Introduction

Lipases (EC 3.1.1.3) are a type of lipolytic hydrolases that specifically catalyze the hydrolysis of triglycerides producing fatty acids and glycerol in an oil-water interface (Javed et al. 2018). They are also multifunctional catalysts capable of catalyzing a wide variety of reactions, such as esterification, interesterification, acidolysis, aminolysis and deacetylation (Hasan et al. 2009). Lipases have been widely and extensively applied in various industrial fields, such as food processing, biodiesel production, organic synthesis, environment, detergents, pharmaceutical, and cosmetics production etc. (Casas-Godoy et al. 2018; Chen et al. 2020). Oleates synthesis mediated by lipases is a research hotspot in recent years, because that oleates can be used in various industrial, including biodiesel, emulsifying agents and emollient esters (Khan et al. 2015; Mayilvahanan et al. 2019; Yan et al. 2016). Therefore, identification of novel lipases with high potency in oleates synthesis is of great significance from an industrial point of view.

Cold-active lipases exhibit high catalytic activities at relatively low temperatures when compared to mesophilic and thermophilic lipases (Yan et al. 2016). They have been considered to be more advantageous in esters synthesis over other lipases, mainly due to that they can reduce energy consumption and minimize side reactions (Duan et al. 2016). To date, some cold-active lipases have been isolated from various microorganisms, such as Acinetobacter radioresistens (Gupta et al. 2018), Bacillus licheniformis (Malekabadi et al. 2018), Pseudomonas sp. (Salwoom et al. 2019), Psychrobacter sp. (Zhang et al. 2018), Rhodotorula sp. (Maharana and Singh 2018) and Salinisphaera sp. (Kim et al. 2019). While most of them are derived from bacteria, and only few fungal cold-active lipases have been ever identified (Duan et al. 2016; Yan et al. 2016). Generally, the productions of cold-active lipases from wild-type strains are usually low, with productions in the range of 0.35 to 36.0 U/mL, which could not satisfy the needs for commercial production (Jain et al. 2019; Salwoom et al. 2019). Heterologous expression is a promising way to improve lipase yield (Duan et al. 2016). P. pastoris is a widely used host for the production of recombinant proteins (Borrelli and Trono 2015). Up to now, several cold-active/adapted lipases have been heterogeneously expressed in P. pastoris, such as the lipases from R. endophyticus (Yan et al. 2016) and the metagenomic library (Yi et al. 2015). However, the yields of cold-active/adapted lipases are still too low to meet the commercial production requirement.

The mesophilic fungus, Rhizopus microsporus, has been reported to secrete multiple kind of enzymes, such as lipases (Martinez-Ruiz et al. 2018), and its genome and transcriptome have been sequenced and annotated in our lab recently. In this study, a novel lipase gene (RmLipA) from R. microsporus was cloned and heterogeneously expressed in P. pastoris. The recombinant lipase was characterized and its application potential in oleates synthesis and bread making were further evaluated.

Materials and methods

Reagents

p-Nitrophenol (pNP), p-nitrophenol acetate (pNPA), p-nitrophenol butyrare (pNPB), p-nitrophenol caprylate (pNPC), p-nitrophenol decanoate (pNPD), p-nitrophenol laurate (pNPL), p-nitrophenol myristate (pNPM) and p-nitrophenol palmitate (pNPP) were bought from Sigma-Aldrich (St. Lous, MO, USA). p-Nitrophenol hexanoate (pNPH) and all tested triglycerides were the products of TCI (Tokyo, Japan). Glyceryl trioleate, oleic acid, 1,2-dioleoyl-sn-glycerol, 1,3-dioleoyl-sn-glycerol and 1-oleoyl-rac-glycerol were purchased from Sigma-Aldrich (St. Lous, MO, USA). All other regents were of analytical grade unless otherwise stated.

Strains, plasmids and media

Escherichia coli DH5α and pMD18-T vector used for plasmids construction and propagation, respectively, were the products of Biomed (Beijing, China). pPIC9K and P. pastoris GS115 (his4) (San Diego, CA, USA) were used as vector and expression host, respectively. R. microsporus was previously isolated and deposited in our laboratory.

