSL proteins from breast milk or producing them using lactating cells; however, these methods are prohibitively expensive for large-scale market demands. While transgenic animals can provide a near-natural post-translational modification environment for certain complex macromolecular drugs and achieve high-yield humoral expression, their long development cycles, uncontrollable expression, unstable yields, high ethical and regulatory burdens, complex purification processes, and limited scalability significantly limit their application in industrial biopharmaceuticals. Mammalian cell expression systems offer significant advantages in complex glycosylation, correct folding, and bioactivity, but also face significant disadvantages such as high cost, low expression levels, complex processes, genetic instability, and safety risks. Therefore, microbial expression systems are generally more advantageous for applications requiring large-scale, low-cost production or with low post-translational modification requirements; while mammalian cells are better suited for the production of high-value products such as pharmaceutical proteins, antibodies, or complex membrane proteins. Prokaryotic expression systems (such as Escherichia coli) lack the endoplasmic reticulum-Golgi apparatus processing mechanisms found in eukaryotic cells, thus hindering crucial post-translational modifications such as N- and O-glycosylation, proper disulfide bond formation, and complex folding. The absence of these modifications typically leads to misfolding, aggregation, inclusion body formation, decreased stability, and loss of activity or biological function in recombinant glycoproteins. Furthermore, the few artificially introduced glycosylation pathways in prokaryotic systems (such as the N-glycosylation pathway in engineered E. coli) still cannot fully mimic the complex glycan structures of eukaryotic cells, limiting their application in the production of functional glycoproteins. Therefore, for proteins whose structure or activity depends on glycosylation, prokaryotic systems are generally considered unsuitable or extremely inefficient expression platforms.

[0007] Therefore, some scholars have turned their attention to Pichia pastoris strains, which have shown excellent performance in expressing exogenous proteins. Sahasrabudhe et al. first expressed hBSSL and a truncated hBSSL-C (retaining two repeats at the carboxyl terminus) in Pichia pastoris strains (GS115, PPF1) using the expression plasmid pHILD4. Both proteins were secreted into the culture medium at a yield of 45-50 mg / L under shake-flask expression conditions, and the yield of hBSSL reached 300 mg / L under fermenter culture conditions. Simultaneously, comparisons revealed that recombinant hBSSL, even hBSSL-C with only two repeats, possessed biochemical characteristics, activity, and stability very similar to natural hBSSL. In 2001, Murasugi et al. integrated four copies of the hBSSL cDNA sequence with five repeat units into Pichia pastoris GS115 for expression and optimized fermenter conditions, enabling the secretion of rhBSSL to reach 0.8-1.0 g / L.

[0008] The existing research has developed an expression scheme based on the methanol-utilizing yeast Pichia pastoris, which has been proven to achieve efficient secretion of hBSSL. The core technical steps include: (1) Gene construction: The cDNA encoding the full-length hBSSL (containing a natural signal peptide of 23 amino acids) is cloned into the pHILD4 vector, placed under the regulation of the AOX1 methanol-inducible promoter, and integrated into the chromosome of the GS115 host bacteria after linearization by BglII; at the same time, the pARC5797 vector is constructed to express hBSSL-C, which is fused with the S. cerevisiae convertase signal peptide (SUC2) to achieve secretion. (2) Strain screening: The high-expression clones are initially screened by the glycerol tartrate plate hydrolysis circle method, and the gene copy number (about 2-3 copies) is determined by dot hybridization, and it is verified that it is a methanol-utilizing wild type (Mut + Phenotype. (3) Fermentation process: A three-step strategy was adopted - in the batch culture stage, glycerol was used as the carbon source, and the cell density reached 150-175 g / L (wet weight) by limiting the addition of glycerol; in the induction stage, methanol was used as the only carbon source and inducer, and the flow rate was gradually increased from 2 mL / L / h to 9.5 mL / L / h for 110-120 hours; the temperature was controlled at 25℃ and pH 5.9 throughout the process, and yeast extract (0.24%) and peptone (0.48%) were added every 24h to inhibit protease activity. (3) Purification process: After centrifugation and microfiltration clarification, the fermentation broth was purified in one step by heparin-agarose affinity chromatography, and recombinant hBSSL with a purity of >90% was obtained by elution with 0-1M NaCl gradient, and the total recovery rate was about 68.5%. The achievable technical effects include: the secretion of recombinant hBSSL at the shake-flask level is 45-50 mg / L, and the yield is increased to 300 mg / L after optimization by high-density fermentation; the protein correctly cleaves the N-terminal signal peptide; and the enzymatic properties (optimal bile salt concentration, inhibitor sensitivity, etc.) are basically consistent with those of natural hBSSL.

[0009] Although existing Pichia pastoris expression protocols have achieved milligram-level preparation of hBSSL, those skilled in the art may still face the following insurmountable technical defects when implementing industrial production: (1) Insufficient reliability of screening methods leads to low R&D efficiency. Existing technologies use the glycerol tartrate plate hydrolysis circle method for initial screening, but subsequent experiments have confirmed that the screening results of this method are not correlated with the expression level in liquid culture. This defect leads to low screening efficiency of high-expression clones and a high false positive rate, increasing the R&D cycle and cost. (2) Low secretion efficiency leads to intracellular protein retention. In existing technologies, about 30% of recombinant hBSSLs remain in the cell in active form and fail to be effectively secreted into the culture medium. This phenomenon indicates that there is a bottleneck in the signal peptide processing and secretion pathway, resulting in product loss and reduced yield in downstream processes. Existing studies have not explored the retention mechanism and lack specific technical means to improve secretion efficiency, resulting in the inability to further optimize specific productivity (protein yield per unit cell weight). (3) Lack of systematic optimization of key process parameters leads to scale-up risks. In existing technologies, the methanol flow acceleration rate is adjusted based on experience, and the induction strategy is conservative, which poses a risk of excessive methanol accumulation leading to cytotoxicity. Temperature optimization is limited to a single dimension; the carbon source feeding strategy is conservative and lacks synergistic optimization, relying entirely on methanol as the sole carbon source during the induction period, without utilizing the "co-feeding effect" of mixed carbon sources to balance energy metabolism and protein synthesis.

[0010] References:

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[0012] Citation 2: Hui DY, Hayakawa K, Oizumi J. Lipoamidase activity innormal and mutagenized pancreatic cholesterol esterase (bile salt-stimulatedlipase). Biochem J. 1993 Apr 1;291 ( Pt 1)(Pt 1):65-9.

