Production Of Recombinant Enzymes Research Articles

Beyond expectations: the development and biological activity of cytokinin oxidase/dehydrogenase inhibitors.

Cytokinins are one of the main groups of plant hormones that regulate growth and development of plants. Cytokinin oxidase/dehydrogenase (CKX) is an enzyme that rapidly and irreversibly degrades cytokinins and thus directly affects their concentration and physiological effect. Genetically modified plants with reduced CKX activity in the shoot, i.e. with a higher concentration of cytokinins, showed e.g. increased tolerance to drought stress, formed larger inflorescences and had higher grain yield. For these reasons, chemical compounds capable of inhibiting the CKX activity (CKX inhibitors) were sought. First, they were identified among strong synthetic cytokinins, but their inhibitory activity was low. The trend has been to develop potent CKX inhibitors with minimal intrinsic cytokinin activity in the hope of avoiding the negative effect of cytokinins on root growth. Cloning CKX, production of key recombinant enzymes from Arabidopsis (AtCKX2) and maize (ZmCKX1 and ZmCKX4a), development of screening bioassays and progress in X-ray crystallography and synthetic organic chemistry led to extensive progress in the development of these compounds. Currently, the most suitable CKX inhibitors are seeking their application in research and the commercial sphere in two main areas - plant tissue cultures and agriculture. The key milestones that preceded it are summarized in this review.

Obtaining and Characterizing Thermostable α-Amylases Secreted by Bacillus subtilis, Originating from Bacillus amyloliquefaciens and Bacillus subtilis

The production of recombinant enzymes, primarily used for obtaining pure and functional target molecules, holds significant importance in modern biotechnology. This study aimed to obtain and characterize recombinant, extracellularly expressed α-amylases (Amy3500 and Amy1974), derived from B. amyloliquefaciens MDC1974 and B. subtilis MDC3500, respectively, using the pBE-S shuttle vector. Both α-amylase genes were molecularly cloned into the E. coli/B. subtilis pBE-S shuttle vector, both with (Amy1974sig and Amy3500sig) and without their signal peptides (Amy1974 and Amy3500), along with a signal peptide originating from the plasmid, and tested in flask fermentations. For recombinant Amy3500, the amylase variants resulted in similar levels of volumetric activity (700–750 U/mL). In contrast, the expression of Amy1974 nearly doubled compared to Amy1974sig with double signal peptides, reaching 2000 U/mL. SDS-PAGE estimated the molecular weight of Amy1974 α-amylase to be 54.6 kDa, which is consistent with the theoretical molecular mass calculations. However, the estimated molecular weight of Amy3500 α-amylase was significantly lower upon exiting the producer cells. Ca2⁺ ions exhibit a modest activating effect on the activities of Amy1974 and Amy3500 amylases, likely due to their tight binding to the protein scaffold. Both enzymes exhibited broad activity peaks between 45 and 70 °C, with a maximum at 65 °C. The Amy1974 and Amy3500 α-amylases demonstrated broad pH optima and pH-dependent thermostability, with optimum pH values at 6.5 and 5.8, and thermal stability peaks at pH 7.6 and 5.9, respectively. Both α-amylases displayed high relative activity against various starches, including corn amylopectin and potato amylose, while showing comparatively lower activity towards α-, β-, and γ-cyclodextrins. The Amy1974 amylase is effective in converting starch into dextrins of varying lengths, while Amy3500 primarily converts starch into glucose. These characteristics make them promising candidate enzymes for industrial applications.

CRISPR-Cas9 knockout screen informs efficient reduction of the Komagataella phaffii secretome

BackgroundThe yeast Komagataella phaffii is widely used for manufacturing recombinant proteins, but secreted titers of recombinant proteins could be improved by genetic engineering. In this study, we hypothesized that cellular resources could be redirected from production of endogenous proteins to production of recombinant proteins by deleting unneeded endogenous proteins. In non-model microorganisms such as K. phaffii, however, genetic engineering is limited by lack gene annotation and knowledge of gene essentiality.ResultsWe identified a set of endogenous secreted proteins in K. phaffii by mass spectrometry and signal peptide prediction. Our efforts to disrupt these genes were hindered by limited annotation of essential genes. To predict essential genes, therefore, we designed, transformed, and sequenced a pooled library of guide RNAs for CRISPR-Cas9-mediated knockout of all endogenous secreted proteins. We then used predicted gene essentiality to guide iterative disruptions of up to 11 non-essential genes. Engineered strains exhibited a ~20× increase in the production of human serum albumin and a twofold increase in the production of a monoclonal antibody.ConclusionsWe demonstrated that disruption of as few as six genes can increase production of recombinant proteins. Further reduction of the endogenous proteome of K. phaffii may further improve strain performance. The pooled library of secretome-targeted guides for CRISPR-Cas9 and knowledge of gene essentiality reported here will facilitate future efforts to engineer K. phaffii for production of other recombinant proteins and enzymes.