PDA medium was used to cultivate R. microsporus. MD medium was used to select transformants. Yeast peptone dextrose-geneticin 418 (YPD-G418) medium was used to screen transformants with multiple copies. BMGY and BMMY media were used for the growth and induction of transformants, respectively. Fermentation basal salts (FBS) medium and PTM1 trace salts used for high cell density fermentation were prepared according to the method of Duan et al. (2013).

Cloning of a lipase gene from R. microsporus

The extraction of genomic DNA of R. microsporus was performed as described by Lodhi et al. (1994). A first strand of cDNA was synthesized using PrimeScript™ RT-PCR Kit (TaKaRa, Tokyo, Japan). To amplify the encoding gene, the specific primer pair RmLipAF (5’-AAATACGTAGTTCCTGTTGCTGGTCATAAAGG-3’) and RmLipAR (5’-AAAGCGGCCGCTTAAAGACAGCTGCCTTCGTTAATAC-3’) were designed based on the genomic sequence of R. microsporus. Polymerase chain reaction (PCR) was performed as described by Duan et al. (2019). The product was purified, cloned into pMD19-T vector and verified by sequencing.

Sequence alignments were done using BLAST server (http://www.ncbi.nlm.nih.gov/blast). The theoretical molecular weight and isoelectric point (pI) were predicted at ExPASy server using ProtParam tool (http://web.expasy.org/protparam/).

Heterologous expression of the lipase gene in P. pastoris

The coding sequence of RmLipA was transferred into P. pastoris expression vector pPIC9K using EcoRI and NotI sites. Electroporation and transformation of P. pastoris GS115 cells were conducted according to the operation manual of Invitrogen. The tansformants were coated on YPD-G418 plates (containing 1, 2, 4 and 6 mg/mL of G418, respectively) at 30 °C for 3–5 days. The colonies with G418 resistance were picked out, inoculated into 5 mL BMGY medium, and then cultured at 30 °C and 200 rpm. After cultured for 24 h, 1 mL medium was transferred into 10 mL BMMY medium and 50 µL of methanol was added every 24 h. The culture broth was centrifuged at 10,000×g for 10 min after 72 h incubation, and the lipase activity of the supernatant was determined.

High-cell density fermentation

The transformant with the highest lipase activity was selected and inoculate into a 5-L fermentor for high-cell density fermentation (Version B, 053002, Invitrogen). To produce the target enzyme efficiently, a two-step cultivation strategy as described by Yan et al. (2016) was adopted. Briefly, a glycerol fed-batch phase was used to obtain high cell density in the first step, and methanol fed-batch phase was then started to induce accumulation of the target enzyme in the second step. The wet cell mass, protein concentration and lipase activity of samples collected every 12 h were assayed. Meanwhile, the proteins in the culture were analyzed by SDS-PAGE.

Purification of the recombinant enzyme

The crude enzyme solution was collected by centrifugation at 10, 000 × g (4 °C for 10 min), dialyzed against 20 mM Tris-HCl buffer (pH 7.5, buffer A) over light, and then subjected to a Q sepharose fast flow (QSFF) column (1 × 10 cm) chromatography. The unbound or weakly bound impurities were washed off by buffer A, and the bound proteins were then eluted by buffer A containing 150 mM NaCl. The fractions displaying lipase activity were collected, and the purity was examined by SDS-PAGE. The flow rate in the whole purification process was maintained at 1 mL/min.

Enzyme activity assay

Lipase activity was determined according to the method of Duan et al. (2019). Namely, 50 µL suitably diluted enzyme solution was mixed with 400 µL 50 mM Tris-HCl buffer (pH 8.0) and 50 µL 10 mM pNPL, and incubated at 25 °C for 10 min. Then, the reaction was stopped by the adding 500 µL moving alkaline copper phosphate suspension. After centrifugation at 10,000×g for 5 min, the absorbance of the solution at 410 nm was measured. One unit of lipase activity was defined as the amount of enzyme capable of releasing 1 µmol pNP per minute at 25 °C, pH 8.0.

The protein concentration was measured by the method of Lowry et al. (1951) using bovine serum albumin as the standard.