[0013] Citation 3: Kolar MJ, Kamat SS, Parsons WH, Homan EA, Maher T, PeroniOD, Syed I, Fjeld K, Molven A, Kahn BB, Cravatt BF, Saghatelian A. BranchedFatty Acid Esters of Hydroxy Fatty Acids Are Preferred Substrates of theMODY8 Protein Carboxyl Ester Lipase. Biochemistry. 2016 Aug 23;55(33):4636-41.

[0014] Citation 4: Tanaka H, Mierau I, Ito F. Purification andcharacterization of bovine pancreatic bile salt-activated lipase. J Biochem.1999 May;125(5):883-90.

[0015] Citation 5: Xiao X, Jones G, Sevilla WA, Stolz DB, Magee KE, HaughneyM, Mukherjee A, Wang Y, Lowe ME. A carboxyl ester lipase (CEL) mutant causeschronic pancreatitis by forming intracellular aggregates that activateapoptosis. J Biol Chem. 2017 May 12;292(19):7744.

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[0018] Citation 8: Lombardo D, Guy O. Studies on the substrate specificity of a carboxyl ester hydrolase from human pancreatic juice. II. Action on cholesterol esters and lipid-soluble vitamin esters. Biochim Biophys Acta. 1980 Jan 11;611(1):147-55.

[0019] Citation 9: Bläckberg L, Angquist KA, Hernell O. Bile-salt-stimulated lipase in human milk: evidence for its synthesis in the lactating mammary gland. FEBS Lett. 1987 Jun 8;217(1):37-41.

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[0027] Citation 17: Bernbäck S, Bläckberg L, Hernell O. The complete digestion of human milk triacylglycerol in vitro requires gastric lipase, pancreatic colipase-dependent lipase, and bile salt-stimulated lipase. J Clin Invest. 1990 Apr;85(4):1221-6.

[0028] Citation 18: Casper C, Carnielli VP, Hascoet JM, Lapillonne A, Maggio L, Timdahl K, Olsson B, Vågerö M, Hernell O. rhBSSL improves growth and LCPUFA absorption in preterm infants fed formula or pasteurized breast milk. J Pediatr Gastroenterol Nutr. 2014 Jul;59(1):61-9.

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[0030] Citation 20: Terzyan S, Wang CS, Downs D, Hunter B, Zhang XC. Crystal structure of the catalytic domain of human bile salt activated lipase. Protein Sci. 2000 Sep;9(9):1783-90.

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[0032] Citation 22: Bläckberg L, Strömqvist M, Edlund M, Juneblad K, Lundberg L, Hansson L, Hernell O. Recombinant human-milk bile-salt-stimulated lipase. Functional properties are retained in the absence of glycosylation and the unique proline-rich repeats. Eur J Biochem. 1995 Mar 15;228(3):817-21.

[0033] Citation 23: Sahasrabudhe AV, Solapure SM, Khurana R, Suryanarayan V, Ravishankar S, deSousa SM, Das G. Production of recombinant human bile salt stimulated lipase and its variant in Pichia pastoris. Protein Expr Purif. 1998 Dec;14(3):425-33.

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[0035] Citation 25: Strömqvist M, Törnell J, Edlund M, Edlund A, Johansson T, Lindgren K, Lundberg L, Hansson L. Recombinant human bile salt-stimulated lipase: an example of defective O-glycosylation of a protein produced in milk of transgenic mice. Transgenic Res. 1996 Nov;5(6):475-85.

[0036] Citation 26: Wang Y, Sheng Z, Wang Y, Li Q, Gao Y, Wang Y, Dai Y, Liu G, Zhao Y, Li N. Transgenic mouse milk expressing human bile salt-stimulated lipase improves the survival and growth status of premature mice. Mol Biotechnol. 2015 Mar;57(3):287-97.

[0037] Citation 27: Dalrymple, M., Lundberg, L., & Stroemqvist, M. (2001). HUMAN BILE SALT-STIMULATED LIPASE (BSSL) OBTAINABLE FROM TRANSGENIC SHEEP.

[0038] Cited literature 28: Wang Y, Ding F, Wang T, Liu W, Lindquist S, Hernell O, Wang J, Li J, Li L, Zhao Y, Dai Y, Li N. Purification and characterization of recombinant human bile salt-stimulated lipase expressed in milk of transgeniccloned cows. PLoS One. 2017 May 5;12(5):e0176864. Summary of the Invention

[0039] The problem the invention aims to solve

[0040] This invention addresses the problems existing in the prior art by providing a method for preparing recombinant human bile salt-activated lipase and its application in food preparation. The aim is to achieve rapid and accurate identification of high-expression clones, improve the secretion efficiency and product yield of Pichia pastoris recombinant human bile salt-activated lipase, and construct a fermentation process platform that can be linearly scaled up.

[0041] Solution for solving the problem

[0042] [1]. A method for preparing recombinant human bile salt-activated lipase, characterized in that the method comprises the following steps:

[0043] S1: Construct a recombinant expression vector containing a codon-optimized human bile salt-activated lipase encoding gene;

[0044] S2: The recombinant expression vector was introduced into Pichia pastoris strain KM71 to obtain recombinant host cells;

[0045] S3: Fermentation culture of the recombinant host cells and obtaining fermentation supernatant; the fermentation culture includes stage I, fermentation using glycerol as a carbon source, and stage II, fermentation using methanol and sorbitol as carbon sources;

[0046] S4: Separate and purify the recombinant human bile salt-activated lipase from the fermentation supernatant.

[0047] [2]. According to the preparation method described in [1], the characteristic is that, in S1, in the recombinant expression vector, the codon-optimized human bile salt-activated lipase encoding gene is located downstream of the Saccharomyces cerevisiae α-factor signal peptide encoding gene, and the codon-optimized human bile salt-activated lipase encoding gene is fused with the Saccharomyces cerevisiae α-factor signal peptide encoding gene in the same frame.

[0048] [3]. The preparation method according to [1] or [2] is characterized in that, in S2, after introducing the recombinant expression vector into Pichia pastoris strain KM71, the method includes the steps of detecting the copy number of the human bile salt activated lipase encoding gene in the genome of the positive clone and / or detecting the activity of the human bile salt activated lipase expressed by the positive clone.