Production of N-glycosylated alcohol dehydrogenase in Escherichia coli

N-glycosylation of recombinant proteins using bacterial glycosylation system has proven to be a valuable although developing tool ultimately applicable to various industries. When used for enzyme engineering, it offers the possibility of increased stability or immobilization route and thus increasing effectiveness of e.g. biotransformation or other biocatalysis procedures. One such promising enzyme is alcohol dehydrogenase (ADH) for use in redox biotransformation reactions. Given the current possibilities of recombinant enzyme production, including major advances in glycoengineering and glycoprotein production in bacterial organisms, the aim of this work was the production of thermotolerant ADH from Rhodococcus ruber (RrADH) in glycosylated form in Escherichia coli. We have successfully developed a dual plasmid expression system enabling glycosylation of target proteins utilizing a glyco-tag approach. We were able to produce RrADH in soluble form and at the same time we detected a bacterial glycan conjugated to RrADH as well as the activity of the enzyme. The glycan bound to recombinant enzyme can be used for oriented covalent immobilization of the enzyme, which would increase the potential for its practical application in biotransformation of various compounds.

Biodegradation of Pharmaceutical Pollutants Using Fungal Enzyme

Abstract: The potential of filamentous fungi and their enzymes are investigated for bioremediating pharmaceutical pollutants from wastewater thereby addressing the growing concern of emerging pollutants in water resources. Among the various microorganisms, the current abstract discusses the role of fungi in degrading antibiotics and persistent pharmaceutical compounds. These fungi allow the biodegradation via the formation of highly oxidative hydroxyl radicals. It summarizes the role of both intra (cytochrome P450) and extracellular (laccase) enzyme systems for the bioremediation of emerging clinical pollutants. It emphasizes the importance of both biosorption and biodegradation for effective pollutant removal by breaking down pharmaceuticals (e.g. antibiotics) and other hazardous compounds (e.g. endocrine-disrupting compounds, polycyclic aromatic hydrocarbons). Although the enzymes CYP450 are not directly engaged, their metabolic pathways execute mechanisms such as oxidation, the formation of reactive intermediates, and chemical modification, all of which result in the degradation of clinical pollutants. Amongst all enzyme systems in the case of laccase which oxidise a wide spectrum of substrates, converts them to free radicals. Additionally, the role of recombinant enzyme production and transgenic strains for pollutant degradation has also been elaborated. These considerations emphasise the need for ongoing research and biotechnological advancements to address the growing concern of pharmaceuticals and personal care product pollutants in water systems.

Enzymes as indispensable markers in disease diagnosis.

Enzymes have been usedfor disease diagnosis for many decades; however, advancements in technology like ELISA and flow cytometry-based detection havesignificantlyincreased their use and have increased the sensitivity of detection. Technological advancements in recombinant enzyme production haveincreased enzymatic stability,and the use ofcolorimetric-based and florescence-based assays has led to their increased use as biomarkers for disease detection. Enzymes like acid phosphatase, cathepsin, lactate dehydrogenase, thymidine kinaseand creatine kinase are indispensable markers for diagnosing cancer, cardiovasculardiseasesand others. This minireview summarizes various enzymes used in disease diagnosis, their metabolic role, market valueand potential as disease markers across various metabolic and other disorders.