Molecular mass determination

The denatured molecular mass of RmLipA was determined by SDS-PAGE using 12.5% separating gel. Proteins were stained with coomassie brilliant blue solution, and decolorized with methanol/acetic acid/water (50/14/136, v/v/v) solution. The native molecular mass of RmLipA was estimated by molecular sieve chromatography using a Sephacryl S100 column (1 × 40 cm, GE Healthcare). 400 µL sample or standard protein solutions (about 4 mg/mL) were loaded onto the column, separately, and eluted by 20 mM phosphate buffer pH 7.0 containing 150 mM NaCl at a flow velocity of 0.33 mL/min. The protein molecular mass calibration kit contained phospholipase b (97.2 kDa), bovine serum albumin (66.4 kDa), fetuin from fetal bovine serum (48.9 kDa), α-chymotrypsinogen A (25.6 kDa) and cytochrome C (12.4 kDa).

Biochemical characterization of RmLipA

The effect of pH on the activity of RmLipA was investigated by measuring its activity at 25 °C in various buffers, namely, glycine-HCl (pH 2.0–3.5), citrate (pH 3.0–6.0), phosphate (pH 6.0–8.0), Tris-HCl (pH 7.0–9.0), tricine (pH 7.0–9.0), 2-(cyclohexylamino) ethanesulfonic acid (CHES, pH 8.0–10.0), and 3-(cyclohexylamino)-1-propanesulphonic acid (CAPS, pH 10.0–11.0). The pH stability of RmLipA was evaluated by measuring the residual activity under the standard conditions after incubating the enzyme at 25 °C for 30 min in the above mentioned buffers. To investigate the optimal temperature, the lipase activity of RmLipA was assayed at a temperature range of 0–50 °C in 50 mM pH 8.0 Tris-HCl buffer. For thermal stability determination, RmLipA was incubated in 50 mM pH 8.0 Tris-HCl buffer at 0–60 °C for 30 min, and the retained activity was then assayed under the standard conditions. To determine the thermal denaturing half-lives, RmLipA was incubated in 50 mM pH 8.0 Tris-HCl buffer at 45 °C, 50 °C, and 55 °C for 8 h, respectively, and the retained activities of the samples taken at different times were then determined by the standard enzyme assay.

To evaluate the influence of surfactants and organic solvents on the enzyme activity, RmLipA was incubated in 50 mM Tris-HCl buffer (pH 8.0) with 30% (v/v) different surfactants or organic solvents at 25 °C for 1 h, and the retained activities were then assayed. The effect of mental ions was investigated by determining the residual enzyme activity of RmLipA after treatment in 50 mM pH 8.0 Tris-HCl buffer with 1 mM various mental ions at 25 °C for 1 h.

Substrate specificity RmLipA

Substrate specificity of RmLipA was determined using different substrates at 25 °C. For pNP esters, the enzyme activity was assayed by measuring the amount of pNP released according to the method of Duan et al. (2019) in 50 mM Tris-HCl buffer pH 8.0. For triglycerides, the enzyme activity was determined by measuring the amount of fatty acid released. Namely, 50 µL of appropriately diluted enzyme solution was mixed with 20 mL 2.5 mM Tris-HCl buffer (pH 8.0) containing 10 mM triglycerides, 0.1% (w/v) arabic gum and 0.1% (w/v) Triton X-100, and incubated at 25 °C for 15 min with a shaking speed of 180 rpm. The reaction was terminated by the addition of 95% (v/v, 15 mL) ethanol solution. After that, the reaction mixture was titrated using 10 mM NaOH solution with phenolphthalein as indicator. One unit of enzyme activity was defined as the amount of enzyme requiring to release1 µmol pNP or free fatty acid per minute under the conditions mentioned above.

The positional specificity of RmLipA was analyzed by identifying the hydrolysis products of triolein. 1 mL reaction solution containing 100 U RmLipA and 100 mM triolein in 50 mM Tris-HCl buffer (pH 8.0) were incubated at 25 °C for 12 h, and then extracted with 1 mL n-hexane. After centrifugation (4000×g) at 4 °C for 5 min, 3 µL of solution in upper layer was subjected to TLC analysis. A mixture of petroleum ether/ether/acetic acid (16/4/0.1) was used as developing solvent. The products were visualized by immersing the plate in methanol/sulfuric (95/5) solvent, followed by heating at 180 °C for a few seconds.