[0049] [4]. According to the preparation method described in [3], the step of detecting the copy number of the human bile salt activated lipase encoding gene in the positive clone genome is completed by real-time quantitative PCR.

[0050] [5]. The preparation method according to [3] or [4] is characterized in that, in the step of detecting the activity of human bile salt-activated lipase expressed by positive clones, p-nitrophenyl myristate is used as a substrate and sodium taurocholate is used as an activator.

[0051] [6]. The preparation method according to any one of [1] to [5] is characterized in that, in S3, the fermentation temperature of stage I is 28 to 30°C and the fermentation temperature of stage II is 20 to 30°C.

[0052] [7]. The preparation method according to any one of [1] to [6] is characterized in that, in S3, in stage II, a mixture of methanol and sorbitol is added once every 11 to 13 hours to maintain the methanol concentration in the fermentation broth at 0.75% (v / v) to 2% (v / v), and in the mixture of methanol and sorbitol, the carbon molar mass ratio of methanol to sorbitol is 15:1 to 40:1.

[0053] [8]. The preparation method according to any one of [1] to [7] is characterized in that, in S4, the separation and purification are carried out by means of Ni-NTA affinity chromatography and ultrafiltration.

[0054] [9]. A method for preparing a food product, characterized in that the method for preparing the food product includes a step of preparing recombinant human bile salt-activated lipase according to any one of [1] to [8].

[0055]

[10] . According to the preparation method described in [9], the food is characterized in that it is infant food, children's food, adult food, pregnant and postpartum food, and / or elderly food.

[0056] The effects of the invention

[0057] This invention provides a method for preparing recombinant human bile salt-activated lipase, comprising the steps of constructing a recombinant expression vector containing a codon-optimized human bile salt-activated lipase encoding gene and introducing it into *Pichia pastoris* for fermentation. The specific *Pichia pastoris* strain selected in this invention, combined with signal peptide optimization and a mixed carbon source feeding strategy, significantly improves the secretion efficiency and product yield of recombinant human bile salt-activated lipase. Simultaneously, this invention develops a positive clone screening technology based on real-time quantitative fluorescence PCR and a targeted screening method based on high-throughput enzyme activity detection of specific substrates, enabling rapid and accurate identification of high-expression clones. Furthermore, this invention synergistically optimizes parameters in the fermentation process, such as induction temperature, carbon source type and concentration, and constructs a linearly scalable fermentation process platform. Attached Figure Description

[0058] Figure 1 Schematic diagram of the hBSSL protein variant and recombinant expression vector pPIC9K-hBSSL in Example 1.

[0059] Figure 2 hBSSL production capacity of different strains in Example 3.

[0060] Figure 3 The effect of different treatment combinations on hBSSL activity in fermentation broth in Example 4.

[0061] Figure 4 The effect of fermentation conditions on hBSSL expression in strain KM71-27 in Example 5.

[0062] Figure 5 SDS-PAGE analysis results of hBSSL in Example 6. Detailed Implementation

[0063] 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.

[0064] 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.

[0065] In this invention, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0066] In this invention, "optional" or "optionally" means that certain substances, components, execution steps, application conditions, etc., are used or not used.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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*.

[0072] 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.

[0073] 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.

[0074] This invention provides a method for preparing recombinant human bile salt-activated lipase, the method comprising the following steps:

[0075] S1: Construct a recombinant expression vector containing a codon-optimized human bile salt-activated lipase encoding gene;

[0076] S2: The recombinant expression vector was introduced into Pichia pastoris strain KM71 to obtain recombinant host cells;

[0077] S3: Fermentation culture of the recombinant host cells and obtaining fermentation supernatant; the fermentation culture includes stage I, fermentation using glycerol as a carbon source, and stage II, fermentation using methanol and sorbitol as carbon sources;

[0078] S4: Separate and purify the recombinant human bile salt-activated lipase from the fermentation supernatant.

[0079] In some embodiments, in S1, the coding sequence of the human bile salt-activated lipase is artificially designed and optimized based on the codon preference of the target host cell (Pichia pastoris) to obtain an optimized nucleotide sequence, which is then cloned into a vector to construct a recombinant expression vector. This invention does not impose any particular limitation on the codon-optimized human bile salt-activated lipase coding gene, as long as it encodes human bile salt-activated lipase (GenBank: AAA63211.1). In some specific embodiments, the codon-optimized human bile salt-activated lipase coding gene does not contain all the recognition sequences of SacI, EcoRI, NotI, and SalI restriction endonucleases.

[0080] In some embodiments, the recombinant expression vector is a recombinant expression plasmid, preferably pPIC9K.

[0081] In some embodiments, in the recombinant expression vector, the codon-optimized human bile salt-activated lipase encoding gene is located downstream of the *Saccharomyces cerevisiae* α-factor signal peptide encoding gene, and the codon-optimized human bile salt-activated lipase encoding gene is fused in the same frame with the *Saccharomyces cerevisiae* α-factor signal peptide encoding gene. This invention selects the most commonly used secretion signal peptide in *Pichia pastoris*, specifically the α-factor pre-pro signal peptide (pre-pro-α signal sequence) from *Saccharomyces cerevisiae*, rather than an endogenous signal peptide. The pro-region has a chaperone-like auxiliary function, improving the folding efficiency, transport speed, and secretion stability of most heterologous proteins. Many studies have confirmed that removing the pro-region significantly reduces secretion levels. Numerous studies and commercial products (such as enzyme preparations and recombinant proteins) use the α-factor signal peptide as a core secretory element, demonstrating its reliability and high-yield characteristics in large-scale production.

[0082] Furthermore, this invention selects Pichia pastoris strain KM71 as the host cell and introduces the recombinant expression vector into the Pichia pastoris strain KM71 to obtain recombinant host cells. In the prior art, Pichia pastoris strain GS115 is commonly used to express human bile salt-activated lipase, but results show that approximately 30% of the recombinant human bile salt-activated lipase remains in its active form intracellularly and fails to be effectively secreted into the culture medium. It is speculated that the cells may accumulate in the endoplasmic reticulum due to excessive folding pressure, affecting protein secretion. This invention uses Mut... S The KM71 strain exhibits a mild methanol-inducible strength, reducing folding stress and oxidative stress, thereby supporting higher secretion efficiency.