State of the Art Technologies for High Yield Heterologous Expression and Production of Oxidoreductase Enzymes: Glucose Oxidase, Cellobiose Dehydrogenase, Horseradish Peroxidase, and Laccases in Yeasts P. pastoris and S. cerevisiae

Oxidoreductase (OXR) enzymes are in high demand for biocatalytic applications in the food industry and cosmetics (glucose oxidase (GOx) and cellobiose dehydrogenase (CDH)), bioremediations (horseradish peroxidase (HRP) and laccase (LAC)), and medicine for biosensors and miniature biofuel cells (GOx, CDH, LAC, and HRP). They can be used in a soluble form and/or within the yeast cell walls expressed as chimeras on the surface of yeast cells (YSD), such as P. pastoris and S. cerevisiae. However, most of the current studies suffer from either low yield for soluble enzyme expression or low enzyme activity when expressed as chimeric proteins using YSD. This is always the case in studies dealing with the heterologous expression of oxidoreductase enzymes, since there is a requirement not only for multiple OXR gene integrations into the yeast genome (super transformations), and codon optimization, but also very careful design of fermentation media composition and fermentation conditions during expression due to the need for transition metals (copper and iron) and metabolic precursors of FAD and heme. Therefore, scientists are still trying to find the optimal formula using the above-mentioned approaches; most recently, researcher started using protein engineering and directed evolution to increase in the yield of recombinant enzyme production. In this review article, we will cover all the current state-of-the-art technologies and most recent advances in the field that yielded a high expression level for some of these enzymes in specially designed expression/fermentation systems. We will also tackle and discuss new possibilities for further increases in fermentation yield using cutting-edge technologies such as directed evolution, protein and strain engineering, high-throughput screening methods based on in vitro compartmentalization, flow cytometry, and microfluidics.

Cultivation of recombinant Aspergillus niger strains on dairy whey as a carbohydrate source.

Agricultural waste valorisation provides a sustainable solution to waste management, and combining waste utilisation with commodity production allows for responsible production processes. Recombinant Aspergillus niger D15 strains expressing fungal endoglucanases (Trichoderma reesei eg1 and eg2 and Aspergillus carneus aceg) were evaluated for their ability to utilise lactose as a carbon source to determine whether dairy waste could be used as a feedstock for enzyme production. The recombinant A. niger D15[eg1]PyrG, D15[eg2]PyrG, and D15[aceg]PyrG strains produced maximum endoglucanase activities of 34, 54, and 34 U/mL, respectively, on lactose and 23, 27, and 22 U/mL, respectively, on whey. The A. niger D15[eg2]PyrG strain was used to optimise the whey medium. Maximum endoglucanase activity of 46 U/mL was produced on 10% whey medium containing 0.6% NaNO3. The results obtained indicate that dairy whey can be utilised as a feedstock for recombinant enzyme production. However, variations in enzyme activities were observed and require further investigation.

Development of a continuous fermentation process for the production of recombinant uricase enzyme by Pichia pastoris.

Recent studies in the biopharmaceutical industry have shown an increase in the productivity and production efficiency of recombinant proteins by continuous culture. In this research, a new upstream fermentation process was developed for the production of recombinant uricase in the methylotrophic yeast Pichia pastoris. Expression of recombinant protein in this system is under the control of the AOX1 promoter and therefore requires methanol as an inducing agent and carbon/energy source. Considering the biphasic growth characteristics of conventional fed-batch fermentation, physical separation of the growth and induction stages for better control of the continuous fermentation process resulted in higher dry-cell weight (DCW) and enhanced recombinant urate oxidase activity. The DCW and recombinant uricase activity enzyme for fed-batch fermentation were 79g/L and 6.8u/mL. During the continuous process, in the growth fermenter at a constant dilution rate of 0.025h-1 , DCW increased to 88.39g/L. In the induction fermenter, at methanol feeding rates of 30, 60, and 80mL/h, a recombinant uricase activity was 4.13, 7.2, and 0u/mL, respectively. The optimum methanol feeding regime in continuous fermentation resulted in a 4.5-fold improvement in productivity compared with fed-batch fermentation from 0.04u/mL/h (0.0017mg/mL/h) to 0.18u/mL/h (0.0078mg/mL/h).

In Search of Complementary Extraction Methods for Comprehensive Coverage of the Escherichia coli Metabolome.