Oleates synthesis by RmLipA

The synthesis of oleates was performed as described by Cea et al. (2019) using oleic acid and alcohols with different carbon chain lengths. Esterification reaction was carried out in 1 mL isooctane containing 1 mM oleic acid, 2 mM alcohol, 50 U lipase and 30–50 mg 4Å molecular sieve. The reaction was terminated by adding 15 mL of 95% (v/v) ethanol solution after incubation at 25 °C for 24 h at 180 rpm. The synthesized products were qualitatively analyzed by TLC. 1 µL of samples were spotted onto a TLC silica gel plate, and developed in a solvent system of hexane/ether/formic acid (80/15/1, v/v/v). The products were visualized by heating plates in an oven at 180 °C for a few seconds after immersion in methanol/sulfuric acid (95/5, v/v) solvent.

The oleate yield was expressed as the conversion rate of oleic acid in the reaction system. Oleic acid content was determined by HPLC as described by Khan et al. (2015). Briefly, the samples were collected, dried at room temperature, re-dissolved in 1 mL of acetonitrile/acetone (1/1, v/v), and then subjected to HPLC analysis using a C18 column (4.6 × 250 mm). The column oven was set at 35 °C. The mobile phase used was acetonitrile/acetone (50/50, v/v) at a flow rate of 1.1 mL/min.

Application of RmLipA in bread making

To evaluate the effect of RmLipA on the quality of bread, different amount of RmLipA (75–1000 U kg/flour) was firstly dissolved in water, and the enzyme solution was then added in flour mixture for dough preparation. The bread formulation is 700 g wheat flour, 406 g water, 70 g sugar, 28 g butter, 8.4 g dry yeast and 7 g NaCl. The mass, volume and crumb firmness of bread were measured according to the method of Jiang et al. (2008). Bread staling was analyzed by measuring the crumb firmness of breads after stored at 4 °C for 0, 2 and 4 days. Each sample was measured in triplicate and the results were expressed as means ± SD.

Results

Cloning and sequence analysis of a lipase gene from R. microsporus

A lipase gene (RmLipA) from R. microsporus was obtained (NCBI accession no. ORE19568.1). RmLipA has an open reading frame (ORF) of 1, 170 bp, encoding a protein of 389 amino acids. There is a predicted 26-amino acids signal peptide in its N-terminal. The theoretical pI and molecular weight of the deduced protein were estimated to be 6.78 and 42.1 kDa, respectively. There are three possible N-glycosylation sites (N40, N86 and N289) in the protein sequence (Supplementary Fig. 1).

Sequence alignments revealed that Asp259, Ser265 and His324 formed the catalytic triad, and Ser265 located in the conserved GHSLG pentapeptide motif. RmLipA exhibited the highest similarity of 75.3% with Rhizopus niveus lipase (P61871.1), followed by the lipases from Rhizomucor miehei (49.6%, P19515.2), Aspergillus terreus (35.6%, Q0CBM7.1), Aspergillus awamori (34.8%, Q9P979.3), Aspergillus oryzae (33.2%, Q2UNW5.1) and Thermomyces lanuginosus (33.0%, O59952.1) (Supplementary Fig. 2). Therefore, RmLipA should be a novel lipase.

High-level expression of RmLipA in P. pastoris

A transformant secreted the highest yield of RmLipA was selected (from about 300 positive transformants) and inoculated in a 5-L fermentor. The highest lipase activity of 7, 931 U/mL was obtained after 168 h of methanol induction (protein concentration 10.9 mg/mL, wet cell weight 432 g/L) (Fig. 1A). The target protein accounted for approximately 90% of the total proteins in the culture broth as indicated on SDS-PAGE (Fig. 1B).

Fig. 1
figure 1

Time course profile of RmLipA expression in P. pastoris (A) and SDS-PAGE analysis of the secreted proteins (B) in the culture broth. Symbols represent enzyme activity (black square), protein concentration (white circle) and cell wet weight (black triangle). Lane M, low molecular weight protein markers; lanes 1–9, samples withdrawn at 0, 24, 48, 72, 96, 120, 144, 168 and 192 h after induction, respectively

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Purification of RmLipA

RmLipA was purified to homogeneity with a purification fold of 1.1 and a recovery yield of 64.8% (Supplementary Table 1). The denatured molecular mass of RmLipA was estimated to be 40.2 kDa on SDS-PAGE (Fig. 2), which is consistent with the native molecular mass (42.2 kDa) determined by molecular sieve chromatography, suggesting that the lipase should be a monomeric protein.