[0083] In some embodiments, S2, after introducing the recombinant expression vector into Pichia pastoris strain KM71, includes the steps of detecting the copy number of the human bile salt-activated lipase encoding gene in the genome of the positive clone and / or detecting the activity of the human bile salt-activated lipase expressed by the positive clone.

[0084] In some embodiments, the step of detecting the copy number of the human bile salt-activated lipase-encoding gene in the genome of a positive clone is performed using real-time quantitative PCR. In some embodiments, the TEF1 gene is used as a single-copy reference gene during the real-time quantitative PCR.

[0085] In some embodiments, p-nitrophenyl myristate is used as a substrate and sodium taurocholate as an activator in the step of detecting the activity of human bile salt-activated lipase expressed by positive clones. p-nitrophenyl myristate (pNPM) has been used as a substrate to determine the activity of bile salt-activated lipase derived from the hepatopancreas of red snapper. However, its suitability for determining the activity of human bile salt-activated lipase or its recombinant forms has not been reported. This study is the first to systematically compare the differences in reaction specificity and detection sensitivity between pNPM and the conventionally used substrate p-nitrophenyl acetate (pNPA). The results show that pNPM can effectively distinguish specific recombinant human bile salt-activated lipase activity from nonspecific background. The high signal generated by pNPA in blank culture medium strongly suggests chemical interference from the culture medium components under these assay conditions. Therefore, pNPM was chosen as the substrate based on its advantage of providing objective data with lower background and a higher signal-to-noise ratio. Furthermore, adding sodium taurocholate (NaTC) as an activator to pNPM can significantly improve the catalytic efficiency of the enzyme and help distinguish between specific recombinant human bile salt-activated lipase activity and non-specific background. In some specific embodiments, in the step of detecting the activity of human bile salt-activated lipase expressed by positive clones, the working concentration of nitrophenyl myristate is 3-7 mM, preferably 4-6 mM; the working concentration of sodium taurocholate is 4-8 mM, preferably 5-7 mM. In some specific embodiments, in the step of detecting the activity of human bile salt-activated lipase expressed by positive clones, the working pH is 8.0 ± 0.2, preferably 8.0; the working temperature is 20-40°C, preferably 25-40°C, more preferably 35-40°C; and the working time is 25-35 min, preferably 30 min. In some preferred embodiments, in the step of detecting the activity of human bile salt-activated lipase expressed by positive clones, the buffer is Tris-HCl at pH 8.0, the reaction is carried out at 37°C for 30 minutes, and then the absorbance of the working solution in the OD405 band is measured at room temperature.

[0086] In some embodiments, in S3, stage I is carried out in BMGY medium containing glycerol; preferably, the fermentation temperature of stage I is 25~35°C, more preferably 28~30°C, more preferably 30°C, the glycerol content in BMGY medium is 3%(v / v)~5%(v / v), more preferably 4%(v / v)~5%(v / v), and the fermentation pH of stage I is 4.0-5.2.

[0087] In some embodiments, in S3, stage II is carried out in BMMY medium containing methanol and sorbitol; preferably, the methanol concentration in the fermentation broth is maintained at 0.75% (v / v) to 2% (v / v), for example, 0.75% (v / v), 0.80% (v / v), 0.85% (v / v), 0.90% (v / v), 0.95% (v / v), 1.0% (v / v), 1.3% (v / v), 1.6% (v / v), 1.9% (v / v), 2% (v / v), etc.; preferably, the methanol concentration in the fermentation broth is maintained at 0.90% (v / v) to 1.5% (v / v); more preferably, the methanol concentration in the fermentation broth is maintained at 0.90% (v / v) to 1.3% (v / v). This invention systematically evaluates the effect of methanol concentration on recombinant protein expression through gradient experiments. The total enzyme activity yield of the fermentation broth reaches its maximum when the methanol concentration is 0.90% (v / v) to 1.3% (v / v), preferably 1.0% (v / v). While maintaining a high cell density, it drives a higher protein expression flux through a stronger induction effect, thereby maximizing the total output.

[0088] Furthermore, in some embodiments, during stage II, a mixture of methanol and sorbitol is added every 11-13 hours to maintain the methanol concentration in the fermentation broth within the aforementioned range. In this methanol-sorbitol mixture, the carbon-to-molar mass ratio of methanol to sorbitol is 15:1 to 40:1, for example, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, etc.; preferably, the carbon-to-molar mass ratio of methanol to sorbitol is 15:1 to 30:1; more preferably, the carbon-to-molar mass ratio of methanol to sorbitol is 15:1 to 25:1. This invention conducted gradient experiments to explore the optimal methanol-to-sorbitol feeding ratio suitable for recombinant host cells expressing recombinant human bile salt-activated lipase. When the ratio of methanol to sorbitol is 15:1 to 25:1, preferably 20:1, the enzyme activity and YP / X of the fermentation broth reach their highest values. This indicates that appropriate mixed feeding with sorbitol and methanol may reduce the metabolic burden of methanol and oxygen demand, thereby obtaining cells with higher activity and improving the expression level of recombinant human bile salt activated lipase in cells.

[0089] In some embodiments, the fermentation temperature of stage II is 20-30°C, for example, 20°C, 22°C, 24°C, 26°C, 28°C, 30°C, etc.; preferably, the fermentation temperature of stage II is 20-25°C; more preferably, the fermentation temperature of stage II is 20-23°C. Experimental results of this invention show that the yield of recombinant human bile salt-activated lipase products from recombinant host cells was significantly improved under induction conditions of 21-23°C, increasing by 1.31 times compared to 25°C and by 1.59 times compared to 30°C.

[0090] In some embodiments, the duration of stage II is 72-120 hours, preferably 84-108 hours, and more preferably 90-102 hours.

[0091] In some implementations, in S4, the fermentation supernatant is first separated from recombinant human bile salt-activated lipase and impurities by Ni-NTA affinity chromatography, and then desalted and replaced with buffer solution by ultrafiltration.

[0092] II. Food Preparation Methods

[0093] This invention provides a method for preparing a food product, the method comprising the step of preparing recombinant human bile salt-activated lipase according to the above preparation method.

[0094] In some implementations, the food is infant food, children's food, adult food, maternal food, and / or elderly food.

[0095] Example

[0096] 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.