Escherichia coli is an invaluable research tool for many fields of biology, in particular for the production of recombinant enzymes. However, the activity of many such recombinant enzymes cannot be determined using standard biochemical assays, as often, the relevant substrates are not known, or the products produced are not detectable. Today, the biochemical footprints of such unknown enzyme activities can be revealed via the analysis of the metabolomes of the recombinant E. coli clones in which they are expressed, using sensitive technologies such as mass spectrometry. However, before any metabolites can be identified, it is necessary to achieve as high a coverage of the potential metabolites present within E. coli as possible. We have therefore analyzed a wide range of different extraction methods against the cell free extracts of various recombinant E. coli clones. The results were analyzed to determine the minimum number of extractions that achieved high recovery and coverage of metabolites. Two methods were selected for further analysis due to their ability to produce not only high numbers of ions, but also wide mass coverage and a high degree of complementarity. One extraction method uses acetonitrile and water, in a 4:1 ratio, which is then dried down and reconstituted in the chromatography running buffer prior to injection onto the chromatography column, and the other extraction method uses a combination of methanol, water and chloroform, in a 3:1:1 ratio, which is injected directly onto the chromatography column. These two extraction methods were shown to be complementary to each other, as regards the respective metabolites extracted, and to cover a large range of metabolites.

Enzyme Engineering Strategies for the Bioenhancement of L-Asparaginase Used as a Biopharmaceutical.

Over the past few years, there has been a surge in the industrial production of recombinant enzymes from microorganisms due to their catalytic characteristics being highly efficient, selective, and biocompatible. L-asparaginase (L-ASNase) is an enzyme belonging to the class of amidohydrolases that catalyzes the hydrolysis of L-asparagine into L-aspartic acid and ammonia. It has been widely investigated as a biologic agent for its antineoplastic properties in treating acute lymphoblastic leukemia. The demand for L-ASNase is mainly met by the production of recombinant type II L-ASNase from Escherichia coli and Erwinia chrysanthemi. However, the presence of immunogenic proteins in L-ASNase sourced from prokaryotes has been known to result in adverse reactions in patients undergoing treatment. As a result, efforts are being made to explore strategies that can help mitigate the immunogenicity of the drug. This review gives an overview of recent biotechnological breakthroughs in enzyme engineering techniques and technologies used to improve anti-leukemic L-ASNase, taking into account the pharmacological importance of L-ASNase.

SCREENING THE EFFECT OF THE EXPRESSION MEDIUM AND GROWTH CONDITIONS ON THE PERFORMANCE OF ENGINEERED XYLANASE PRODUCED BY IMMOBILIZED RECOMBINANT E. COLI

Escherichia coli is the most prevalent host organism for the production of recombinant en-zymes. This was feasible due to the possibility of genetic modification and the availability of multiple E. coli strains as recombinant systems. The primary disadvantage of using E. coli as a host, however, is bacterial cell lysis due to tension build-up in the periplasmic space caused by the overexpression of the recombinant enzyme. Therefore, immobilization is preferable to cytoplasmic excretion for directing the expression of recombinant enzymes into the culture medium. This research investigated the effect of graphene oxide (GO) on the xylanase and β-galactosidase activity of immobilized recombinant E. coli. The effect of culture conditions (expression medium, IPTG, post induction temperature, post induction duration, agitation rate, and pH) on xylanase excretion and cell survival of an immobilized cell was studied using the one factor at a time (OFAT) method. After 24 hours of induction, using terrific broth (TB) as a medium increased xylanase excretion to 0.060 U/ml and resulted in decreased β-galactosidase activity (1.218 U/ml). Apart from that, a lower concentration of isopropyl -D-1-thiogalactopyranoside (IPTG) at 0.01 mM, a lower post-induction temperature (25°C), a 5-hour post-induction time, neutral pH, and 150 rpm significantly increased the xylanase excretion of immobilized cells with low β-galactosidase activity. This study established that immobilizing recombinant E. coli on GO may be advantageous for the excretion of recombinant proteins with a high cell viability.

Application of Milk Permeate as an Inducer for the Production of Microbial Recombinant Lipolytic Enzymes