Fig. 2
figure 2

Purification of RmLipA. Lane M, low molecular weight protein calibration kit; line 1, crude enzyme; line 2, purified enzyme

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Biochemical characterization of RmLipA

RmLipA was optimally active at pH 8.0 in 50 mM Tris-HCl buffer (Fig. 3A), and showed excellent stability within the pH range of 2.0–11.0 as it retained > 80% of its maximal activity (Fig. 3B). The enzyme was most active at 20–25 °C, and showed 81% of its maximum activity at 0 °C (Fig. 3C). It was stable at the temperatures ≤ 50 °C (Fig. 3D). The thermal denaturing half-lives of RmLipA at 45 °C, 50 and 55 °C were calculated to be 582 min, 268 min, and 28 min, respectively (Fig. 3E).

Fig. 3
figure 3

Biochemical characterization of RmLipA. A Optimal pH, the optimal pH was determined by measuring the enzyme activity at 25 °C in 50 mM different buffers with different pHs. B pH stability, for pH stability, the enzyme was incubated at 25 °C for 30 min in different buffers, and the residual activities were then measured at 25 °C in 50 mM Tris-HCl buffer pH 8.0. C Optimal temperature, the optimal temperature was determined in 50 mM Tris-HCl buffer pH 8.0 at a temperature range of 0–50 °C. D Thermostability, the residual activities were measured after the enzyme was incubated in 50 mM Tris-HCl pH 8.0 at different temperatures. E Thermal denaturation half-lives, the enzyme was incubated in 50 mM Tris-HCl buffer pH 8.0 at 45 °C (black square), 50 °C (black circle), 55 °C (black triangle) for 8 h, and the residual activities of the samples withdrawn at different time intervals were then determined. The buffers used were glycine-HCl (white circle, pH 2.0–3.5), citrate (black triangle, pH 3.0–6.0), sodium phosphate (white square, pH 6.0–8.0), Tris-HCl (black circle, pH 7.0–9.0), Tricine (black square, pH 7.0–9.0), CHES (white diamond, pH 8.0–10.0) and CAPS (black down-pointing triangle, pH 10.0–11.0)

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The lipase activity of RmLipA was significantly activated by Tween 80 (191.1%), Triton X-100 (166.2%) and Tween 60 (147.3%). In contrast, RmLipA was strongly inhibited by formic acid (28.9%), acetic acid (20.0%) and propionic acid (9.9%). RmLipA was completely inactivated by ethanol, n-propanol, isopropanol and butyric acid (Supplementary Table 2).

Substrate specificity and positional specificity of RmLipA

RmLipA exhibited high hydrolytic activity towards pNP esters with long and medium chain fatty acids (Table 1). It exhibited the highest specific activity of 750 U/mg (100%) towards pNPC and pNPL, followed by pNPH (82%), pNPD (65%), pNPB (41%), pNPP (37%) and pNPM (18%), while could hardly hydrolyze pNPA (1%) (Table 1). For triglycerides, RmLipA preferentially hydrolyzed tricaprylin with a maximal activity of 852 U/mg (100%), followed by tricaproin (31%) and tributyrin (21%) (Table 1). The enzyme activity of RmLipA declined sharply with the increase of acyl carbon chain lengths of the substrates from 8 to 16, and almost negligible activity was detected towards tripalmitin. In addition, RmLipA displayed relatively high specific activity towards (251 U/mg).

Table 1 Substrate specificity of RmLipA from R. microsporus
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RmLipA catalyzed the hydrolysis of triolein to release only 1,2-diolein without 1,3-diolein (Supplementary Fig. 3), indicating that RmLipA should be a sn-1,3 regiospecific lipase.

Synthesis of oleates

RmLipA catalyzed the synthesis of oleates using oleic acid and various alcohols as the substrates (Fig. 4). The yields of all formed oleates reached > 95% (Table 2), suggesting that RmLipA should be a good candidate in oleate synthesis.