[0097] Example 1: Construction of recombinant expression vector

[0098] To express the hBSSL protein, a suitable template was first screened. All relevant full-length sequences in the UniProt database were compared with human milk-derived BSSL sequences. Based on this comparison, the protein sequence AAA63211.1 was determined to be the optimal choice. Although the UniProtKB / Swiss-Prot entry P19835 is recorded as a standard sequence containing 17 VNTR repeats, AAA63211.1 was selected as the expression template because it is directly derived from human milk and represents the 16 most common VNTR alleles. Figure 1 The sequence A corresponds to the nucleotide sequence M54994.1 (2229 bp).

[0099] Furthermore, the above nucleotide sequence was codon-optimized to adapt to the *Pichia pastoris* (K. phaffii) expression system. Simultaneously, to avoid potential enzymatic cleavage risks at internal sites, all recognition sequences of SacI, EcoRI, NotI, and SalI restriction endonucleases were excluded from the codon-optimized sequence. The optimized sequence was synthesized by BGI (Shenzhen, China). Additionally, to facilitate protein purification, a six-histidine tag was added to the C-terminus of the hBSSL sequence. The resulting fragment was inserted into the pPIC9K vector (Invitrogen) via the EcoRI / NotI restriction site, and fused co-frame downstream of the α-factor signal peptide in pPIC9K, ultimately obtaining the recombinant plasmid pPIC9K-hBSSL. Figure 1 (B) pPIC9K-hBSSL was transformed into Escherichia coli DH5α strain and cultured in lysogenic broth at 37°C to prepare large quantities of the recombinant expression vector pPIC9K-hBSSL.

[0100] Figure 1 Figure A shows a schematic diagram of the structures of two major hBSSL variants, with the functional domains labeled as follows: signal peptide (yellow), conserved N-terminal domain containing bile salt binding sites, N-glycosylation sites, and a catalytic triplet (white), and C-terminal domain consisting of variable-number tandem repeats (VNTRs, dark blue). The dashed box marks the deletion of 11 amino acids between the 3rd and 4th repeat units in the highly variable VNTR region of the AAA63211.1 sequence (compared to the P19835 sequence). This deletion leads to repeat unit remodeling, forming the classic 16-VNTR allele most commonly found in human milk. Figure 1 Figure B shows the genetic map of the pPIC9K-hBSSL recombinant expression plasmid. The codon-optimized synthetic hBSSL sequence was inserted downstream of the *Saccharomyces cerevisiae* α-factor secretion signal via in-frame fusion, and its expression is regulated by the potent methanol-inducible AOX1 promoter. The AmpR and KanR genes confer resistance to ampicillin and kanamycin, respectively, in *E. coli*, enabling bacterial selection. The KanR gene also exhibits G418 resistance and can be used for *Pichia pastoris* selection. The plasmid was linearized using Sal I restriction sites and then integrated into the *Pichia pastoris* genome using the HIS4 marker.

[0101] Example 2: Construction of recombinant host cells

[0102] Komagataella phaffii (Pichia pastoris) KM71 (his4, Mut s The strain was engineered to serve as a BSSL production platform and cultured in yeast extract peptone glucose (YPD) medium under aerobic conditions at 30°C.

[0103] The Sal I linearized plasmid DNA (total DNA 10 μg) from Example 1 was mixed with yeast competent cells and incubated in a pre-cooled 0.2 mm Gene Pulser electroporator for 5 minutes on ice. Electroporation was then performed at 1500 V, 25 μF, and 200 Ω. Immediately after electroporation, 200 mL of filtered sterile sorbitol (1M) was added to a rotating cuvette, mixed thoroughly, and transferred to a sterile centrifuge tube containing 700 mL of sorbitol. The cells were cultured in a constant temperature water bath with shaking at 300 rpm for two hours. After incubation, cells were collected by centrifugation at 4°C and 5000 rpm for 5 minutes. After centrifugation, all cells were resuspended in retention medium (200 mL), plated on MD plates (histidine-deficient selection), and incubated upside down at 30°C for 2–4 days.

[0104] Clones obtained from multiple MD plates were washed with sterile water, merged, and thoroughly mixed. Yeast cell density was determined by measuring OD600. The yeast cell mixture (≤10) was then... 5 CFU / mL was spread onto YPD plates containing 1 mg / mL, 2 mg / mL, 3 mg / mL and 4 mg / mL G418 (genetic mycotoxin) and incubated at 30°C for 2-4 days.

[0105] Previous studies have shown that the copy number of heterologous genes in the Pichia pastoris genome significantly affects its specific productivity and biomass yield per unit (YP / X, defined as ΔP / ΔX, where P represents hBSSL enzyme activity and X represents cell dry weight). Based on the known association between gene copy number and G418 resistance, small-scale expression screening focused on clones selected at 3 mg / mL and 4 mg / mL G418 concentrations to enrich high copy number integrities. Thirty-six clones were isolated from four different concentrations (1–4 mg / mL) of G418 culture plates and numbered 1–36 in ascending order of antibiotic concentration. Strains 1–10 were selected from 1 mg / mL G418 culture plates, strains 11–20 from 2 mg / mL G418 culture plates, strains 21–30 from 3 mg / mL G418 culture plates, and strains 31–36 from 4 mg / mL G418 culture plates.

[0106] The following primers were used for colony PCR screening of positive transformants:

[0107] Forward primer: 5'-TCCTGCTCTAGCCAGTTTGC-3' (SEQ ID NO.12);

[0108] Reverse primer: 5'-GATGTTGCTGTTTTGCCATTTTCC-3' (SEQ ID NO.13).

[0109] PCR analysis confirmed that 29 out of 36 clones were positive, with a positive rate of 80.56%. All positive colonies were screened, numbered, and then amplified in YPD medium.

[0110] To identify high-expression clones, primary tube fermentation assays were performed on these 29 strains. Based on the assay results, 12 strains (21, 22, 23, 26, 27, 28, 29, 31, 32, 33, 34, and 35) were selected for subsequent experiments, with the original KM71 strain serving as a negative control.

[0111] Example 3: Small-scale expression test

[0112] In Example 2, the 12 selected clonal strains were first cultured in YPD medium for 24 h, then transferred to BMGY medium (containing 5% glycerol, w / v) for 36 h to accumulate biomass. The culture temperature was 30°C for all strains. After harvesting, the cultures were resuspended in BMMY medium at a 4:1 (BMGY:BMMY medium volume ratio) for methanol induction (1% v / v, 22°C). Methanol was added every 24 hours, and samples were taken for enzyme activity analysis to determine the optimal fermentation time. The control group included a blank yeast strain and an empty vector strain (denoted as KB).