Recombinantly produced enzymes are applied in many fields, ranging from medicine to food and nutrition, production of detergents, textile, leather, paper, pulp, and plastics. Thus, the cost-effectiveness of recombinant enzyme synthesis is an important issue in biotechnological industry. Isopropyl-β-D-thiogalactoside (IPTG), an analog of lactose, is currently the most widely used chemical agent for the induction of recombinant enzyme synthesis. However, the use of IPTG can lead to production of toxic elements and can introduce physiological stress to cells. Thus, this study aims to find a simpler, cheaper, and safer way to produce recombinant enzymes. In this study, production of several previously designed recombinant lipolytic enzymes (GDEst-95 esterase, GD-95RM lipase, fused GDEst-lip lipolytic enzyme, and putative cutinase Cut+SP from Streptomyces scabiei 87.22) is induced in E. coli BL21 (DE3) using 4 mM milk permeate, a type of waste of the milk manufacturing process possessing >82% lactose. The SDS-PAGE analysis clearly indicates synthesis of all target enzymes during a 2–12 h post-induction timeframe. Further investigation of GDEst-95, GD-95RM, GDEst-lip, and Cut+SP biocatalysts was carried out spectrophotometrically and using zymography method, confirming production of fully active enzymes.

Plastid Transformation: New Challenges in the Circular Economy Era

In a circular economy era the transition towards renewable and sustainable materials is very urgent. The development of bio-based solutions, that can ensure technological circularity in many priority areas (e.g., agriculture, biotechnology, ecology, green industry, etc.), is very strategic. The agricultural and fishing industry wastes represent important feedstocks that require the development of sustainable and environmentally-friendly industrial processes to produce and recover biofuels, chemicals and bioactive molecules. In this context, the replacement, in industrial processes, of chemicals with enzyme-based catalysts assures great benefits to humans and the environment. In this review, we describe the potentiality of the plastid transformation technology as a sustainable and cheap platform for the production of recombinant industrial enzymes, summarize the current knowledge on the technology, and display examples of cellulolytic enzymes already produced. Further, we illustrate several types of bacterial auxiliary and chitinases/chitin deacetylases enzymes with high biotechnological value that could be manufactured by plastid transformation.

Elimination of biodiesel contaminants by recombinant glucoside hydrolase produced from crude glycerol

This work presents the direct crude glycerol use by high cell-density fermentation of Escherichia coli expressing recombinant glycoside hydrolase that cleaves steryl glucosides, one of the main biodiesel contaminants. Optimized fermentation with crude glycerol reached the biomass concentration of 30 g/l with the 0.285 U/mgDCW specific activity, which is comparable with the pure glycerol production. The expression of the inclusion bodies was eliminated by the increase of the induction temperature to 40 °C. The produced enzyme was able to hydrolyze 300 ppm of steryl glucosides, the major biodiesel contaminants, and reached 100 % conversion. This model approach of waste carbon recycling (crude glycerol) for biorefinery-beneficial recombinant enzyme production is in line with the sustainable circular bioeconomy concept.

Production of functional recombinant prolyl hydroxylase-2 enzyme in insect cells for small molecule inhibitor screening studies

Prolyl hydroxylase-2 (PHD2) is a dioxygenase enzyme that specifically hydroxylates the hypoxia inducible factor (HIF) which then targets it for degradation in oxygenated cells. Inhibition of the activity of the PHD2 enzyme under hypoxic environmental conditions acts to upregulate HIF. Thus, PHD2 inhibitors may serve as a promising treatment for HIF-dependent diseases. In this study, recombinant PHD2 protein was successfully expressed using a baculovirus-insect cell expression secretory system. PHD2 was purified and in combination with bacterially expressed functional von Hippel Lindau protein-elongin B-elongin C (VBC) protein complex was used to successfully develop a fluorescence-based PHD2 activity assay. Myricetin was identified as a novel potent PHD2 inhibitor by high-throughput screening of a natural compound library. Further studies showed that treatment of human neuroblastoma SH-SY5Y cells with Myricetin increased HIF-1α protein levels. These results indicate that the insect cell expression system is capable of producing highly active recombinant PHD2 protein from which a fluorescence-based activity assay can be developed for high-throughput screening applications.

Studies on the recombinant production and anticancer activity of thermostable L- asparaginase I from Pyrococcus abyssi.