Fig. 4
figure 4

TLC analysis of the synthesized oleates by RmLipA. Lanes S, oleic acid standard; lane S1, control carried out without enzyme; lane S2, reactions with RmLipA. The reactions were performed in 1 mM alcohol/acid (molar ratio, 2:1) solution at 25 °C for 24 h

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Table 2 Synthesis of oleates by RmLipA from R. microsporus
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Application of RmLipA in bread making

Addition of RmLipA (250 U/kg flour) resulted in a maximal increase of 21.7% in specific volume (Fig. 5A) and a decrease of 28.6% in crumb firmness (Fig. 5B) compared to the control. Besides, RmLipA also significantly decreased the crumb firmness of the bread during the 4-days storage period (Fig. 5C), suggesting that RmLipA delayed the aging of bread.

Fig. 5
figure 5

Effect of RmLipA dosages on the specific volume (A) and hardness (B) of fresh bread, and the hardness changes of bread in the storage process (C). The hardness of the bread was measured at 0 day (white square), 2 days (grey square) and 4 days (black square), respectively

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Discussion

Lipases have attracted great attention in recent years owing to their diverse applications in various fields, including food processing, ester synthesis, biodiesel production, detergents and pharmaceutical industries (Casas-Godoy et al. 2018). To date, many lipases have been identified and biochemically characterized, while few of them are cold-active lipases (Yan et al. 2016). In this study, we describe gene cloning, heterologous expression, characterization and application of a novel cold-active lipase (RmLipA) from R. microsporus.

Generally, the enzyme yield is a key factor affecting its industrial applications, but the enzyme yields from wide-type strains are relatively low (Jain et al. 2019; Salwoom et al. 2019). In order to increase lipase yield for commercial production, several cold-active/adapted lipase genes have been heterologously expressed. For example, the cold-adapted lipases ReLipA and ReLipB from R. endophyticus were expressed in P. pastoris, with yields of 1961 U/mL (Yan et al. 2016) and 1395 U/mL (Duan et al. 2016), respectively. A cold-lipase gene from the metagenomic library derived from deep sea was heterologously expressed in P. pastoris, with the highest yield of 2760 U/mL, which is the highest yield for active/adapted lipase before this study (Yi et al. 2015). In the present study, RmLipA was heterologously expressed in P. pastoris, and the highest lipase yield of 7931 U/mL was achieved (Fig. 1). The value is much higher than that of most other known cold-active lipases (Duan et al. 2016; Yan et al. 2016; Yi et al. 2015), but still lower than that of several mesophilic lipases, such as the lipases from R. oryzae (12, 019 U/mL, Yu et al. 2013; 33,900 U/mL, Jiao et al. 2018). For R. oryzae lipases production, the yields were significantly increased by using the efficient expression strategies of co-expression of molecular chaperones (Jiao et al. 2018) and optimization of nitrogen source (NH4+) concentration ( Yu et al. 2013), respectively.

Generally, cold-active lipases exhibit the highest activities at the temperatures ≤ 30 °C (Cai et al. 2009). RmLipA was most active at 20–25 °C (Fig. 3 C), consisting with that of Lipase-A from Geotrichum sp. (Cai et al. 2009), lipase-B from Rhizomucor endophyticus (Duan et al. 2016) and the lipase from Y. enterocolitica (Ji et al. 2015). It is interesting that RmLipA displayed 81% of its maximum activity (observed at 25 °C) at 0 °C (Fig. 3 C). This value is similar to that of lipase-B from Geotrichum sp. (80%, Cai et al. 2009) and the cold-active lipase from P. cryohalolentis (80%, Novototskaya-Vlasova et al. 2013), but significantly higher than that of most other cold-active lipases, such as the lipases from Aspergillus nidulans (30%, Sugihara et al. 1991), Geotrichum sp. (58%, lipase-A, Cai et al. 2009) and R. endophyticus (75%, Yan et al. 2016). In addition, RmLipA exhibited good stability up to 50 °C (Fig. 3D). The temperature is higher than that of most other reported cold-active lipases, only next to that of the cold-active lipases from R. endophyticus (55 °C, Yan et al. 2016) and Geotrichum sp. P7 (100 °C, Florczak et al. 2013). These excellent properties may make RmLipA an attractive candidate in commercial uses, especially those should be carried out in relatively low temperatures, such as dairy production, cheese making and detergents.