[0113] The protein expression capacity of the strains was assessed using the product-to-biomass ratio (YP / X, defined as ΔP / ΔX, where P represents hBSSL enzyme activity and X represents cell dry weight). Significant differences in hBSSL production were observed among the positive recombinant strains. Figure 2 ). Figure 2 The graph shows the enzyme activity (red left-hand diagonal line), cell dry weight per milliliter of culture (blue right-hand diagonal line), and the product / biomass yield coefficient YP / X (dotted line graph) of the positive strains after 96 hours of induction in 20 mL of BMMY medium under standard conditions. Statistical analysis was performed using one-way ANOVA (*p < 0.05). The results showed that even among strains (21-29) screened at the same antibiotic concentration (3 mg / mL), there were still significant differences in YP / X values. Furthermore, a combined analysis of the screening results at 3 mg / mL (21-29) and 4 mg / mL (31-35) revealed that increasing the antibiotic screening concentration did not always correlate with increased yield. After 96 hours of methanol induction, the recombinant strain KM71-27 achieved a supernatant enzyme activity of 276.9 U / L and a YP / X value of 1.75, demonstrating significantly superior human BSSL production capacity.

[0114] Real-time quantitative PCR (qPCR) was used to detect the clones validated in the above experiments, and to analyze whether the differences in expression levels among different strains were due to variations in gene copy number, with the original KM71 strain as a control. Based on the reported high amplification efficiency (Eref) and geNorm stability of the TEF1 gene (translation elongation factor) in *K. phaffii* (Büchner K, Vidal L, Kerpes R, Becker T. Establishing Reference Genes for *Pichia pastoris* Quantitative Real-Time Polymerase Chain Reaction Analyses. Methods Mol Biol. 2026;2697:119-135.), this gene was selected as a single-copy reference gene, which participates in the elongation of nascent polypeptides at the ribosomal A site. First, plasmids containing the hBSSL or TEF1 gene (pMD-19T-hBSSL and pMD-19T-TEF1) were constructed. Their concentrations were determined using a micronucleic acid analyzer, and the copy number concentration (N, copies / μL) was calculated according to Formula 1 to generate a plasmid standard curve. Subsequently, using yeast genomic DNA as a template, the Ct values ​​of the hBSSL and TEF1 genes in each sample were determined by qPCR. Finally, the measured Ct values ​​were substituted into the corresponding plasmid standard curve equation to calculate the copy number concentrations of hBSSL and TEF1 in the genomic DNA. The hBSSL genome copy number was determined using the NBSSL / NTEF1 ratio.

[0115] Formula 1: N (copy number / μL) = (6.02 × 10⁻⁶) 23 )×(C×10 -9 ) / (L×660;

[0116] Where C = plasmid concentration, ng / μL; L = total plasmid length, bp.

[0117] The two plasmid standards (pMD-19T-hBSSL and pMD-19T-TEF1) showed consistent amplification efficiencies. Therefore, a standard curve was plotted based on the amplification curve of the pMD-19T-hBSSL plasmid standard, with the equation y = -3.0039x + 31.861 (R²). 2=0.9957). Using this standard curve, the copy number concentrations of hBSSL and TEF1 genes, NhBSSL and NTEF1, were calculated based on sample concentration and amplification cycle number. Table 1 lists the hBSSL copy number in the genome of the tested strains. The results showed that there were significant differences in the hBSSL gene copy number among positive recombinant clones screened at the same antibiotic concentration. The KM71-27 strain, which exhibited the highest product / biomass yield (YP / X) in the fermentation experiment, had a relative hBSSL gene content of 1.04 (±0.65) × 10⁻⁶. 4 The hBSSL copy number in this strain was 192-fold higher than that of the negative control strain (KM71 strain carrying an empty plasmid, denoted as KB). After reference gene normalization, the hBSSL copy number in this strain was estimated to be 1.96. The relative hBSSL gene contents of KM71-26 and KM71-29 strains obtained under the same selection pressure were 6.58 (±0.27) × 10⁻⁶. 2 copies / μL and 1.10 (±0.48) × 10 3 The hBSSL gene copy number increased by 12-fold and 20-fold respectively compared to the negative control. Observations showed that the variation trend of hBSSL gene copy number among different strains was consistent with the trend of YP / X values ​​obtained from their fermentation experiments.

[0118] Table 1. hBSSL gene copy number detected by real-time quantitative PCR

[0119]

[0120] Example 4: Optimized detection of hBSSL lipase activity

[0121] To establish a rapid detection method for human hBSSL lipase activity, this study systematically compared two substrates—5 mM p-nitrophenyl myristate (pNPM) and 5 mM p-nitrophenyl acetate (pNPA)—and evaluated the effect of adding 6 mM sodium taurocholate (NaTC).

[0122] Depending on whether sodium taurocholate was added, p-nitrophenyl myristate (long-chain saturated fatty acid) and p-nitrophenyl acetate (short-chain) were selected as substrates, and five control samples were set up: fermentation supernatant, high-pressure homogenized cell lysis supernatant, ultrasonically lysed cell lysis supernatant, BMMY medium, and BMGY medium. Each sample was added with 200 μL of pH buffer (Tris-HCl, pH 8.0) and 20 μL of substrate, and incubated at 37°C for 30 minutes. The absorbance of the supernatant at OD405 was measured at room temperature. After determining the optimal substrate, two variables were set: buffer pH and reaction temperature (Tris-HCl pH 7.5, Tris-HCl pH 8.0; 25°C, 37°C). Four control experiments were conducted using all possible combinations, and lipase activity was measured on day 4 of fermentation induction of recombinant yeast KM71-27 to screen for the optimal reaction conditions.