L-Asparaginase catalysing the breakdown of L-Asparagine to L-Aspartate and ammonia is an enzyme of therapeutic importance in the treatment of cancer, especially the lymphomas and leukaemia. The present study describes the recombinant production, properties and anticancer potential of enzyme from a hyperthermophilic archaeon Pyrococcus abyssi. There are two genes coding for asparaginase in the genome of this organism. A 918 bp gene encoding 305 amino acids was PCR amplified and cloned in BL21 (DE3) strain of E. coli using pET28a (+) plasmid. The production of recombinant enzyme was induced under 0.5mM IPTG, purified by selective heat denaturation and ion exchange chromatography. Purified enzyme was analyzed for kinetics, in silico structure and anticancer properties. The recombinant enzyme has shown a molecular weight of 33 kDa, specific activity of 1175 U/mg, KM value 2.05mM, optimum temperature and pH 80°C and 8 respectively. No detectable enzyme activity found when L-Glutamine was used as the substrate. In silico studies have shown that the enzyme exists as a homodimer having Arg11, Ala87, Thr110, His112, Gln142, Leu172, and Lys232 being the putative active site residues. The free energy change calculated by molecular docking studies of enzyme and substrate was found as ∆G - 4.5 kJ/mole indicating the affinity of enzyme with the substrate. IC50 values of 5U/mL to 7.5U/mL were determined for FB, caco2 cells and HepG2 cells. A calculated amount of enzyme (5U/mL) exhibited 78% to 55% growth inhibition of caco2 and HepG2 cells. In conclusion, the recombinant enzyme produced and characterized in the present study offers a good candidate for the treatment of cancer. The procedures adopted in the present study can be prolonged for in vivo studies.

A Protocol to Enhance Soluble Protein Expression in the Cytoplasm of Bacillus subtilis.

Bacillus subtilis is a generally regarded as safe (GRAS) microorganism, which has been used for industrial production of recombinant enzymes. Many inducible and inducer-free expression vectors have been developed for intracellular production; some of those demonstrated the capability for protein expression up to 42% of total cellular proteins. In this chapter, we introduce the method to enhance the expression of soluble protein in B. subtilis. It includes the construction of vectors, the transformation of a plasmid into B. subtilis, and checking the expression of the protein.

Enhancing the recombinant protein productivity of Yarrowia lipolytica using in situ fibrous bed bioreactor

In this study, the ability of Yarrowia lipolytica to produce the recombinant lipase CalB from Candida antarctica, used as a model protein has been compared across different bioreactor processes using glycerol, a byproduct from the biodiesel industry as the main carbon source. Batch, pulsed fed-batch (PFB), and continuous fed-batch (CFB) strategies were first compared using classical stirred tank (STR) bioreactors in terms of biomass production, carbon source uptake, and lipase production. Additionally, an in situ fibrous bed bioreactor (isFBB) was developed using sugarcane bagasse as a cell immobilization support. The maximum lipase titer achieved using the isFBB culture mode was 38%, 33%, and 49% higher than those obtained using the batch, PFB, and CFB cultures, respectively. The lipase productivity in isFBB mode (142U/mL/h) was 1.4-fold higher than that obtained using batch free cell cultures. These results highlight that isFBB is an efficient technology for the production of recombinant enzymes.

Evaluation of artificial signal peptides for secretion of two lysosomal enzymes in CHO cells

Enzyme replacement therapy (ERT) is a scientifically rational and clinically proven treatment for lysosomal storage diseases. Most enzymes used for ERT are purified from the culture supernatant of mammalian cells. However, it is challenging to purify lysosomal enzymes with sufficient quality and quantity for clinical use due to their low secretion levels in mammalian cell systems. To improve the secretion efficiency of recombinant lysosomal enzymes, we evaluated the impact of artificial signal peptides on the production of recombinant lysosomal enzymes in Chinese hamster ovary (CHO) cell lines. We engineered two recombinant human lysosomal enzymes, N-acetyl-α-glucosaminidase (rhNAGLU) and glucosamine (N-acetyl)-6-sulfatase (rhGNS), by replacing their native signal peptides with nine different signal peptides derived from highly secretory proteins and expressed them in CHO K1 cells. When comparing the native signal peptides, we found that rhGNS was secreted into media at higher levels than rhNAGLU. The secretion of rhNAGLU and rhGNS can, however, be carefully controlled by altering signal peptides. The secretion of rhNAGLU was relatively higher with murine Igκ light chain and human chymotrypsinogen B1 signal peptides, whereas Igκ light chain signal peptide 1 and human chymotrypsinogen B1 signal peptides were more effective for rhGNS secretion, suggesting that human chymotrypsinogen B1 signal peptide is the most appropriate for increasing lysosomal enzyme secretion. Collectively, our results indicate that altering signal peptide can modulate the secretion of recombinant lysosome enzymes and will enable lysosomal enzyme production for clinical use.