Lipases are distinct from esterases in their substrate specificity: lipases preferentially hydrolyze water-insoluble triacylglycerols with long chains (C8–C18), while esterases tend to exhibit high activity on water-soluble triacylglycerols with short chain (< C8) (Jain et al. 2019; Yan et al. 2016). RmLipA exhibited the highest activity towards tricaprylin (C8), pNPC (C8) and pNPL (C12) (Table 1), suggesting that it should be a true lipase. RmLipA showed relatively high activities towards substrates with long and medium carbon chain lengths, which is in accordance to most other known cold-active lipases, such as the lipases from R. endophyticus (C8, Yan et al. 2016), P. palleroniana (C10, Jain et al. 2019), B. licheniformis (C10, Malekabadi et al. 2018) and Pseudomonas sp. (C12, Salwoom et al. 2019). However, there are also several cold-active lipases showed maximal activity toward pNP esters with relatively short carbon chain lengths (Cai et al. 2009). Lipases from different sources exhibited regiosepcificity on the ester bonds of glycerides. RmLipA is a sn-1,3 regiospecific lipase, which is similar to that of most other known cold-active lipases (Yan et al. 2016). Several lipases have reported to exhibit sn-2 regiospecificity and non-regiospecificity. For instance, the lipase from M. cinnamomea is a non-regiospecific lipase (Duan et al. 2019), while the lipase from Geotrichum candidum is sn-2 regiospecific lipase (Sugihara et al. 1991). It has been reported that the lipases with different regiosepcificity are preferred in the synthesis of functional structured lipids (Stergiou et al. 2013). Therefore, RmLipA may be an attractive candidate in fat and oil modification industries.

Oleates have exhibited great application potential in various industries, such as biodiesel, emulsifying agents and emollient esters, attracting more and more attention in recent years (Andualema and Gessesse 2012; Marin-Suarez et al. 2019). Up to now, many novel lipases with high oleates synthesis ability have been identified and put into commercial uses. For example, the lipase from R. microsporus was identified and used to catalyze the synthesis of ethyl oleate, and the yield reached up to 90% (Martinez-Ruiz et al. 2018). A lipase from Rhizomucor endophyticus was identified and found to be able to catalyze the synthesis of butyl oleate, with a maximum yield of 82.2% (Yan et al. 2016). A commercial lipase from Candida antarctica (CALB) was used to catalyze the esterification reaction using oleic acid and n-pentanol as the substrates, and the highest pentyl oleate yield of 39.9% was obtained (Cavallaro et al. 2019). In the present study, RmLipA efficiently catalyzed the synthesis ten kind of oleates, with conversion ratios all over 95% (Table 2). Therefore, RmLipA may have great application potential in oleate synthesis in different industries.

It has been concluded that lipases can degrade the major lipids in wheat flour (monogalactosyldiacylglycerols and N-acyl phosphatidylethanolamines) into polar lipids, thus enhance the gluten-starch plasticization and increase the gas retention of bread (Melis et al. 2019; Serventi et al. 2019). Hence, the application of RmlipA in bread making was further assessed. The addition of RmlipA in bread dough significantly increased the specific volume of the bread by 21.7%, and decreased the crumb firmness by 28.6% under the optimal enzyme dosage (250 U/kg) (Fig. 5 A and B). In addition, RmlipA obviously retarded the staling of bread in the storage period (Fig. 5C). Similar results have also been reported in previous studies. For example, a lipase from Fusarium oxysporum was applied in bread making, and the specific volume of the bread was increased by approximately 15% with 200–700 ppm enzyme dosage (Melis et al. 2019). A similar conclusion was also obtained in the study of Serventi et al. (2019) that addition of lipase can significantly improve the loaf volume, crumb softness and elasticity of bread. Therefore, RmlipA may be a good candidate in bread making as an efficient green biological additive.

Conclusions

A novel cold-active lipase (RmLipA) from R. microsporus was identified and high-level expressed in P. pastoris, and characterized. The highest lipase yield of 7931 U/mL was obtained in a 5-L fermentor. RmLipA was optimally active at pH 8.0 and 20–25 °C, and it retained over 80% of its maximal activity at 0 °C. RmLipA exhibited a sn-1,3 regiospecificity manner and could efficiently synthesize various oleates from oleic acid and different alcohols. RmLipA significantly increased the quality of the bread by increasing its specific volume and decreasing its crumb firmness. The excellent properties of RmLipA may possess it great potential in oleates synthesis and bread making fields.