[0123] The results are as follows Figure 3 As shown. Figure 3 Figure A shows the evaluation results of different substrate-activator NaTC combinations. Four treatment groups were designed: Group A, p-nitrophenyl acetate + sodium taurocholate; Group B, p-nitrophenyl myristate + sodium taurocholate; Group C, p-nitrophenyl acetate + pH buffer (no activator control); Group D, p-nitrophenyl myristate + pH buffer (no activator control). The figure shows the enzyme activity of five types of samples (GQ: BMGY medium; MQ: BMMY medium; CP: sonicated cell lysis supernatant; JP: homogenized cell lysis supernatant; Q: supernatant from day 4 of fermentation) under each treatment condition. Under treatment group B (p-nitrophenyl myristate + sodium taurocholate), the activity of sample Q was significantly higher than that of all other samples (***p < 0.001), indicating that this substrate-activator combination has excellent catalytic specificity. Statistical analysis was performed using one-way ANOVA. Figure 3 Figure B in the diagram presents the results of optimized reaction conditions for human BSSL. The activity response of purified samples (JP) and hBSSL fermentation supernatant (Q) was evaluated using combinations of pH (7.5 vs. 8.0) and temperature (25℃ vs. 37℃). For the target hBSSL fermentation supernatant (Q), the enzyme activity at pH 8.0 was significantly higher than that at pH 7.5 at both test temperatures (p < 0.0001, paired-samples t-test), thus determining the optimal pH conditions.

[0124] The results showed that, regarding substrate specificity, when pNPM was used in combination with 6 mM NaTC ( Figure 3In groups A and B, the enzyme activities of cell lysis supernatants (CP, JP) were not significantly different from those of the blank medium control (MQ), and were significantly lower than those of the fermentation supernatant (Q) (p < 0.001). Conversely, when pNPA was used as a substrate, high absorbance signals were detected in both blank medium samples (GQ, MQ). Regarding the effect of NaTC, quantitative analysis showed that in the presence of 6 mM NaTC (group B, containing pNPM), the lipase activity in the fermentation supernatant was 1.75 times higher than that in the NaTC-free control group (group D). In summary, these results indicate that pNPM can serve as a specific substrate for hBSSL activity in the presence of NaTC, and that NaTC can significantly improve the catalytic efficiency of this enzyme.

[0125] The enzyme activity results also showed a correlation with reaction temperature and buffer pH. For example... Figure 3 As shown in Figure B, when the pH of the reaction system is 8.0, the hBSSL enzyme activity is significantly increased by 2.62 times compared to the pH 7.5 condition. However, the enzyme activity measured at 37℃ is only slightly increased compared to the condition at 25℃.

[0126] Therefore, preferably, the enzyme activity assay for human bile salt-stimulated lipase uses 5 mM p-nitrophenyl myristate as the substrate, with the addition of 6 mM sodium taurocholate. The buffer solution is Tris-HCl at pH 8.0, and the reaction is carried out at 37°C for 30 minutes.

[0127] Example 5: Optimization of fermentation conditions

[0128] Single colonies of the recombinant KM71-27 strain were pre-cultured in 20 mL of YPD medium. The culture was incubated overnight at 30°C and 250 rpm. Subsequently, 1% (v / v) inoculum was transferred to a 1 L flask, and 250 mL of BMGY medium containing 5% glycerol (pH 4.0) was added. The flask was sealed with gauze and incubated aerobically at 30°C for 36 h. After incubation, cells were collected by centrifugation, the medium was discarded, and cells were resuspended in BMMY induction medium containing 1% (v / v) methanol to initiate induction. Induction was maintained at 22°C and 250 rpm for 96 h, with a methanol:sorbitol (20:1, C-mol) mixture added every 12 hours to maintain a 1% (v / v) methanol concentration. The supernatant was finally collected by centrifugation.

[0129] To maximize the yield and quality of recombinant proteins, fermentation optimization is a key strategy. This strategy focuses on regulating cellular physiological states and optimizing metabolic pathways to overcome the limiting factors that restrict yield.

[0130] (1) Effect of induced temperature

[0131] BMGY cultures were resuspended in BMMY medium. Expression was then induced by incubation at different induction temperatures (20℃, 22℃, 25℃, and 30℃) for 96 hours, after which hBSSL yield was measured.

[0132] (2) Effect of inducer concentration

[0133] Shake flask cultures were incubated for 96 hours at the optimized temperature and in different concentrations of inducer (methanol) (0.25%, 0.5%, 0.75%, 1% or 2%), and hBSSL products were measured.

[0134] (3) Carbon source co-feeding effect

[0135] Shake flask cultures were performed for 96 hours in mediums with different methanol-sorbitol ratios (carbon molar ratios: 1:0, 1:1, 2:1, 3:1, 5:1, 10:1, 20:1, 40:1). Biomass and hBSSL activity were measured every 24 hours. Biomass was expressed as stem cell weight (gX), and the specific method can be found in other literature (Resina D, Serrano A, Valero F, Ferrer P. Expression of a Rhizopus oryzae lipase in Pichia pastoris under control of the nitrogen source-regulated formaldehyde dehydrogenase promoter. J Biotechnol. 2004 Apr 8;109(1-2):103-13.).

[0136] The core achievements of fermentation optimization include Figure 4 As shown, Figure 4 A in the diagram shows the results of determining the optimal induction time. Figure 4 B in the figure shows the effect of induction temperature on hBSSL expression. Figure 4 C in the figure shows the effect of methanol concentration on hBSSL expression. Figure 4 Figure D illustrates the effect of sorbitol concentration on hBSSL expression. Data are presented as mean ± standard deviation (n=3). Statistical significance was determined using one-way ANOVA combined with Tukey's post-hoc test (*p<0.005, **p<0.01, ***p<0.001). Overall results show that optimization significantly improved enzyme activity and the product-to-biomass ratio.

[0137] In the early stages of methanol induction, the enzyme activity of rhBSSL gradually increased with increasing induction time. Figure 4(A) The enzyme activity reached its peak at 96 hours, with the enzyme activity in the culture supernatant reaching 602.8 ± 41.13 U / L. Thereafter, extending the induction period actually led to a decrease in enzyme activity.

[0138] When the induction temperature was lowered to 23℃, the fermentation performance of strain KM71-27 was significantly improved. Figure 4 (B in the text). It also successfully solved the problem of excessive foaming during fermentation at 30℃. After centrifugation, the cells showed a whiter color and no longer produced green precipitate.

[0139] K. phaffii KM71 strain exhibits slow methanol utilization (Mut S To achieve optimal expression of the recombinant protein phenotype, the addition of methanol needs to be controlled. Figure 4 Figure C illustrates the effect of methanol concentration on extracellular enzyme activity. Methanol concentration significantly regulated hBSSL expression yield in the recombinant KM71-27 strain. The highest specific enzyme activity (YP / X) measured at a methanol concentration of 0.75% was 9.3 ± 1.1 U / g stem cell weight, reflecting peak cellular biosynthetic efficiency. Figure 4 (C in the text). In contrast, total enzyme activity peaked at 1.0% methanol (689.8 ± 27.8 U / L), a 1.39-fold increase compared to the 0.75% condition. This indicates that a 0.75% methanol concentration maximizes specific productivity, while a 1.0% concentration achieves a higher overall titer by balancing cell density with strongly inducible enhanced expression.

[0140] Figure 4 Figure D illustrates the effect of different sorbitol to methanol ratios on hBSSL yield. When methanol was used alone as the carbon source (ratio 1:0), the enzyme activity was 506.8 ± 25.1 U / L. As the sorbitol ratio decreased (methanol:sorbitol ratio from 1:1 to 10:1), the enzyme activity showed a non-significant decrease. As the sorbitol ratio gradually decreased, the activity gradually recovered, reaching a peak of approximately 699.8 ± 35 U / L at a ratio of 20:1, significantly higher than when methanol was used alone. When the ratio was further increased to 40:1, the enzyme activity decreased. The YP / X ratio showed a similar trend: it decreased significantly at high sorbitol ratios, attributed to the reduction in enzyme titer. The YP / X ratio peaked at a ratio of 20:1, and then decreased slightly at a ratio of 40:1.

[0141] Example 6: Purification and Validation of Recombinant Protein

[0142] Recombinant proteins from the fermentation supernatant of strain KM71-27 were analyzed using His GraviTrap. TMAffinity purification was performed using a Sardorf column, strictly following the manufacturer's instructions. Non-specifically bound impurities were removed by elution with elution buffer (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 20 mM imidazole). hBSSL was eluted with elution buffer containing 300 mM imidazole (50 mM Tris-HCl, pH 8.0), followed by desalting and buffer replacement using an Amicon 30 kDa centrifuge filter (EMD Millipore), and then transferred to storage buffer containing 50 mM Tris (pH 7.5), 200 mM NaCl, 1 mM DTT, and 10% glycerol. All purified proteins were aliquoted and stored at -80°C to inhibit protein degradation.

[0143] Furthermore, protein purity >90% was confirmed by SDS-PAGE (4-12%) and Coomassie Brilliant Blue staining. Protein concentration was quantified using a BSA protein assay kit (Solarbio) according to the manufacturer's instructions. Protein identification was performed by in-gel digestion of the excised protein bands with trypsin. Extracted peptides were analyzed using a Thermo Obirtap Fusion mass spectrometer (coupled with an EASY-nLC 1200 system, Thermo Fisher Scientific). The obtained MS / MS spectra were compared to the Swiss-Prot database using PEAKSStudio X software.

[0144] Recombinant human BSSL protein was obtained through a two-step purification procedure: firstly, Ni... 2+ -NTA affinity chromatography, followed by buffer replacement via ultrafiltration. For example... Figure 5As shown, SDS-PAGE analysis revealed a single main band at approximately 115 kDa, with a purity exceeding 95%, consistent with the previously reported molecular weight of human BSSL (Sahasrabudhe, AV, Solapure, SM, Khurana, R., Suryanarayan, V., Ravishankar, S., Desousa, SM, & Das, G.(1998). Production of recombinant human bile salt stimulated lipase and its variant in Pichia pastoris. Protein Expression and Purification, 14(3), 425-433.). To confirm its identity, the corresponding band in the SDS-PAGE gel was sequenced by LC-MS / MS. The obtained peptide sequence completely matched the amino acid sequence of the target hBSSL protein, thus verifying the identity of the purified protein (Table 2). The concentration of the purified protein was determined by the BCA method to be 111.3 ± 30.1 mg / L, equivalent to a concentration of 61.2 ± 17 mg / L in the original fermentation supernatant.

[0145] Table 2 Protein mass spectrometry results

[0146]

Claims

1. A method for preparing recombinant human bile salt-activated lipase, characterized in that, The method includes the following steps: S1: Construct a recombinant expression vector containing a codon-optimized human bile salt-activated lipase encoding gene; S2: The recombinant expression vector was introduced into Pichia pastoris strain KM71 to obtain recombinant host cells; S3: Fermentation culture of the recombinant host cells and obtaining fermentation supernatant; the fermentation culture includes stage I, fermentation using glycerol as a carbon source, and stage II, fermentation using methanol and sorbitol as carbon sources; S4: Separate and purify the recombinant human bile salt-activated lipase from the fermentation supernatant.

2. The preparation method according to claim 1, characterized in that, In S1, in the recombinant expression vector, the codon-optimized human bile salt-activated lipase encoding gene is located downstream of the Saccharomyces cerevisiae α-factor signal peptide encoding gene, and the codon-optimized human bile salt-activated lipase encoding gene is fused in the same frame with the Saccharomyces cerevisiae α-factor signal peptide encoding gene.

3. The preparation method according to claim 1 or 2, characterized in that, In S2, after introducing the recombinant expression vector into Pichia pastoris strain KM71, the steps include detecting the copy number of the human bile salt-activated lipase encoding gene in the genome of the positive clone and / or detecting the activity of the human bile salt-activated lipase expressed by the positive clone.

4. The preparation method according to claim 3, characterized in that, The step of detecting the copy number of the human bile salt-activated lipase-encoding gene in the genome of positive clones was performed using real-time quantitative PCR.

5. The preparation method according to claim 3 or 4, characterized in that, In the step of detecting the activity of human bile salt-activated lipase expressed by positive clones, p-nitrophenyl myristate was used as a substrate and sodium taurocholate was used as an activator.

6. The preparation method according to any one of claims 1 to 5, characterized in that, In S3, the fermentation temperature of stage I is 28~30℃, and the fermentation temperature of stage II is 20~30℃.

7. The preparation method according to any one of claims 1 to 6, characterized in that, In S3, during stage II, a mixture of methanol and sorbitol is added every 11 to 13 hours to maintain the methanol concentration in the fermentation broth at 0.75% (v / v) to 2% (v / v). In the mixture of methanol and sorbitol, the carbon molar mass ratio of methanol to sorbitol is 15:1 to 40:

1.

8. The preparation method according to any one of claims 1 to 7, characterized in that, In S4, the separation and purification are performed using Ni-NTA affinity chromatography and ultrafiltration.

9. A method for preparing a food product, characterized in that, The method for preparing the food includes the step of preparing recombinant human bile salt-activated lipase according to any one of claims 1 to 8.

10. The preparation method according to claim 9, 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.

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